@article {pmid41364162,
year = {2025},
author = {Murtaza, M and Gupta, P and Choudhary, P and Manzoor, M and Sharma, S and Jaglan, S},
title = {Strategies to decipher silent biosynthetic gene clusters in actinomycetes.},
journal = {Archives of microbiology},
volume = {208},
number = {1},
pages = {53},
pmid = {41364162},
issn = {1432-072X},
mesh = {*Actinobacteria/genetics/metabolism ; *Multigene Family/genetics ; *Biosynthetic Pathways/genetics ; Anti-Bacterial Agents/biosynthesis ; CRISPR-Cas Systems ; },
abstract = {Actinobacteria have a huge, mainly untapped potential for the production of secondary metabolites. These metabolites are an important source of bioactive compounds. However, a majority of biosynthetic gene clusters (BGCs) are either under-expressed or fully silent under standard laboratory conditions, limiting their potential. The present review article aims to explore the biosynthetic gene clusters (BGCs) of actinobacteria using strategies that aid in unlocking these silent BGCs. The strategies discussed are PCR-Targeted Gene Replacement (PCR-TR); Cre-LoxP recombination system; Transcription factor decoys, Ribosome engineering, and CRISPR/Cas technologies. Besides, elicitors also helped with the identification of these cryptic or silent BGCs and advanced our ability to explore these natural products. Combining experimental and computational platforms provides an opportunity to unlock the hidden chemical diversity in nature, thereby accelerating the identification of new bioactive substances. The new antibiotics explored by all the strategies could help in the fight against antimicrobial resistance (AMR).},
}
@article {pmid41362674,
year = {2026},
author = {Toofan, P and Singh, M and Brooks, A and McLuckie, K},
title = {Non-clinical safety considerations on genome editing using the CRISPR/Cas system.},
journal = {Genes & diseases},
volume = {13},
number = {2},
pages = {101785},
pmid = {41362674},
issn = {2352-3042},
abstract = {Recent advances in gene editing using the CRISPR/Cas system have revolutionized genome editing, opening new horizons for human cellular and gene therapy products. Genome editing technologies are rapidly being adopted in clinical trials. However, critical non-clinical safety considerations are required to address challenges in translating research to the clinic. Here, we review current ex vivo and in vivo genome editing approaches using the CRISPR/Cas system and discuss the practical use of these methods in pre-clinical studies and in the clinic. We also discuss known limitations of genome editing in humans and the mitigation of risk factors associated with it from a non-clinical safety perspective. This review aims to aid researchers in acquiring a perspective that is essential for the safe translation of genome editing to the clinic.},
}
@article {pmid41361988,
year = {2025},
author = {Min, YH and Lee, DG and Lee, HY and Yoo, JH and Lee, KH and Shin, YB and Byun, JY},
title = {CRISPR/Cas12a with Antisense Oligonucleotide-Regulated Translational Amplification for Ultrasensitive Nucleic Acid Detection.},
journal = {ACS sensors},
volume = {},
number = {},
pages = {},
doi = {10.1021/acssensors.5c03081},
pmid = {41361988},
issn = {2379-3694},
abstract = {Highly sensitive nucleic acid testing-assisted early disease detection is crucial for effective disease prevention and management, particularly when targeting low-abundance genetic materials in molecular diagnostics. This study describes CRATE (CRISPR/Cas controlled antisense oligonucleotide (ASO)-mediated translational signal enhancement), a novel ultrasensitive approach for nucleic acid detection by integrating Cas12a trans-cleavage, ASO-controlled gene expression, and cell-free signal protein amplification. This assay leverages the target-induced trans-cleavage of ASO-controlled gene expression for the amplification of signal proteins, with luminescent signal allowing for attomolar-level target DNA detection, as well as antigenic protein application enabling visual detection by lateral flow assay. The CRATE assay improves sensitivity using ASO-modified locked nucleic acid, achieving a 10-aM-level DNA detection. The proof of concept demonstrates 0.1 copies/μL detection of HPV genomic DNA from HPV-positive cancer cells as well as colorimetric lateral flow tests with ∼10 copies/μL sensitivity. The CRATE assay can detect the HBV target in plasma from HBV-positive patients with 100% sensitivity and specificity. With high specificity and accuracy, the CRATE assay retains the potential for detecting any nucleic acid of interest. By integration of precise CRISPR-based cleavage, ASO regulation, and efficient protein signal amplification, this approach provides a promising solution for highly selective and sensitive nucleic acid detection and potential applications in clinical diagnostics and point-of-care testing.},
}
@article {pmid41361700,
year = {2025},
author = {Ain, QU and McCarthy, A and Nadeem, A and Javed, M and Niakan, K and Nashta, AF},
title = {CRISPR/Cas9-mediated generation of GATA3 knockout in Bovine Fibroblast and MDBK cell lines to assess sgRNAs targeting efficiency.},
journal = {Functional & integrative genomics},
volume = {25},
number = {1},
pages = {269},
pmid = {41361700},
issn = {1438-7948},
mesh = {Animals ; Cattle ; *CRISPR-Cas Systems ; *GATA3 Transcription Factor/genetics/metabolism ; Fibroblasts/metabolism/cytology ; Cell Line ; *Gene Knockout Techniques ; *RNA, Guide, CRISPR-Cas Systems/genetics ; Gene Editing/methods ; },
abstract = {GATA3 is expressed in the outer cells of the morula stage during embryonic development and is considered a key driver of the regulation of early lineage development in bovines. This research presents an optimised somatic cell validation resource, successfully generating GATA3 knockout (KO) Bovine Fetal Fibroblasts and MDBK cells using CRISPR/Cas9-mediated genome editing for their future implications in vivo studies designed to definitively understand the role of GATA3 in cell lineage specification and bovine embryo development. This involved designing single-guide RNAs (sgRNAs) targeting different regions of the GATA3 gene, cloning them into the px459 plasmid, delivering the CRISPR clone into bovine fibroblast cells and the MDBK cell line, screening for successful targeting and knockouts, and MiSeq analysis to verify successful disruption of the GATA3 gene. A total of eleven guides were designed targeting the functional domains in Exons 4 and 5 and the transcription initiation site in Exon 2. Designed guides were first optimized and screened using an in vitro cleavage assay. The guides with the best cutting efficiencies were then tested in vivo by targeting bovine fetal fibroblast (BFFs) and MDBK cell line followed by MiSeq analysis to verify the successful knockouts. A total of two effective guides were identified targeting the zinc-finger (ZnF) functional domains of the GATA3 gene (sgRNA#5 and sgRNA#8 in Exon 4 and Exon 5, respectively) and one in Exon 2 (sgRNA#1) targeting the transcription initiation site of the GATA3 gene. MiSeq data from targeted bovine cells showed indel frequency of 47.40%, 55.5%, and 42.4% in bovine fetal fibroblasts, 11.03%, 28.9% and 7.3% for MDBK cells for top three sgRNAs. Overall, MiSeq data for 3 selected sgRNAs showed successful disruption of the GATA3 gene, inserting a base pair 2-3 bp upstream of the PAM site, ultimately resulting in a premature stop codon TAA in the downstream region. This study established and validated highly efficient sgRNAs targeting the GATA3 gene, forming a molecular basis for forthcoming functional investigations in bovine embryos to explore gene function and protein-level effects.},
}
@article {pmid41361167,
year = {2025},
author = {Fast, L and Omar, M and Kanis, P and Schaffer, T and Chowdhury, D and Rakava, E and Pääbo, S and Riesenberg, S},
title = {Search-and-remove genome editing allows selection of cells by DNA sequence.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {10985},
pmid = {41361167},
issn = {2041-1723},
mesh = {*Gene Editing/methods ; Humans ; Animals ; CRISPR-Cas Systems/genetics ; Neanderthals/genetics ; Mutation, Missense ; CRISPR-Associated Protein 9/metabolism ; Base Sequence ; Cell Line, Tumor ; },
abstract = {The selection of cells that have acquired a desired gene edit is often done by the introduction of additional genes that confer drug resistance or encode fluorophores. However, such marker genes can have unintended physiological effects and are not compatible with editing of single nucleotides. Here, we present SNIPE, a method that allows the marker-free selection of edited cells based on single nucleotide differences to unedited cells. SNIPE drastically enriches for cells, which have been precisely edited (median 7-fold). We validate the approach for 42 different edits using Cas9 or Cas12a in different cell types and species. We use it to enrich for combinations of substitutions that change missense mutations carried by all people today back to the ancestral state seen in Neandertals and Denisovans. We also show that it can be used to kill cultured tumor cells with aberrant genotypes and to repair heterozygous tumorigenic mutations.},
}
@article {pmid41359835,
year = {2025},
author = {Roura-Martinez, D and Popa, N and Jaouen, F and Rombaut, C and Lepolard, C and Bachar, D and Borges, A and Cazorla, M and Villet, M and Moreno, S and Marie, H and Gascon, E},
title = {Combination of Cas9 and adeno-associated vectors enables efficient in vivo knockdown of precise miRNAs in the rodent and primate brain.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {122},
number = {50},
pages = {e2513076122},
doi = {10.1073/pnas.2513076122},
pmid = {41359835},
issn = {1091-6490},
support = {ANR-22-CE17-0034//Association Nationale de la Recherche et de la Technologie (ANRT)/ ; 6239//Fondation France Alzheimer/ ; 2022//Fondation Recherche Alzheimer/ ; },
mesh = {Animals ; *MicroRNAs/genetics/metabolism ; *CRISPR-Cas Systems/genetics ; *Brain/metabolism ; Mice ; Genetic Vectors/genetics ; *Gene Knockdown Techniques/methods ; *Dependovirus/genetics ; Rats ; RNA, Guide, CRISPR-Cas Systems/genetics ; Neural Stem Cells/metabolism ; Neurons/metabolism ; Primates ; Receptors, AMPA/metabolism ; Olfactory Bulb/metabolism ; CRISPR-Associated Protein 9/metabolism ; },
abstract = {microRNAs (miRNAs) are key regulators of multiple biological functions. Although intensively studied, inactivating miRNAs in vivo is particularly challenging, especially in the brain. Here, we designed cell-specific tools aiming at downregulating defined miRNA species in vivo and investigating their function in discrete neuronal networks. Focusing on miR-124, a miRNA highly expressed in the mammalian brain and transcribed from three independent chromosomal loci, we designed and validated different guide RNAs. In vivo, our CRISPR-Cas9 designs strongly downregulate miR-124 levels without affecting the expression of other miRNAs. As a result, levels of endogenous miR-124 targets exhibit a significant increase supporting the release of its silencing activity. We provide evidence that specific deletion of miR-124 in neural stem cells of the subventricular zone altered migration of newly generated neurons into the olfactory bulb. We also showed that our vectors modified the Ca[2+] permeability of AMPA receptors, a robust functional output downstream of miR-124. We also extended our approach to other miRNAs, mammalian species, and Cas9 proteins, confirming the versatility of CRISPR-Cas9. These tool properties support their potential for elucidating miRNA functions in complex experimental in vivo settings such as brain networks.},
}
@article {pmid41359384,
year = {2025},
author = {Ichinose, M and Ohta, M and Shimajiri, Y and Akaiwa, Y and Nakamura, I and Shimamoto, M and Makinoda, R and Ozaki, S and Tamai, T and Maekawa, N and Tonomoto, M and Nakamura, T and Yagi, Y and Gutmann, B},
title = {RECODE: a programmable guide-free C-to-U RNA editing tool.},
journal = {Nucleic acids research},
volume = {53},
number = {22},
pages = {},
pmid = {41359384},
issn = {1362-4962},
support = {//EditForce, Inc/ ; },
mesh = {*RNA Editing ; Humans ; Animals ; Mice ; *Cytidine Deaminase/genetics/metabolism/chemistry ; RNA, Guide, CRISPR-Cas Systems/genetics ; CRISPR-Cas Systems ; *Uridine/genetics/metabolism ; HEK293 Cells ; *Cytidine/metabolism/genetics ; RNA-Binding Proteins/genetics/metabolism ; Gene Editing/methods ; },
abstract = {Programmable RNA cytidine deaminase tools have been developed to convert cytidine-to-uridine (C-to-U) using CRISPR systems with guide RNAs. These tools, however, have limitations such as low editing efficiency, limited targetable sequence flexibility, and off-target RNA editing. Here, we present a novel guide-free C-to-U editing tool, named RECODE (RNA Editor for C-to-U with an Optimized DYW Enzyme), based on the RNA-binding pentatricopeptide repeat proteins, naturally fused to a C-terminal DYW cytidine deaminase domain. The RECODE specificity domain was engineered to enable retargeting, while its length and sequence were optimized to reduce off-target effects. Further optimization of the C-terminal catalytic region increased both the editing activity and the translation of the edited RNA. We showed that RECODE efficiently edits a wide range of targets in human cells, without affecting adjacent cytidines. It achieved over 50% editing efficiency for most sites, except those with an upstream guanine. Furthermore, we showed that RECODE is functional in mice, with high editing efficiency observed in specific tissues such as skeletal muscles using an AAV delivery system, suggesting its therapeutic potential for various diseases.},
}
@article {pmid41308487,
year = {2026},
author = {Rodríguez-Estévez, D and Gil-Durán, C and Silva, R and Palma, D and Vaca, I and Chávez, R},
title = {CRISPR/Cas9-mediated development of Penicillium roqueforti strains deficient in roquefortine C and mycophenolic acid enables toxin-free blue cheese production.},
journal = {International journal of food microbiology},
volume = {446},
number = {},
pages = {111535},
doi = {10.1016/j.ijfoodmicro.2025.111535},
pmid = {41308487},
issn = {1879-3460},
mesh = {*Penicillium/genetics/metabolism ; *Cheese/microbiology/analysis ; *Mycophenolic Acid/metabolism ; *CRISPR-Cas Systems ; *Mycotoxins/biosynthesis ; Food Microbiology ; *Indoles/metabolism ; Heterocyclic Compounds, 4 or More Rings ; Piperazines ; },
abstract = {Penicillium roqueforti, a key fungus in the manufacture of blue-veined cheeses, can produce mycotoxins such as roquefortine C and mycophenolic acid. The production of these metabolites is highly strain- and condition-dependent. In industrial manufacture, hypotoxigenic P. roqueforti strains are typically used as controlled adjunct starters under standardized conditions, resulting in minimal mycotoxin accumulation, whereas naturally matured or artisan cheeses display more variable strain composition and ripening environments, which can elevate risk. In this context, the development of strains incapable of mycotoxin biosynthesis represents an important step toward safer cheese products. Here, we report the generation of P. roqueforti strains lacking the ability to synthesize roquefortine C and mycophenolic acid using CRISPR/Cas9. Single and double mutants deficient in one or both mycotoxins were obtained. Laboratory-scale cheeses produced under artisan-like conditions with these engineered strains contained no detectable levels of the target mycotoxins, in contrast to cheeses made with the wild-type strain. All mutants retained the ability to colonize cheese but displayed altered fungal biomass production compared to the native strain. These differences were consistent in curd and laboratory media and were not associated with changes in lipolytic or proteolytic activities. Further analyses revealed that while the absence of mycophenolic acid did not affect NaCl sensitivity, the lack of roquefortine C increased sensitivity to salt. Collectively, these results demonstrate the feasibility of producing mycotoxin-free blue cheeses using strains deficient in roquefortine C and mycophenolic acid biosynthesis, thereby laying the foundation for developing mycotoxin-free cheeses with engineered atoxigenic P. roqueforti strains.},
}
@article {pmid41271253,
year = {2025},
author = {Li, L and Xiong, Y and Guo, Y and Duan, H and Leng, Y and Huang, X and Chen, G and Xiong, Y},
title = {G-Quadruplex-Enhanced DNA Silver Nanoclusters Enable CRISPR/Cas12a System for Ultrasensitive Detection of Salmonella typhimurium.},
journal = {Journal of agricultural and food chemistry},
volume = {73},
number = {49},
pages = {31603-31610},
doi = {10.1021/acs.jafc.5c13828},
pmid = {41271253},
issn = {1520-5118},
mesh = {G-Quadruplexes ; *CRISPR-Cas Systems ; *Silver/chemistry ; *Salmonella typhimurium/genetics/isolation & purification ; Metal Nanoparticles/chemistry ; *Biosensing Techniques/methods/instrumentation ; Limit of Detection ; Milk/microbiology ; Animals ; *DNA/chemistry/genetics ; Bacterial Proteins/genetics/metabolism ; CRISPR-Associated Proteins/genetics/metabolism ; Endodeoxyribonucleases ; },
abstract = {DNA-templated silver nanoclusters (tDNA-AgNCs) show considerable promise as fluorescence reporters for the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) system because of their ease of synthesis, strong resistance to photobleaching, and large Stokes shift. However, the weak luminous intensity and low cleavage efficiency of tDNA-AgNCs limit the sensitivity of CRISPR assays. In this study, we developed a novel approach by introducing activator DNA (aDNA) with a G-quadruplex structure to enhance the luminous intensity of the tDNA-AgNCs. As a result of the high cleavage efficiency of free aDNA by activated CRISPR/Cas12a, the G-quadruplex-enhanced tDNA-AgNCs (GED-AgNCs) were integrated into recombinase polymerase amplification and CRISPR/Cas12a system for the ultrasensitive detection of Salmonella typhimurium. By optimizing the synthesis of tDNA-AgNCs and GED-AgNCs, our developed G-quadruplex-enhanced DNA-AgNC CRISPR assay (G-DACA) platform enabled the sensitive determination of S. typhimurium with a detection limit as low as 1 CFU/mL and a wide dynamic range of 10-10[8] CFU/mL. Moreover, our proposed method demonstrated good accuracy and reliability for the quantitative analysis of S. typhimurium in real pasteurized milk samples, with recovery rates ranging from 81.06% to 102.33% and relative standard deviations between 7.17% and 14.84%. Overall, our innovative G-DACA platform offers an economical and versatile solution for food safety and clinical diagnostics.},
}
@article {pmid41270744,
year = {2025},
author = {Pei, Y and Li, S and Garipler, G and Kamimoto, K and Mazzoni, EO},
title = {Stem cell-based approach to identify regulatory TFs during mammalian cell differentiation.},
journal = {Stem cell reports},
volume = {20},
number = {12},
pages = {102716},
doi = {10.1016/j.stemcr.2025.102716},
pmid = {41270744},
issn = {2213-6711},
mesh = {*Cell Differentiation/genetics ; Animals ; Humans ; Mice ; *Transcription Factors/metabolism/genetics ; Motor Neurons/cytology/metabolism ; *Pluripotent Stem Cells/metabolism/cytology ; Transcriptome ; Single-Cell Analysis ; CRISPR-Cas Systems ; },
abstract = {Cell differentiation is regulated by transcription factors (TFs), but specific TFs needed for mammalian differentiation pathways are not fully understood. For example, during spinal motor neuron (MN) differentiation, 1,370 TFs are transcribed, yet only 55 have reported functional relevance. We developed a method combining pluripotent stem cell differentiation, single-cell transcriptomics, and a CRISPR-based TF loss-of-function screen and applied it to MN differentiation. The CRISPR screen identified 245 genes important for mouse MN differentiation, including 116 TFs. This screen uncovered important genes not showing differential transcription and identified a regulatory hub at the MN progenitor (pMN) stage. A secondary human screen of 69 selected candidates revealed a conservation between mouse pMN and human pMN and ventral pMN (vpMN) regulations. The validation of three hits required for efficient human MN differentiation supported the effectiveness of our approach. Collectively, our strategy offers a framework for identifying important TFs in various differentiation pathways.},
}
@article {pmid41270297,
year = {2025},
author = {Kraiczy, J and Yu, B},
title = {Human fallopian tube epithelial organoids with TP53 mutation recapitulate features of serous tubal intraepithelial carcinoma (STIC).},
journal = {Gynecologic oncology},
volume = {203},
number = {},
pages = {198-208},
doi = {10.1016/j.ygyno.2025.10.038},
pmid = {41270297},
issn = {1095-6859},
mesh = {Humans ; Female ; *Tumor Suppressor Protein p53/genetics ; *Organoids/pathology ; *Fallopian Tubes/pathology ; *Fallopian Tube Neoplasms/genetics/pathology ; Mutation ; *Carcinoma in Situ/genetics/pathology ; *Ovarian Neoplasms/genetics/pathology ; DNA Copy Number Variations ; *Cystadenocarcinoma, Serous/genetics/pathology ; CRISPR-Cas Systems ; Gene Editing ; },
abstract = {OBJECTIVE: Serous tubal intraepithelial carcinoma (STIC) is the immediate precursor lesion for high-grade serous ovarian carcinoma (HGSOC) and harbors universal TP53 mutations. The lack of an appropriate in vitro model for STIC presents a major challenge in studying its pathogenesis. We aimed to develop a human in vitro model that mimics STIC lesions.<br><br>METHODS: Using CRISPR-Cas9 gene editing, we generated human fallopian tube epithelial organoids with TP53 loss-of-function mutations (TP53[-/-] FTOs). We characterized TP53[-/-] FTOs on a cellular and molecular level using immunofluorescence confocal imaging, copy number variation (CNV) analysis, and RNA sequencing.<br><br>RESULTS: TP53[-/-] FTOs recapitulated key features of STIC lesions. They exhibited increased proliferation and nuclear abnormalities, including nuclear enlargement and atypical mitotic figures. Copy number variation analysis revealed aneuploidy in some TP53[-/-] FTOs. Compared to unedited controls, TP53[-/-] FTOs demonstrated significant transcriptomic changes, including the downregulation of DNA repair genes and upregulation of epithelial-mesenchymal transition (EMT) pathways. Similar to STIC lesions, TP53[-/-] FTOs showed a marked reduction in ciliated cells and ciliogenesis-associated gene expression.<br><br>CONCLUSIONS: These findings suggest that p53 loss in FTOs promotes a proliferative and genomically unstable state that is conducive to carcinogenesis. The TP53[-/-] FTO model we have generated provides a valuable tool for studying early events in ovarian carcinogenesis and for developing new strategies for the early detection and prevention of ovarian cancer.},
}
@article {pmid41251373,
year = {2025},
author = {Pérez Antón, E and Dujeancourt-Henry, A and Rotureau, B and Glover, L},
title = {A CRISPR-based diagnostic tool to survey drug resistance in human African trypanosomiasis.},
journal = {Antimicrobial agents and chemotherapy},
volume = {69},
number = {12},
pages = {e0093325},
doi = {10.1128/aac.00933-25},
pmid = {41251373},
issn = {1098-6596},
support = {ANR-PRC 2021 SherPa//Agence Nationale de la Recherche/ ; LabEx IBEID//Labex/ ; },
mesh = {Humans ; *Trypanosomiasis, African/drug therapy/parasitology/diagnosis ; *Trypanocidal Agents/pharmacology ; *Drug Resistance/genetics ; *Trypanosoma brucei gambiense/drug effects/genetics ; Pentamidine/pharmacology/therapeutic use ; Melarsoprol/pharmacology/therapeutic use ; *CRISPR-Cas Systems/genetics ; Mutation ; Protozoan Proteins/genetics ; },
abstract = {The World Health Organization aims to eliminate human African trypanosomiasis caused by Trypanosoma brucei gambiense (gHAT) by 2030. With the decline of reported cases, maintaining active surveillance is essential, including for the potential emergence of drug-resistant parasites. We have developed new highly specific diagnostic tools, using the Cas13a-based Specific High-Sensitivity Reporter Enzymatic UnLOCKing (SHERLOCK) technology, for the detection of drug-resistant genotypes that (i) are already circulating, such as the AQP2/3(814) chimera providing resistance to pentamidine and melarsoprol or (ii) could emerge, such as the TbCPSF3 (N[232]H) mutation, associated with acoziborole resistance under laboratory conditions. The AQP2/3(814) SHERLOCK assay detected RNA from both cultured parasites and field strains isolated from gHAT patients who relapsed following melarsoprol or pentamidine treatment. The CPSF3(SNV) SHERLOCK assay discriminated between wild-type CPSF3 RNA and CPSF3 bearing a single A-C mutation that confers resistance to acoziborole in vitro. These SHERLOCK assays are amenable for use as a high-throughput screening method to monitor for drug-resistant-associated mutations in Trypanosoma brucei, providing a new molecular tool for epidemiological surveillance during the gHAT elimination phase.},
}
@article {pmid41237780,
year = {2025},
author = {Sivakumar, S and Wang, Y and Goetsch, SC and Pandit, V and Wang, L and Zhao, H and Sundarrajan, A and Armendariz, D and Takeuchi, C and Deng, M and Nzima, M and Chen, WC and Dederich, AE and El Hayek, L and Gao, T and Gogate, A and Kaur, K and Kim, HB and McCoy, MK and Niederstrasser, H and Oura, S and Pinzon-Arteaga, CA and Sanghvi, M and Schmitz, DA and Yu, L and Zhang, Y and Zhou, Q and Kraus, WL and Xu, L and Wu, J and Posner, BA and Chahrour, MH and Hon, GC and Munshi, NV},
title = {Benchmarking and optimizing Perturb-seq in differentiating human pluripotent stem cells.},
journal = {Stem cell reports},
volume = {20},
number = {12},
pages = {102713},
doi = {10.1016/j.stemcr.2025.102713},
pmid = {41237780},
issn = {2213-6711},
mesh = {Humans ; *Cell Differentiation/genetics ; *Pluripotent Stem Cells/cytology/metabolism ; Benchmarking ; Gene Regulatory Networks ; Myocytes, Cardiac/cytology/metabolism ; CRISPR-Cas Systems/genetics ; High-Throughput Nucleotide Sequencing/methods ; RNA, Guide, CRISPR-Cas Systems/genetics ; },
abstract = {Perturb-seq is a powerful approach to systematically assess how genes and enhancers impact the molecular and cellular pathways of development and disease. However, technical challenges have limited its application in stem-cell-based systems. Here, we benchmarked Perturb-seq across multiple CRISPRi modalities, on diverse genomic targets, in multiple human pluripotent stem cells, during directed differentiation to multiple lineages, and across multiple single guide RNA (sgRNA) delivery systems. To ensure cost-effective production of large-scale Perturb-seq datasets as part of the Impact of Genomic Variants on Function (IGVF) consortium, our optimized protocol dynamically assesses experiment quality across the weeks-long procedure. Our analysis of 1,996,260 sequenced cells across benchmarking datasets reveals shared regulatory networks linking disease-associated enhancers and genes with downstream targets during cardiomyocyte differentiation. This study establishes open tools and resources for interrogating genome function during stem cell differentiation.},
}
@article {pmid41218364,
year = {2025},
author = {Huang, X and Xiao, T and Zhao, X and Yang, Z and Sun, Z and Lu, K},
title = {Olfactory perception of trifluralin by GOBP2 decreases the susceptibility of Spodoptera litura to insecticides through modulation of 20E signaling pathway-mediated metabolic detoxification.},
journal = {Journal of hazardous materials},
volume = {500},
number = {},
pages = {140417},
doi = {10.1016/j.jhazmat.2025.140417},
pmid = {41218364},
issn = {1873-3336},
mesh = {Animals ; *Insecticides/toxicity/pharmacology ; *Spodoptera/drug effects/metabolism/genetics ; Signal Transduction/drug effects ; Inactivation, Metabolic ; *Trifluralin/pharmacology/toxicity ; Larva/drug effects/metabolism ; *Insect Proteins/genetics/metabolism ; Chlorpyrifos/toxicity ; CRISPR-Cas Systems ; *Herbicides/toxicity/pharmacology ; Oxazines ; },
abstract = {Herbicide contamination has emerged as a critical ecological concern due to its persistent environmental accumulation and unintended impacts on non-target species. This study reveals that olfactory exposure to the volatile herbicide trifluralin enhances metabolic detoxification in Spodoptera litura larvae, significantly reducing their susceptibility to indoxacarb and chlorpyrifos. Building on our previous discovery of GOBP2's trifluralin-binding capacity, CRISPR/Cas9-generated GOBP2 knockout (GOBP2[KO]) larvae exhibited compromised loss of cross-tolerance, accompanied by impaired induction of carboxylesterases (COEs) and glutathione S-transferases (GSTs). Mechanistically, trifluralin perception through GOBP2 activated a 20-hydroxyecdysone (20E)-dependent pathway, upregulating both 20E biosynthesis genes and ecdysone receptor (EcR)/ultraspiracle (USP) complexes in wild-type insects. Systemic disruption of this signaling axis via RNAi-mediated EcR/USP co-silencing abolished trifluralin-induced detoxification enzyme activation and restored insecticide vulnerability. Molecular validation through dual-luciferase assays and yeast-one hybrid systems confirmed EcR/USP-mediated transactivation of COE/GSTe genes via conserved response elements. Functional characterization identified GSTe1 and GSTe16 as key effectors, with GSTe16 exhibiting effective metabolic efficiency against indoxacarb (19.25 % degradation) and chlorpyrifos (13.04 % degradation), while GSTe1 showed significant activity toward indoxacarb metabolism (14.96 %) and chlorpyrifos degradation (10.17 %). Genetic evidence from CRISPR/Cas9-ablated S. litura and transgenic Drosophila models further established causal relationships between GSTe expression levels and insecticide tolerance. Our integrated analysis establishes that GOBP2-mediated endocrine signaling constitutes a central axis in trifluralin-induced insecticide tolerance, directly bridging herbicide perception to metabolic resistance through 20E-dependent regulatory cascades.},
}
@article {pmid41055127,
year = {2025},
author = {Lin, Y and Ye, X and Zeng, L and Luo, Y and Feng, Y and Zhang, Y},
title = {Heat shock-optimized CRISPR/Cas9 system for visible clonal analysis and mutant generation in Drosophila.},
journal = {G3 (Bethesda, Md.)},
volume = {15},
number = {12},
pages = {},
doi = {10.1093/g3journal/jkaf236},
pmid = {41055127},
issn = {2160-1836},
support = {LY21C070003//Natural Science Foundation of Zhejiang Province/ ; },
mesh = {Animals ; *CRISPR-Cas Systems ; *Heat-Shock Response/genetics ; *Mutation ; RNA, Guide, CRISPR-Cas Systems/genetics ; *Drosophila/genetics ; Drosophila Proteins/genetics ; Clone Cells ; *Drosophila melanogaster/genetics ; Gene Editing ; },
abstract = {In Drosophila genetic studies, clonal analysis such as mosaic and Mosaic Analysis with a Repressible Cell Marker has been widely used to investigate gene function. Recently, the CRISPR/Cas9 system has been established as a powerful tool for efficient mutant generation; however, its application in clonal analysis has been rarely reported. Here, we present a suite of Gal4/UAS-Cas9 binary expression systems that integrate UAS-Cas9 and multiple-sgRNAs (single-guide RNAs) into a single plasmid. These systems facilitate versatile applications, enabling Gal4-driven direct phenotypic studies, approximate clonal analysis, in vitro cell transfection, and stable mutant generation, among which, the third-generation constructs: G3a/b incorporate visible labeling strategies for marking approximate clonal regions. In addition, compared to continuously active drivers, we found that the short-pulse-induced heat shock-Gal4 (hs-Gal4) was sufficient to induce high clonal efficiency and generate larger clones. In the germline, short-pulse heat shock is also effective. It reduces residual Cas9 activity in the germline stem cells, thereby minimizing the risk of affecting germline stem cell survival and improving mutant acquisition.},
}
@article {pmid41052774,
year = {2025},
author = {Salama, R and Peet, E and Morrione, TL and Durant, S and Seager, M and Rennie, M and Scarlata, S and Nechipurenko, I},
title = {Functional classification of GNAI1 disorder variants in Caenorhabditis elegans uncovers conserved and cell-specific mechanisms of dysfunction.},
journal = {Genetics},
volume = {231},
number = {4},
pages = {},
doi = {10.1093/genetics/iyaf216},
pmid = {41052774},
issn = {1943-2631},
support = {//Charles H. Hood Foundation/ ; //Child Health Research Award/ ; R35 GM155316/NH/NIH HHS/United States ; },
mesh = {Animals ; *Caenorhabditis elegans/genetics/metabolism ; Cilia/metabolism/genetics ; *GTP-Binding Protein alpha Subunits, Gi-Go/genetics/metabolism ; Humans ; *Caenorhabditis elegans Proteins/genetics/metabolism ; Neurons/metabolism ; CRISPR-Cas Systems ; Mutation ; Phenotype ; },
abstract = {Heterotrimeric G proteins transduce signals from G protein-coupled receptors, which mediate key aspects of neuronal development and function. Mutations in the GNAI1 gene, which encodes Gαi1, cause a disorder characterized by developmental delay, intellectual disability, hypotonia, and epilepsy. However, the mechanistic basis for this disorder remains unknown. Here, we show that GNAI1 is required for ciliogenesis in human cells and use Caenorhabditis elegans as a whole-organism model to determine the functional impact of 7 GNAI1-disorder patient variants. Using CRISPR-Cas9 editing in combination with robust cellular (cilia morphology) and behavioral (chemotaxis) assays, we find that T48I, K272R, A328P, and V334E orthologous variants impact both cilia assembly and function in AWC neurons, M88V and I321T have no impact on either phenotype, and D175V exerts neuron-specific effects on cilia-dependent sensory behaviors. Finally, we validate in human ciliated cell lines that D173V, K270R, and A326P GNAI1 variants disrupt ciliary localization of the encoded human Gαi1 proteins similarly to their corresponding orthologous substitutions in the C. elegans ODR-3 (D175V, K272R, and A328P). Overall, our findings determine the in vivo effects of orthologous GNAI1 variants and contribute to the mechanistic understanding of GNAI1-disorder pathogenesis as well as neuron-specific roles of ODR-3 in sensory biology.},
}
@article {pmid40991383,
year = {2025},
author = {Suter, A and Graham, A and Kuah, JY and Crisologo, J and Gunatilake, C and Sourris, K and See, M and Rossello, FJ and Ramialison, M and Vlahos, K and Howden, SE},
title = {Efficient Installation of Heterozygous Mutations in Human Pluripotent Stem Cells Using Prime Editing.},
journal = {The CRISPR journal},
volume = {8},
number = {6},
pages = {401-411},
doi = {10.1177/25731599251380122},
pmid = {40991383},
issn = {2573-1602},
mesh = {Humans ; *Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; *Mutation ; *Pluripotent Stem Cells/metabolism/cytology ; Heterozygote ; Induced Pluripotent Stem Cells/metabolism/cytology ; Fibroblasts/metabolism/cytology ; Leukocytes, Mononuclear/cytology ; Gene Knock-In Techniques ; Cellular Reprogramming ; },
abstract = {The utility of human pluripotent stem cells (hPSCs) is greatly enhanced by the ability to introduce precise, site-specific genetic modifications with minimal off-target effects. Although Cas9 endonuclease is an exceptionally efficient gene-editing tool, its propensity for generating biallelic modifications often limits its capacity for introducing heterozygous variants. Here, we use prime editing (PE) to install heterozygous edits in over 10 distinct genetic loci, achieving knock-in efficiencies of up to 40% without the need for subsequent purification or drug selection steps. Moreover, PE enables the precise introduction of heterozygous edits in paralogous genes that are otherwise extremely challenging to achieve using endonuclease-based editing approaches. We also show that PE can be successfully combined with reprogramming to derive heterozygous induced pluripotent stem cell clones directly from human fibroblasts and peripheral blood mononuclear cells. Our findings highlight the utility of PE for generating hPSCs with complex edits and represent a powerful platform for modeling disease-associated dominant mutations and gene-dosage effects in an entirely isogenic context.},
}
@article {pmid40971219,
year = {2025},
author = {Basharat, R and Rizzo, G and Zoodsma, JD and Wollmuth, LP and Sirotkin, HI},
title = {Optimizing Prime Editing in Zebrafish.},
journal = {The CRISPR journal},
volume = {8},
number = {6},
pages = {426-435},
doi = {10.1177/25731599251380500},
pmid = {40971219},
issn = {2573-1602},
mesh = {*Zebrafish/genetics ; Animals ; *Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; Mutation, Missense ; RNA, Guide, CRISPR-Cas Systems/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; RNA, Messenger/genetics ; },
abstract = {Prime editing is a clustered regularly interspaced short palindromic repeats-based approach that enables the introduction of precise genetic modifications, including missense mutations, making it valuable for generating disease models. The comparative performance of novel prime editor (PE) variants in zebrafish remains largely unexplored. Here, we systematically evaluated the efficiency of five PEs-PE2, PE6b, PE6c, PEmax, and PE7-in zebrafish. We tested mRNA encoding for each of these PEs with prime editing guide RNAs (pegRNAs) designed to install five missense mutations. Efficient editing was achieved at four of the five sites with multiple PEs. Among these, PEmax emerged as the most efficient editor for introducing pure prime edits, with rates reaching 15.34%. We found that strategies proposed to block 3' degradation of pegRNAs (epegRNAs and addition of a La RNA binding motif to the PE) did not improve performance in our assays. Together, these findings establish PEmax as a robust tool to introduce missense mutations into zebrafish.},
}
@article {pmid40810623,
year = {2025},
author = {Salemdawod, A and Cooper, B and Liang, Y and Walczak, P and Vatter, H and Maciaczyk, J and Janowski, M},
title = {CRISPR-Cas9 Single Nucleotide Editing of Tuberous Sclerosis Complex 2 Gene in Mesenchymal Stem Cells.},
journal = {The CRISPR journal},
volume = {8},
number = {6},
pages = {412-425},
doi = {10.1177/25731599251367059},
pmid = {40810623},
issn = {2573-1602},
mesh = {Humans ; *Mesenchymal Stem Cells/metabolism ; *Tuberous Sclerosis Complex 2 Protein/genetics ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; Point Mutation ; Tuberous Sclerosis ; },
abstract = {The tuberous sclerosis complex (TSC)2 gene regulates the mammalian target of rapamycin (mTOR) pathway, impacting cell proliferation and growth. The loss-of-function mutations, especially in mesenchymal progenitors, drive the development multiple benign and malignant tumors. TSC2 mutations in certain cancer types, e.g., breast cancer, are also associated with poorer prognosis. The databases of TSC2-mutations report point mutations as the most prevalent. We aimed to test the feasibility of inducing point mutations in mesenchymal stem cells (MSCs), targeting the most frequent point mutations of the TSC2 gene, TSC2. c.1864 C>T (p.Arg622Trp), TSC2. c.1832 G>A (p.Arg611Glu), and TSC2. c.5024 C>T (p.Pro1675Leu) using two delivery methods for CRISPR-Cas9. We report a high editing efficiency of up to 85% inducing TSC2 point mutations in hMSCs using lipofectamine-based transfection. Overall, the high editing efficiency of some TSC2 mutations enables the induction and reversal of mutations in primary hMSCs without needing resource-consuming derivation of cell lines frequently distinct from their primary counterparts.},
}
@article {pmid41359128,
year = {2025},
author = {Li, Z and Cheng, Y and Li, C and Wu, Q and Xin, Y},
title = {Harnessing microalgae for bioproducts: innovations in synthetic biology.},
journal = {World journal of microbiology &amp; biotechnology},
volume = {41},
number = {12},
pages = {500},
pmid = {41359128},
issn = {1573-0972},
support = {32560020 and 31600059//National Natural Science Foundation of China/ ; RZ2300002678//Start-Up Funds of Hainan University/ ; DC2300001799//Open Project of State Key Laboratory of Marine Resource Utilization in South China Sea/ ; 2018YFA0902500//National Key Research and Development Program of China/ ; },
mesh = {*Microalgae/metabolism/genetics ; *Synthetic Biology/methods ; Biofuels ; *Metabolic Engineering/methods ; Gene Editing ; Lipids/biosynthesis ; CRISPR-Cas Systems ; Metabolic Networks and Pathways ; Photobioreactors ; },
abstract = {Microalgae are increasingly recognized as versatile platforms for sustainable production of biofuels and high-value bioproducts such as lipids, carotenoids and polyunsaturated fatty acids. Rapid progress in synthetic biology is transforming microalgal engineering by enabling precise rewiring of metabolic pathways and overcoming long-standing technical bottlenecks, particularly those related to transformation efficiency, genetic stability and strain scalability. Recent innovations (including CRISPR/Cas genome editing, modular cloning systems, synthetic promoter libraries and dynamic, environment-responsive regulatory circuits) have greatly expanded the genetic toolset available for both model and recalcitrant species. These advances support targeted control of lipid and pigment biosynthesis, improved flux distribution and more robust performance under industrially relevant conditions. When integrated with progress in photobioreactor design, automated cultivation, and process intensification, synthetic biology unlocks new potential for scalable, economically viable microalgal biomanufacturing. This review summarizes these developments, highlights remaining challenges in strain robustness and bioprocess translation, and outlines future pathways toward high-performance microalgal biofactories that can contribute meaningfully to a low-carbon, bio-based economy.},
}
@article {pmid41356798,
year = {2026},
author = {Birappa, G and Perumalsamy, H and Hong, SH and Gowda, DAA and Chandrasekaran, AP and Karapurkar, JK and Rajkumar, S and Balusamy, SR and Jayachandran, A and Baek, KH and Lee, J and Matam, V and Kim, WJ and Kim, KS and Ramakrishna, S and Suresh, B},
title = {Single-cell RNA sequence analysis reveals USP32 as a therapeutic target to mitigate PD-L1-driven colorectal tumorigenesis in vitro and in vivo.},
journal = {Theranostics},
volume = {16},
number = {2},
pages = {986-1005},
pmid = {41356798},
issn = {1838-7640},
mesh = {Humans ; Animals ; *B7-H1 Antigen/metabolism/genetics ; *Colorectal Neoplasms/genetics/pathology/metabolism ; Mice ; *Ubiquitin Thiolesterase/genetics/metabolism ; Single-Cell Analysis/methods ; Ubiquitination ; *Carcinogenesis/genetics ; Cell Line, Tumor ; Gene Expression Regulation, Neoplastic ; CRISPR-Cas Systems ; Sequence Analysis, RNA ; },
abstract = {Background: The expression levels of the programmed death-ligand 1 (PD-L1) protein serves as a prognostic indicator for patients with colorectal cancer (CRC). Advancement of CRC is facilitated by deubiquitinating enzymes (DUBs), which regulate oncoprotein levels via the ubiquitin-proteasomal pathway. The post-translational regulatory mechanisms governing PD-L1 protein abundance on CRC, in relation to different tumor grades and their clinical relevance, remains unknown. Methods: We analyzed single-cell RNA sequencing (scRNA-seq) data to identify DUB genes associated with PD-L1 expression in CRC. We used a loss-of-function-based CRISPR/Cas9 library to identify putative DUB genes that regulate the PD-L1 protein level. Immunoprecipitation was used to confirm the interaction between the USP32 and PD-L1 along with its ubiquitination status. A series of in vitro and in vivo carcinogenesis-related experiments were conducted to determine the clinical relevance between USP32 and PD-L1 expression in CRC progression. Results: In this study, we analyzed scRNA-seq data from extensive cohorts of human and mice at the single-cell level to identify DUB genes associated with PD-L1 expression in CRC. Our analysis identified multiple putative DUBs, including USP32 and USP12, as prognostic markers associated with PD-L1 expression, which was found to be elevated in T cells, macrophages, and classical monocytes cell types in patients with CRC. A secondary screening using CRISPR/Cas9-mediated loss-of-function analysis for DUBs found that USP32 modulates PD-L1 protein levels in CRC. Furthermore, we demonstrated that USP32 interacts with, stabilizes, and extends the half-life of PD-L1 by preventing its K-48-linked polyubiquitination as an underlying mechanism that contributes for tumorigenesis. Conclusion: A combination of scRNA-seq analysis and wet-lab experimental validation confirmed that USP32 mediates PD-L1 protein stabilization in colon cancer, identifying it as a potential therapeutic target for CRC. CRISPR/Cas9-mediated targeted knockout of the USP32 gene reduced PD-L1 protein levels and significantly mitigated colorectal cell proliferation and tumorigenesis, both in vitro and in vivo, in a xenograft mouse model, underscoring a novel and alternative approach to the treatment of CRC.},
}
@article {pmid41356473,
year = {2025},
author = {Wei, C and Chen, Z},
title = {Comprehensive analysis of phage genomes from diverse environments reveals their diversity, potential applications, and interactions with hosts and other phages.},
journal = {Frontiers in microbiology},
volume = {16},
number = {},
pages = {1686402},
pmid = {41356473},
issn = {1664-302X},
abstract = {Phages are ubiquitous and diverse, playing a key role in maintaining microbial ecosystem balance. However, their diversity, potential applications, and their interactions with hosts and other phages remain largely unexplored. To address this, we collected 59,652,008 putative viral genomes from our laboratory, 45 public viral datasets, and an integrated public viral genome database (IGN), covering seven habitats. We obtained 741,692 phage genomes with completeness ≥50% (PGD50), and most (93.83%, 695,938/741,692) of these phage genomes were classified into the Caudoviricetes class. We found that 158,522 species-level viral clusters that contained 28.96% (214,814/741,692) phage genomes without any known phage genomes in the IGN, indicating substantial novelty. Global phylogenetic trees for five iterations based on complete phage genomes significantly expanded the known diversity of the virosphere. Genome analysis revealed phage potential divergence with habitat types and highlighted the utilization of alternative genetic codes. Furthermore, 3D structural similarity searches demonstrated significant potential for annotating previously uncharacterized viral proteins. Analysis of CRISPR spacer inferred potential hosts of phages and competitive networks among phages, highlighting virulent phages as promising candidates for phage therapy against pathogenic bacteria. Intriguingly, diverse CRISPR-Cas systems were detected within phage genomes themselves, suggesting their enormous potential as novel gene editing tools. Collectively, this study provides a comprehensive phage genome resource, foundational for future research into phage-host and phage-phage interactions, phage therapy development, and the mining of next-generation genetic tools.},
}
@article {pmid41356196,
year = {2026},
author = {Zhang, Y and Deng, Q and Xu, Y and Wu, W and Wu, T and Huang, J and Hu, Y and Lin, W and Xu, X and Wu, J},
title = {ROS-responsive cellular vesicles with ferroptosis-targeting siACMSD delivery for acute kidney injury therapy.},
journal = {Theranostics},
volume = {16},
number = {4},
pages = {1941-1958},
pmid = {41356196},
issn = {1838-7640},
mesh = {*Ferroptosis/drug effects ; Animals ; *Reactive Oxygen Species/metabolism ; *Acute Kidney Injury/therapy/metabolism/drug therapy/pathology ; Mice ; Humans ; Cisplatin/pharmacology ; *Carboxy-Lyases/genetics/metabolism ; Disease Models, Animal ; Cell Line ; Male ; Mice, Inbred C57BL ; CRISPR-Cas Systems ; Mitochondria/metabolism/drug effects ; },
abstract = {Background: Acute kidney injury (AKI) is a severe and prevalent nephrotic syndrome which lack of definitive therapies. Alpha-amino-β-carboxymuconic acid-ε-semialdehyde decarboxylase (ACMSD) is a metabolic enzyme mainly expressed in the kidney which exacerbated AKI injury by promoting TCA cycle and inhibiting nicotinamide adenine dinucleotide (NAD[+]) production, whereas lack of effective intervention strategies for ACMSD-targeted therapy. Methods: Herein, we knocked out ACMSD in vitro through CRISPR-Cas9 method, and developed a reactive oxygen species (ROS)-responsive neutrophil-derived cellular vesicles (CVs) drugs (RNAi@ROS-CVs), which efficiently mediated ACMSD knockdown in vivo, exploring the mechanism of ACMSD-induced ferroptosis process in AKI. Results: ACMSD knockout effectively alleviated cisplatin (CP)-induced mitochondrial damage, suppressed TCA cycle progression, promoted NAD[+] synthesis, and inhibited ferroptosis in HK2 cells. In mice AKI model, RNAi@ROS-CVs effectively targeted the injured kidneys, downregulated ACMSD expression in renal tubular epithelial cells, reduced ROS production and lipid peroxidation, and alleviated CP or ischemia/reperfusion (I/R)-induced ferroptosis. Conclusion: These findings highlight the therapeutic potential of ACMSD-targeted knockout in AKI intervention and introduce a versatile and efficient controlled-release drug delivery platform for AKI-targeted therapy, with potential applicability to other acute renal diseases.},
}
@article {pmid41356190,
year = {2026},
author = {Zhong, XY and Yang, YX and Xiong, YF and Ye, GC and Gong, X and Zhong, ML and He, HD and Wang, SG and Xia, QD},
title = {Programmable molecular microscopy: CRISPR/Cas fluorescent probes revolutionizing spatiotemporal genomic imaging.},
journal = {Theranostics},
volume = {16},
number = {4},
pages = {1877-1904},
pmid = {41356190},
issn = {1838-7640},
mesh = {*CRISPR-Cas Systems/genetics ; *Fluorescent Dyes ; Humans ; Animals ; *Molecular Imaging/methods ; *Genomics/methods ; Gene Editing/methods ; },
abstract = {Bioimaging technologies visually resolve spatiotemporal dynamics of biomolecules, cells, and tissues, enabling essential insights into gene regulation, disease mechanisms, and drug metabolism. CRISPR/Cas-based fluorescent probes transform CRISPR from "genetic scissors" into "molecular microscopes," providing an indispensable tool for in situ decoding of molecular events in living systems. Their high nucleic acid specificity establishes CRISPR/Cas as a pivotal technology for dynamically monitoring genomic and transcriptomic events at live-cell and in vivo levels. This work systematically outlines design strategies and functional mechanisms of mainstream CRISPR/Cas fluorescent probes for bioimaging, encompassing five categories: fluorescent proteins, synthetic dyes, smart gated probes, nanomaterials, and multimodal integrated probes. Recent advances and persistent challenges in achieving high-sensitivity targeted imaging, effective signal amplification, and precise delivery control are comprehensively examined, including analysis of their advantages, limitations, and adaptability in complex biological environments. Building on breakthroughs in in vivo delivery systems, diverse carriers demonstrate significant potential for enhancing CRISPR/Cas transport efficiency, improving tissue penetration, and enabling spatiotemporal controlled release. Continued innovation drives CRISPR/Cas imaging platforms toward higher sensitivity, enhanced biocompatibility, and multifunctional integration, thereby fostering the convergence and broad application of gene editing and molecular diagnostics.},
}
@article {pmid41354981,
year = {2025},
author = {Das, T and Barman, T and Prasad, A},
title = {Precision editing to improve fruit traits: CRISPR/Cas into the picture.},
journal = {Protoplasma},
volume = {},
number = {},
pages = {},
pmid = {41354981},
issn = {1615-6102},
abstract = {Crop growth, quality, and yield can be adversely affected by various biotic and abiotic stresses. Crop characteristics can be improved with conventional breeding and other variation-based breeding strategies. However, these strategies are time as well as resource consuming and to overcome this, novel approaches are necessary. CRISPR/Cas technique allows to improve desired traits more efficiently and accurately by targeting specific genes. Genome editing has become more versatile with CRISPR/Cas systems and is a valuable tool to protect food security by developing commercial crops optimized for yield and nutritional quality. Researchers are able to target and edit stress response pathway genes to develop crops with increased tolerance to stress. A lack of regeneration protocols and sufficient genome sequencing data has restricted fruit editing to only a few fruits (tomatoes, citrus, apple, kiwi, banana, grapes, strawberries, watermelon, etc.). This review is focused on CRISPR/Cas applications on the nutritional aspects of fruit engineering along with the challenges and opportunities. Another aspect which will be covered is the use of CRISPR/Cas technology to improve fruit resilience to biotic and abiotic stress, but not at the cost of yield. We discuss the pros and cons of using this technology, such as unintended effects on fruit traits or public concerns about GMOs. We conclude that the application of CRISPR/Cas9 technology has the potential to be of great benefit to the agricultural industry not only to improve nutritional aspects but also to help reduce crop losses.},
}
@article {pmid41354953,
year = {2025},
author = {Nguyen, VT and Van, BTT and Uyen, TN and Tong, NX and Pham, TL and Vy, NHT and Thuy, DT and Thuy, NP and Kobayashi, M},
title = {Functional divergence of zebrafish keap1 paralogs revealed by CRISPR/Cas9-mediated gene editing: a specialized role for keap1b in inflammation.},
journal = {Transgenic research},
volume = {34},
number = {1},
pages = {53},
pmid = {41354953},
issn = {1573-9368},
support = {108.06-2020.19//National Foundation for Science and Technology Development/ ; },
mesh = {Animals ; *Zebrafish/genetics ; *Zebrafish Proteins/genetics/metabolism ; *CRISPR-Cas Systems/genetics ; Oxidative Stress/genetics ; *Inflammation/genetics ; Kelch-Like ECH-Associated Protein 1/genetics ; NF-E2-Related Factor 2/genetics ; Signal Transduction/genetics ; Gene Editing ; Carrier Proteins ; },
abstract = {The Keap1/Nrf2 signaling pathway is a master regulator of cellular defense against oxidative and electrophilic stress. In teleosts like zebrafish (Danio rerio), whole-genome duplication resulted in two keap1 paralogs, keap1a and keap1b, whose functional specificities remain incompletely understood. This study investigates the divergent roles of these paralogs by comparing the responses of established keap1a and novel keap1b knockout larvae to distinct chemical stressors. By comparing the responses of keap1b[dl40], keap1a[dl07], and nfe2l2a[dl703] (Nrf2a) larvae to these stressors, we uncovered a striking functional dichotomy. While loss of either paralog conferred resistance to H2O2-induced oxidative stress, keap1b[dl40] larvae, unlike their keap1a[dl07] counterparts, exhibited extreme sensitivity to the lethal effects of CuSO4 exposure, with survival rates plummeting to ~ 25%. This heightened sensitivity to copper sulfate was associated with a blunted transcriptional response of inflammatory markers tnf-a and c3a, suggesting that Keap1b is critical for modulating the Nrf2a-mediated response to inflammatory stress in orchestrating a viable inflammatory response. This work clarifies the non-redundant, vital function of Keap1b in the response to heavy metal-induced stress and provides a valuable genetic resource (keap1b[dl40] null allele) for future studies.},
}
@article {pmid41353974,
year = {2025},
author = {Madny, MA and Yadav, KS},
title = {Biomimetic oral drug delivery: Translating nature's design into therapeutic innovation.},
journal = {Colloids and surfaces. B, Biointerfaces},
volume = {259},
number = {},
pages = {115348},
doi = {10.1016/j.colsurfb.2025.115348},
pmid = {41353974},
issn = {1873-4367},
abstract = {Oral drug delivery, the most patient friendly administration route offers convenience and compliance but faces formidable biological barriers. Enzymatic degradation, mucosal entrapment, efflux transport and extensive first-pass metabolism drastically reduce the effectiveness of sensitive therapeutics including peptides, proteins, nucleic acids and vaccines. Conventional formulations often fail to overcome these challenges highlighting the need for innovative approaches. Biomimetic drug delivery has emerged as a transformative strategy. By emulating structures and functions from cells, membranes, exosomes, viruses and gut microbiota these systems achieve immune evasion, mucus penetration, site-specific targeting and stimulus-responsive release. Such approaches improve formulation stability and in vivo absorption but also promise precise and patient centric therapies. This review provides a comprehensive overview of biomimetic oral systems highlighting their mechanisms, design principles and translational potential. Recent advances include cell membrane-coated nanoparticles for tumor targeting and immune modulation, exosome-inspired carriers for protein and RNA transport, virus-like particles (VLPs) for oral vaccines, and mucoadhesive or mucus-penetrating polymers modeled on pathogen strategies. Complementary pH, enzyme and redox-responsive platforms exploit gastrointestinal (GI) microenvironments to ensure controlled release. Emerging tools such as bioinspired computational modeling, 3D/4D printing, organoid-on-chip models and CRISPR/Cas-based platforms accelerate optimization and clinical translation. Although most technologies remain in preclinical development, early findings demonstrate superior pharmacokinetics, therapeutic efficacy, and safety over conventional systems. This article critically examines biomimetic oral drug delivery addressing advances and underlying mechanisms including regulatory considerations and future directions. They stand poised to form the foundation of next-generation precision therapeutics.},
}
@article {pmid41352919,
year = {2026},
author = {Guan, X and Wang, S and Wang, P and Zhang, J and Sun, S},
title = {Enhanced chemiluminescence aptasensing with triple cascade amplification for sensitive detection of tumor-derived exosomes.},
journal = {Analytica chimica acta},
volume = {1383},
number = {},
pages = {344873},
doi = {10.1016/j.aca.2025.344873},
pmid = {41352919},
issn = {1873-4324},
mesh = {*Exosomes/chemistry/metabolism ; Humans ; *Aptamers, Nucleotide/chemistry/metabolism ; *Luminescent Measurements/methods ; Gold/chemistry ; Metal Nanoparticles/chemistry ; Limit of Detection ; Mucin-1 ; Alkaline Phosphatase/chemistry/metabolism ; Nucleic Acid Amplification Techniques ; CRISPR-Cas Systems ; *Biosensing Techniques/methods ; Biomarkers, Tumor ; Tetraspanin 30 ; },
abstract = {BACKGROUND: Tumor-associated exosomes hold significant clinical promise as liquid biopsy biomarkers. However, the accurate detection of these rare exosome subpopulations in clinical samples demands analytical platforms with exceptionally high sensitivity and specificity. While conventional nucleic acid amplification-based methods provide considerable detection sensitivity, they are often hampered by time-consuming procedures, operational complexity, and susceptibility to contamination. Therefore, it is imperative to develop practical exosome measurement platforms that combine high sensitivity, robustness, and rapid analysis capabilities to provide reliable evidence-based support for precision oncology.<br><br>RESULTS: In this work, a triple cascade-amplified aptasensor (TCAA) via functionalized gold nanoparticle (fAuNP), CRISPR/Cas12a, and alkaline phosphatase (ALP) was developed for enhanced chemiluminescence (CL) assay of tumor-derived exosomes without nucleic acid amplification. The target exosomes were initially recognized by CD63 and MUC1 aptamers. fAuNP-conjugated Trigger sequences then activated CRISPR/Cas12a to cleave single-stranded DNA and release ALP. Consequently, the ALP catalyzed substrate to produce CL signals correlating with the concentration of the analyte. By simultaneously integrating the signal amplification capabilities of multiple techniques, this TCAA achieved a limit of detection of 44 particles/&#x3bc;L for MUC1-positive exosomes within 60&#xa0;min with excellent robustness. Compared with the single- and dual-amplification methods, the sensitivity was increased by 40-fold and 6-fold, respectively. Clinical trials showed that the area under the curve of this approach was 0.96, which was higher than that of the commercialized chemiluminescence immunoassay and effectively distinguished breast cancer-derived specimens.<br><br>SIGNIFICANCE: These findings indicate that the TCAA strategy provides a highly sensitive, rapid, and robust tool for the detection of low-abundance tumor exosome subpopulations without nucleic acid amplification. It effectively addresses the limitations of conventional methods and demonstrates high clinical utility. This work offers a reliable and practical platform for non-invasive liquid biopsy, holding great potential for trace-level detection of diverse biomarkers.},
}
@article {pmid41330004,
year = {2025},
author = {Coşar, B and Kılıç, P and İşeri, &#xd6;D},
title = {The intersection of CAR-T immunotherapy with emerging technologies.},
journal = {Cytokine &amp; growth factor reviews},
volume = {86},
number = {},
pages = {238-259},
doi = {10.1016/j.cytogfr.2025.11.001},
pmid = {41330004},
issn = {1879-0305},
mesh = {Humans ; *Immunotherapy, Adoptive/methods ; *Receptors, Chimeric Antigen/immunology/genetics ; *Neoplasms/therapy/immunology ; Animals ; Cytokines/immunology ; *T-Lymphocytes/immunology ; CRISPR-Cas Systems ; Gene Editing ; Tumor Microenvironment/immunology ; },
abstract = {Chimeric antigen receptor (CAR) T-cell (CAR-T) therapy is a transformative modality in cancer immunotherapy that employs genetically engineered T-cells to eliminate malignant cells selectively. Its efficacy and limitations are governed by cytokine- and growth factor-mediated signaling networks that shape T-cell activation, proliferation, differentiation, and persistence. This review traces the molecular evolution of CAR-T architecture across generations, highlighting how synthetic modulation of cytokine and co-stimulatory pathways enhances potency while reducing exhaustion and toxicity. We discuss strategies that incorporate cytokine engineering, metabolic reprogramming, and logic-gated activation to counteract the immunosuppressive tumor microenvironment. Recent technological advances-such as clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (CRISPR/Cas9)-based cytokine pathway editing, induced pluripotent stem cell (iPSC)-derived "off-the-shelf" CAR-T platforms, and extracellular vesicle (EV)-mediated cytokine delivery-are reshaping adoptive immunotherapy. Framing CAR-T development through the lens of cytokine and growth factor biology, we outline how integrating these pathways enables safer, more durable, and scalable next-generation therapies for hematologic and solid tumors.},
}
@article {pmid41292433,
year = {2025},
author = {Lan, F and Chen, A and Ding, Y and Yang, C and Zhang, P and Fang, X},
title = {Sensitive and Specific Analysis of miRNAs in Single Tumor-Derived Extracellular Vesicles Using CRISPR-Based Nanoflow Cytometry.},
journal = {Analytical chemistry},
volume = {97},
number = {48},
pages = {26521-26531},
doi = {10.1021/acs.analchem.5c04700},
pmid = {41292433},
issn = {1520-6882},
mesh = {*MicroRNAs/analysis/genetics ; Humans ; *Extracellular Vesicles/chemistry/genetics/metabolism ; *CRISPR-Cas Systems ; *Prostatic Neoplasms/genetics/diagnosis ; *Flow Cytometry/methods ; Male ; Limit of Detection ; Biomarkers, Tumor/genetics ; *Nanotechnology ; },
abstract = {Tumor-derived extracellular vesicle (TEV) microRNAs (miRNAs) are promising cancer biomarkers but pose detection challenges due to their low abundance and sequence homology. Here, we present a CRISPR/Cas13a-based nanoflow cytometry (nFCM) platform integrated with a DNA-guided orthogonal membrane fusion strategy for ultrasensitive miRNA detection of TEVs at the single particle level. TEVs were identified with aptamers against CD63 and EpCAM markers to create an orthogonal barcode-anchored TEV (Orth-TEV). Meanwhile, liposomes preloaded with CRISPR/Cas13a molecular sensing components were modified with cholesterol-tagged DNA probes to produce Tags-CRISPR/Cas13a@Lipo. The complementary DNA sequences on the Orth-TEV and Tags-CRISPR/Cas13a@Lipo vesicles facilitated zipper-like hybridization, thereby achieving specific membrane fusion to effectively eliminate the interference of nontarget vesicles or free molecules. The resulting TEV-CRISPR/Cas13a@Lipo vesicles allow in situ detection of three prostate cancer (PCa)-associated miRNAs in a single TEV via nFCM with a low detection limit (LOD) of 14.7 (miR-153), 16.0 (miR-183), and 23.7 (miR-940) particles/mL, respectively. The approach was further applied to plasma samples from PCa patients and healthy donors, showing significantly elevated miRNA signals in PCa-derived TEV. ROC analysis yielded AUC values of 0.931, 0.923, and 0.869 for the three target miRNAs, confirming excellent diagnostic performance. To enhance classification accuracy, we conducted a statistical multivariate analysis based on the PCA-LDA model, which achieved perfect group separation and a diagnostic accuracy of 91.3%. Overall, this CRISPR/Cas13a-based nFCM platform offers a robust, accurate, and clinically applicable platform for single-vesicle miRNA profiling with broad potential in liquid biopsy-based cancer diagnosis.},
}
@article {pmid41292075,
year = {2025},
author = {Du, J and Hu, J and An, J and Li, H and Chen, B and Luo, J and Li, S and Teng, Y and Yuan, T and Zhu, X and Jiang, L and Xiong, E and Yang, R},
title = {Guanine-Quadruplex-Engineered crRNA Enables Light-Activated CRISPR/Cas12a System for Robust One-Pot Viral Assay.},
journal = {Analytical chemistry},
volume = {97},
number = {48},
pages = {26580-26589},
doi = {10.1021/acs.analchem.5c04848},
pmid = {41292075},
issn = {1520-6882},
mesh = {*G-Quadruplexes ; *CRISPR-Cas Systems/genetics ; Nucleic Acid Amplification Techniques/methods ; Humans ; *Guanine/chemistry ; *Light ; CRISPR-Associated Proteins/metabolism ; Bacterial Proteins ; Endodeoxyribonucleases ; },
abstract = {Conventional one-pot detection platforms integrating CRISPR/Cas12a with isothermal amplification significantly streamline the nucleic acid detection workflow, while minimizing the risk of aerosol contamination. However, the intrinsic cleavage activity of the CRISPR/Cas12a system can substantially interfere with the nucleic acid amplification efficiency, ultimately compromising detection sensitivity. Herein, we develop a light-activated CRISPR/Cas12a system by engineering the crRNA with a guanine-quadruplex (G4) motif at its 3'-terminal, achieving precise regulation of Cas12a activity via photoswitching G4 structure formation. Through coupling with a recombinase polymerase amplification (RPA) reaction, we establish a one-pot detection platform that demonstrates superior detection performance compared to traditional Cas12a-based one-pot systems. The detection sensitivity has been improved by 2 orders of magnitude, reaching a level of 1 copy/μL. Notably, the platform demonstrated comparable sensitivity and specificity to PCR, the gold standard method, in detecting clinical samples, such as Epstein-Barr virus (EBV) and Influenza A virus (IAV), making it a promising technology for clinical diagnostics.},
}
@article {pmid41289351,
year = {2025},
author = {Su, T and Wei, T and Wang, Z and Wu, H and Fan, Y and Su, S and Zhu, D and Wang, L},
title = {A Pre-Amplification-Free Modular Dual-CRISPR System for Enhanced Pathogen Detection Sensitivity.},
journal = {Analytical chemistry},
volume = {97},
number = {48},
pages = {26640-26648},
doi = {10.1021/acs.analchem.5c05145},
pmid = {41289351},
issn = {1520-6882},
mesh = {*CRISPR-Cas Systems/genetics ; Nucleic Acid Hybridization ; *African Swine Fever Virus/genetics/isolation &amp; purification ; Limit of Detection ; Humans ; *DNA, Viral/analysis/genetics ; Animals ; Nucleic Acid Amplification Techniques ; },
abstract = {CRISPR/Cas12a is extensively utilized for pathogen detection owing to its high specificity and efficiency. However, traditional single-CRISPR/Cas12a encounters challenges due to its limited sensitivity, requiring pre-amplification of nucleic acids. This increases the complexity of the procedure and the potential for cross-contamination and false positives. Herein, a modular dual-CRISPR approach was developed coupled with hybridization chain reaction (HCR) for the universal and sensitive detection of pathogen nucleic acids without the need for pre-amplification. The system comprises two core modules: the first CRISPR/Cas12a recognition module specifically identifies pathogen targets and releases the activating agent, while the second CRISPR/Cas12a signal module is activated by this agent to initiate the HCR reaction for generating a strong fluorescent signal through DNA nanostructure self-assembly. Through rational design, we demonstrate the ability of this dual-CRISPR system to achieve attomolar (aM) level sensitivity for pathogen nucleic acid detection without pre-amplification, showing over six-order-of-magnitude higher sensitivity than a traditional single-CRISPR/Cas12a system. Additionally, the flexibility and versatility of the modular dual-CRISPR system have been confirmed for diverse pathogen targets, such as African swine fever virus (ASFV), severe fever with thrombocytopenia syndrome virus (SFTSV), and human papillomavirus type 16 (HPV-16) DNA. The system's practicality was demonstrated by examining ASFV quality control samples in complex environments. The exploration of the pre-amplification-free dual-CRISPR system offers a new perspective on enhancing pathogen nucleic acid detection systems.},
}
@article {pmid41265176,
year = {2026},
author = {Mao, S and Guo, Y and Dong, C and Wang, D and Wang, X and Weng, L and Yang, Y and Li, Y and Niu, T and Wu, Q and Zheng, Z and Shan, Z and Tan, X and Gao, Y and Jin, J and Wang, P and Ge, X and Shen, B and Yao, X and Fang, L},
title = {Loss of cyclin C drives resistance to anti-TIGIT therapy by upregulating CD155-mediated immune evasion.},
journal = {Drug resistance updates : reviews and commentaries in antimicrobial and anticancer chemotherapy},
volume = {84},
number = {},
pages = {101318},
doi = {10.1016/j.drup.2025.101318},
pmid = {41265176},
issn = {1532-2084},
mesh = {Humans ; *Receptors, Immunologic/antagonists &amp; inhibitors/immunology ; Killer Cells, Natural/immunology ; Cell Line, Tumor ; *Drug Resistance, Neoplasm/immunology/genetics ; Up-Regulation ; *Neoplasms/immunology/drug therapy/genetics ; CRISPR-Cas Systems ; T-Lymphocytes/immunology ; Gene Expression Regulation, Neoplastic ; Immune Evasion ; Immune Checkpoint Inhibitors/pharmacology ; Tumor Escape ; Receptors, Virus ; },
abstract = {AIMS: CD155 is an immune checkpoint protein expressed in tumor cells that interacts with its ligand T cell immunoreceptor with immunoglobulin and ITIM domain (TIGIT) on natural killer (NK) cells and T cells, mediating inhibitory regulation on immune cells. Blockade of the CD155-TIGIT interaction has demonstrated clinical benefits in patients with advanced cancers. The transcriptional and post-translational mechanisms governing CD155 expression remain largely unknown.<br><br>METHODS: To identify regulators of CD155, we conducted a genome-wide CRISPR-Cas9 screen in cancer cells. Surface CD155 protein levels were analyzed via flow cytometry. The role of candidate regulators was validated through loss- and gain-of-function experiments with flow cytometry, Western blot, quantitative PCR, and chromatin immunoprecipitation (ChIP) assays. Additionally, ubiquitination assay was performed to examine post-translational modifications. Functional studies, including NK and T cell cytotoxicity assays, were conducted to assess the immune modulatory effects of CD155 regulation. Clinical relevance was evaluated by analyzing Cyclin C (CCNC) and CD155 expression in datasets of cancer patients who underwent immune checkpoint blockade therapy.<br><br>RESULTS: The CRISPR-Cas9 screen identified CCNC as a transcriptional suppressor of CD155. CCNC knockout led to increased surface CD155 expression in cancer cell lines. Mechanistically, CCNC inhibited CD155 transcription by suppressing the activity of the transcription factor FOSL2. Furthermore, CCNC was found to be ubiquitinated and degraded by the E3 ubiquitin ligase FBXO11, suggesting a post-translational regulatory mechanism. Functionally, loss of CCNC promoted CD155 upregulation, thereby enhancing tumor immune evasion from NK and T cell-mediated responses. Clinically, CCNC expression was negatively correlated with CD155 levels in cancer patients, particularly those receiving immune checkpoint blockade therapy.<br><br>CONCLUSION: This study identifies a previously unrecognized master regulator CCNC that functions as a suppressor of CD155-mediated cancer immune evasion. The findings of this study suggest that tumors with low CCNC expression may be resistant to monotherapy and highlight a combination immunotherapy (TIGIT/PD-1 co-blockade) as a promising anti-cancer therapeutic strategy to overcome immune evasion in CCNC-deficient tumors.},
}
@article {pmid41259748,
year = {2025},
author = {Zheng, L and Zhou, X and Zhang, Y and Wang, W and Chen, C and Lin, X and Zheng, Y and Lou, Y},
title = {Rapid Bacterial Identification and Antimicrobial Susceptibility Testing Directly from Urine Samples via an Asymmetric Polymerase Chain Reaction-Cas12a Platform.},
journal = {Analytical chemistry},
volume = {97},
number = {48},
pages = {26466-26474},
doi = {10.1021/acs.analchem.5c04410},
pmid = {41259748},
issn = {1520-6882},
mesh = {Humans ; Microbial Sensitivity Tests ; *Bacteria/drug effects/isolation &amp; purification/genetics ; *Anti-Bacterial Agents/pharmacology ; *Polymerase Chain Reaction/methods ; CRISPR-Cas Systems ; *CRISPR-Associated Proteins/genetics ; *Endodeoxyribonucleases/genetics/metabolism ; Bacterial Proteins ; },
abstract = {Antimicrobial resistance poses a critical global health challenge, largely due to the prolonged turnaround times of conventional pathogen identification (ID) and antimicrobial susceptibility testing (AST). Here, we present a clinically validated diagnostic platform integrating asymmetric polymerase chain reaction (aPCR) with CRISPR/Cas12a for direct bacterial ID and phenotypic AST from urine samples. Unlike traditional multiplex PCR requiring complex primer sets, our platform employs a singleplex aPCR targeting the V3-V4 region of 16S rDNA to generate single-stranded and double-stranded DNA. This design enables protospacer adjacent motif-free activation of Cas12a when required via the ssDNA fraction generated by aPCR, facilitating species-level multiplex detection of six common uropathogens at 10[3] CFU/mL via programmable CRISPR/Cas12a crRNAs. Phenotypic AST is accomplished within 60 min by quantifying nucleic acid changes following antibiotic exposure, allowing accurate discrimination between susceptible and resistant strains. When validated with 86 clinical urine samples, the aPCR-Cas12a platform achieved complete concordance with culture-based identification among the 45 samples carrying target pathogens and demonstrated high accuracy for AST, confirming its reliability for direct pathogen detection and susceptibility assessment from urine. The complete workflow requires only 5.5 h, significantly reducing the diagnostic time compared to standard methods (>48 h). This rapid, cost-effective, and scalable platform offers a promising solution for infection diagnosis and antimicrobial stewardship, with strong potential for integration into routine clinical microbiology and point-of-care settings.},
}
@article {pmid41217936,
year = {2025},
author = {He, X and Deng, L and Zhou, S and Gu, T and Li, X and Zhu, S and Luo, X and Huo, D and Hou, C},
title = {Breaking the PAM Restriction: A Universal Double Stranded DNA Detection Method Based on the Sticky End-Mediated CRISPR/Cas12a Coupled RPA and Its Application to KRAS G12C Single Base Mutations.},
journal = {Analytical chemistry},
volume = {97},
number = {48},
pages = {26886-26896},
doi = {10.1021/acs.analchem.5c05908},
pmid = {41217936},
issn = {1520-6882},
mesh = {*CRISPR-Cas Systems/genetics ; *Proto-Oncogene Proteins p21(ras)/genetics ; *DNA/analysis/genetics ; Humans ; *Nucleic Acid Amplification Techniques/methods ; Point Mutation ; *CRISPR-Associated Proteins/metabolism/genetics ; *Endodeoxyribonucleases/metabolism/genetics ; Limit of Detection ; *Recombinases/metabolism ; Bacterial Proteins ; },
abstract = {The CRISPR/Cas12a system facilitates efficient and specific nucleic acid detection, but its dependence on Protospacer Adjacent Motif (PAM) sequences and the complexity of existing sticky end-based methods pose challenges for stable and portable applications. To address these issues, this study developed a universal dsDNA detection method by integrating the sticky end-mediated CRISPR/Cas12a with recombinase polymerase amplification (RPA). By incorporating NlaIII recognition sites into RPA primers, precise cleavage of amplification products was achieved, generating uniform sticky ends and eliminating reliance on PAM sites. In comparison to flat end dsDNA containing PAM sites, the use of sticky end dsDNA significantly enhanced Cas12a activity. This strategy demonstrated sensitivity and specificity, achieving a detection limit of 40 aM and successfully identifying KRAS G12C mutations at a frequency of 0.1%, with genomic DNA results aligning with those obtained from FastNGS. Furthermore, we preliminarily explored a one-tube detection strategy, which effectively streamlined the operational process and reduced aerosol contamination. In summary, we established a simple, sensitive, and universal PAM-free CRISPR/Cas12a detection platform that integrates the advantages of isothermal amplification with a standardized sticky end design, thereby offering broad application prospects in molecular diagnostics and clinical translation.},
}
@article {pmid40974875,
year = {2026},
author = {Sun, D and Bo, L and Jiang, C and Lan, Y and Zhang, B and Zhang, C and Chen, ZS and Fan, Y},
title = {Beyond the boundary: The emerging roles of ATP-binding cassette transporters in multidrug resistance (MDR) and therapeutic targeting in cancer.},
journal = {Drug resistance updates : reviews and commentaries in antimicrobial and anticancer chemotherapy},
volume = {84},
number = {},
pages = {101310},
doi = {10.1016/j.drup.2025.101310},
pmid = {40974875},
issn = {1532-2084},
mesh = {Humans ; *Drug Resistance, Neoplasm/drug effects/genetics ; *Neoplasms/drug therapy/genetics/pathology ; *Drug Resistance, Multiple/drug effects/genetics ; *ATP-Binding Cassette Transporters/genetics/metabolism/antagonists &amp; inhibitors ; *Antineoplastic Agents/pharmacology/therapeutic use ; Animals ; Gene Editing/methods ; CRISPR-Cas Systems ; Molecular Targeted Therapy ; Immunoconjugates/pharmacology/therapeutic use ; Immunotherapy/methods ; },
abstract = {Multidrug resistance (MDR) remains a primary obstacle to successful cancer chemotherapy, with the overexpression of ATP-binding cassette (ABC) transporters being a principal cause. These transporters actively efflux a wide range of anticancer drugs, reducing their intracellular efficacy. Consequently, targeting ABC transporters represents a critical strategy for overcoming therapeutic resistance. This comprehensive review details the molecular architecture and functional mechanisms of all seven human ABC transporter subfamilies (ABCA-ABCG), elucidating their distinct roles in both cancer progression and the development of MDR. We trace the evolution of therapeutic interventions, from first, second, and third-generation small molecule inhibitors to the potential of natural products. Furthermore, this review explores advanced and emerging strategies designed to circumvent or neutralize ABC transporter activity. These include genetic approaches such as RNA interference and CRISPR-Cas9 gene editing, immunotherapy-based tactics like monoclonal antibodies and antibody-drug conjugates (ADCs), and the application of sophisticated nanoparticle delivery systems designed to bypass efflux mechanisms. By providing a holistic overview of the entire ABC transporter family and the broad array of strategies being developed to counteract their function, this article aims to equip researchers with a full-scope perspective on the field, identifying current challenges and illuminating future directions for combating MDR in cancer.},
}
@article {pmid41352908,
year = {2026},
author = {Li, L and Tang, Z and Xu, H and Zhou, F and Ji, X and He, Z},
title = {Investigation on CRISPR-Cas12a-split crRNA system for successively detecting DNA and RNA in one tube.},
journal = {Analytica chimica acta},
volume = {1383},
number = {},
pages = {344860},
doi = {10.1016/j.aca.2025.344860},
pmid = {41352908},
issn = {1873-4324},
mesh = {*CRISPR-Cas Systems ; *DNA, Viral/analysis/genetics ; *RNA, Viral/analysis/genetics ; Hepatitis B virus/genetics ; *CRISPR-Associated Proteins/metabolism/genetics ; Humans ; *Endodeoxyribonucleases/metabolism/genetics ; *Bacterial Proteins/metabolism/genetics ; HIV/genetics ; },
abstract = {Recently, CRISPR/Cas system has been proposed as a novel tool with simplicity and high accuracy. The CRISPR RNA (crRNA) can be divided into spacer crRNA and handle crRNA without losing its original function. In this work, we have investigated CRISPR Cas12a with split crRNA to detect HBV DNA and HIV RNA in a single tube. In the first step, Cas12a can recognize HBV DNA and initiate its trans-cleavage on FAM-BHQ1 reporter, after 1 h incubation, the fluorescence intensity was correlated with the concentration of HBV DNA. In the second step, the TAMRA BHQ2 ds DNA reporter was introduced in the same tube to bind with remained Cas12a proteins, HIV RNA and handle crRNA. The trans-cleavage from the first step would not interfere with HIV RNA and dsDNA reporter. With the incubation for another hour, HIV RNA can be quantified by the cis-cleavage of TAMRA BHQ2 reporter. we can successively identify the two nucleic acids with the limit of detection of 0.70 pM for HBV DNA, and 0.47 nM for HIV RNA, respectively. This special designed split crRNA can simplify detecting procedure and only need Cas12a protein in a single tube. Next, we expand this strategy in semi-quantifying two kinds of DNA in one tube. Overall, this study overcomes the limitation of conventional CRISPR-based methods and provides a new, inexpensive, and low-threshold approach based on Cas12a with split crRNA.},
}
@article {pmid41352906,
year = {2026},
author = {Fakhr, ZA and Xie, W and Zeng, S and Cai, S},
title = {Site accessibility-driven CRISPR/Cas13a activation for amplification-free RNA biosensing.},
journal = {Analytica chimica acta},
volume = {1383},
number = {},
pages = {344858},
doi = {10.1016/j.aca.2025.344858},
pmid = {41352906},
issn = {1873-4324},
mesh = {*Biosensing Techniques/methods ; *CRISPR-Cas Systems ; *RNA/analysis ; *RNA, Guide, CRISPR-Cas Systems/metabolism/genetics/chemistry ; Kinetics ; *CRISPR-Associated Proteins/metabolism ; },
abstract = {BACKGROUND: CRISPR-Cas13a biosensing enables rapid, amplification-free RNA diagnostics, yet assay sensitivity varies widely because guide RNAs (gRNAs) differ in their ability to activate the enzyme. Two factors, including the gRNA-target binding affinity and the structural accessibility of the target site, have been proposed to govern activation efficiency, but their relative importance remains unclear. In this study, we systematically disentangle these contributions by measuring binding affinities for gRNAs that span a spectrum of site accessibilities and by comparing their Michaelis-Menten kinetic parameters.<br><br>RESULTS: Three ciRS-7-specific gRNAs were designed with high, intermediate, and low spacer accessibility. Isothermal titration calorimetry (ITC) quantified site accessibility through entropy changes (&#x394;S&#xa0;=&#xa0;-862, -813, and -615&#xa0;cal/mol/K), confirming greater structural exposure for less structured spacers, and also determined binding affinity for each gRNA-target pair. Michaelis-Menten analysis showed kcat values of 1.39, 1.31, and 1.16 s[-1] for the high, intermediate, and low-accessibility guides, respectively, establishing a clear relationship between structural accessibility and catalytic turnover. Importantly, the most structured gRNA exhibited lower activation efficiency compared with the gRNA that had higher site accessibility and lower binding affinity, demonstrating that site accessibility drives Cas13a activation. Detection-limit experiments also confirmed these results, showing that gRNAs with greater spacer accessibility yielded stronger signals and superior sensitivity.<br><br>SIGNIFICANCE: Our data establish site accessibility as a critical determinant of Cas13a activation for amplification-free RNA sensing. Prioritizing unstructured spacer regions enables improved enzyme activation efficiency, providing a clear design rule for next-generation CRISPR diagnostics. This accessibility-driven strategy will facilitate the development of faster, simpler, and more sensitive point-of-care assays for diverse RNA biomarkers.},
}
@article {pmid41351274,
year = {2025},
author = {Hou, H and Li, Y and Su, N and Ding, Y and Shang, C and Li, X and Xiong, Z and Sun, Y and Zhan, W and Wang, Y and Zhang, X and Pan, Y and Wu, L and Li, J},
title = {Slmsh1-induced heritable enhancement of traits for tomato breeding improvement.},
journal = {The Plant journal : for cell and molecular biology},
volume = {124},
number = {5},
pages = {e70607},
doi = {10.1111/tpj.70607},
pmid = {41351274},
issn = {1365-313X},
support = {CSTB2023TIAD-KPX0026//Special Key Project of Technological Innovation and Application Development of Chongqing/ ; 31872123//National Natural Science Foundation of China/ ; 32172597//National Natural Science Foundation of China/ ; CARS-23-B08//China Agriculture Research System/ ; SWU-KF25027//Fundamental Research Funds for the Central Universities/ ; },
mesh = {*Solanum lycopersicum/genetics/physiology/growth &amp; development ; *Plant Breeding/methods ; *Plant Proteins/genetics/metabolism ; Fruit/genetics/growth &amp; development ; Droughts ; Quantitative Trait, Heritable ; Phenotype ; CRISPR-Cas Systems ; Gene Expression Regulation, Plant ; },
abstract = {Vegetable grafting is a horticultural technique employed to develop specialized plant varieties by effectively enhancing resistance to both biotic and abiotic stresses, as well as improving fruit quality and yield. However, these advantageous traits are generally non-heritable. The MSH1 gene induced heritable enhancement-through-grafting (HEG) effect on growth vigor, demonstrating promising application potential. In this study, we employed the msh1 mutant tomato as a rootstock to induce heritable superior traits and combined this approach with hybridization techniques to enhance tomato cultivars. Three Slmsh1 mutants were generated using CRISPR/Cas9 which exhibited a dwarf phenotype with whitened spots. By grafting several distinct inbred lines onto Slmsh1, we observed significant HEG, drought stress tolerance, and fruit quality. Under drought conditions, Slmsh1-grafted tomato seedlings exhibited increased biomass and enhanced drought tolerance through the regulation of antioxidant enzyme activities. Differential expression and methylation analyses of the graft progeny revealed that these heritable enhanced traits (HETs) are likely attributable to epigenetic modifications in the expression of ROS-scavenging- and hormone-related genes. Furthermore, to explore practical applications, we crossed inbred lines with HETs and evaluated the growth, yield, and fruit quality of the resulting hybrid combinations. The results indicated that these hybrid combinations improved fruit yield and quality, enhancing the total soluble solids, soluble sugar, and soluble protein content. These findings suggest that Slmsh1-grafted progenies enhanced plant biomass and drought resistance, while their hybrid combinations positively influenced root growth, yield, and fruit quality, providing new insights into the synergistic integration of genome editing and conventional breeding.},
}
@article {pmid41350682,
year = {2025},
author = {Lyu, G and Li, P and Lang, W},
title = {A review of recent studies on CRISPR/Cas9-mediated genome editing in a variety of muscle-related genetic disorders.},
journal = {Journal of translational medicine},
volume = {23},
number = {1},
pages = {1381},
pmid = {41350682},
issn = {1479-5876},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Gene Editing ; Animals ; *Muscular Diseases/genetics/therapy ; *Genetic Diseases, Inborn/genetics ; Genetic Therapy ; Muscular Dystrophies/genetics ; },
abstract = {The human body is capable of mutating a single gene to produce a wide range of debilitating disorders. Genomic editing for disease prevention via phenotypic reversal was a significant challenge prior to the development of clustered regulatory interspaced short tandem repeats (CRISPR) and CRISPR-associated protein (Cas) systems. Gene therapy-editing a patient's DNA to correct a particular mutation-and treating human diseases that have not responded to conventional medicine are two areas where CRISPR/Cas9 technology shows the most promise as a therapeutic tool. This powerful instrument has shown great promise in muscle-related illnesses, offering new insights into muscle biology and developing more effective treatment techniques. Discoveries about the hereditary causes of the majority of inherited myopathies and muscular dystrophies (MDs) have emerged over the last two decades. Additionally, skeletal muscles weaken and degenerate over time due to a group of hereditary disorders known as MDs. The field of skeletal muscle diseases and associated genetic alterations is seeing remarkable progress in developing therapeutic vectors to fix these mutations. Myopathies, MDs, and neuromuscular disorders are just a few examples of the many genetic abnormalities related to muscles that have sparked renewed interest in the potential of genome editing as a therapeutic tool due to its efficiency, adaptability, and relative ease of use in targeted genome editing. Consequently, CRISPR/Cas9 has garnered much interest and is used more often in therapeutic techniques due to its potential capacity to cure various human ailments. To pave the way for more effective and personalized therapies, this review article provides a thorough overview of the revolutionary role of CRISPR/Cas9 in improving our understanding and treatment of genetic disorders related to muscles by combining present knowledge with future perspectives.},
}
@article {pmid41349515,
year = {2025},
author = {Kim, I and Suh, JY},
title = {Capture first, then deliver!.},
journal = {Structure (London, England : 1993)},
volume = {33},
number = {12},
pages = {2008-2009},
doi = {10.1016/j.str.2025.11.001},
pmid = {41349515},
issn = {1878-4186},
mesh = {*CRISPR-Cas Systems ; *CRISPR-Associated Proteins/chemistry/metabolism ; DNA/metabolism/chemistry ; *Integrases/chemistry/metabolism ; Protein Binding ; },
abstract = {In this issue of Structure, Henriques et al.[1] present structural snapshots that capture distinct conformational states of the type I-F Cas1-Cas2/3 integrase complex, illustrating that foreign DNA binding triggers a large-scale domain rearrangement that enables prespacer delivery to the CRISPR array.},
}
@article {pmid41349512,
year = {2025},
author = {Kosaka, Y and Lopez, B and Kishimoto, N and Jacob, S and Montenont, E and Huallanca, R and Coughenour, G and Di Paola, J and Ross, J and Lee, K and Rondina, MT and Bray, PF and Rowley, JW},
title = {Functional classification of platelet gene variants using CRISPR HDR in CD34[+] cell-derived megakaryocytes.},
journal = {American journal of human genetics},
volume = {112},
number = {12},
pages = {2888-2901},
doi = {10.1016/j.ajhg.2025.11.004},
pmid = {41349512},
issn = {1537-6605},
mesh = {Humans ; *Megakaryocytes/metabolism ; *Blood Platelets/metabolism ; *CRISPR-Cas Systems/genetics ; *Antigens, CD34/metabolism/genetics ; Integrin beta3/genetics ; Gene Editing/methods ; Integrin alpha2/genetics ; *Genetic Variation ; Hematopoietic Stem Cells/metabolism ; Thrombasthenia/genetics ; },
abstract = {The interpretation of genetic variants in inherited diseases, such as inherited platelet disorders (IPDs), remains a major clinical challenge, as most are classified as variants of uncertain significance (VUSs). A key barrier to functional evaluation is the lack of accessible, lineage-appropriate assays that reliably reflect native gene regulation and cell-specific biology. To address this gap, we developed CRIMSON HD (CRISPR-edited megakaryocytes [MKs] for surveying platelet variant functions through homology-directed repair [HDR]), a CRISPR-Cas9 HDR-based genome-editing platform applicable to CD34[+] cell-derived blood lineages and optimized for evaluating platelet-associated variants. Using this system, we modeled known and candidate disease-associated variants in integrin alpha 2b (ITGA2B) and integrin beta 3 (ITGB3), which encode the platelet αIIb/β3 integrin and are causative in Glanzmann thrombasthenia (GT). We introduced precise variants into primary human MKs derived from CD34[+] hematopoietic stem and progenitor cells, achieving >90% editing efficiency. Edited MKs faithfully recapitulated both expression and functional phenotypes of known type I, II, and III GT variants. CRIMSON HD enabled functional evaluation and reclassification of several GT VUSs, including αIIb Gly201Ala, a population variant now shown to cause near-complete loss of αIIb/β3 expression; αIIb Ala777Asp, which results in intermediate αIIb/β3 expression and impaired agonist-induced integrin binding; and β3 Arg119Gln, previously linked to the loss of anti-HPA1a antibody binding in fetal and neonatal alloimmune thrombocytopenia (FNAIT), now shown to impair integrin surface expression. These findings demonstrate the importance of lineage-specific, physiologically relevant assays for the functional classification of platelet-related variants, providing mechanistic information and clinically meaningful insights for individuals with IPDs.},
}
@article {pmid41348871,
year = {2025},
author = {Puppala, AK and Nielsen, AC and Regan, M and Mancinelli, GE and De Pooter, RF and Arnovitz, S and Harding, C and McGregor, M and Balanis, NG and Clarke, R and Merrill, BJ},
title = {Programmable multistep CRISPR gene activation via control of RNA polymerase III termination.},
journal = {Science advances},
volume = {11},
number = {49},
pages = {eadt1532},
pmid = {41348871},
issn = {2375-2548},
mesh = {Humans ; *RNA Polymerase III/metabolism/genetics ; *CRISPR-Cas Systems ; *Transcriptional Activation ; Gene Editing/methods ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Induced Pluripotent Stem Cells/metabolism/cytology ; HEK293 Cells ; },
abstract = {Although genomes encode instructions for mammalian cell differentiation with rich syntactic relationships, existing methods for genetically programming cells have only modest capabilities for stepwise gene regulation. Here, we develop a sequential genetic system that transcriptionally activates endogenous genes in a preprogrammed, stepwise manner. This system uses the removal of an RNA polymerase III termination sequence to trigger both the transcriptional activation and DNA endonuclease activities of a Cas9-VPR protein, driving progression through a cascade of gene activation events. The system's functionality in human cells, including iPSCs, enables the development of a path for cellular programming by controlling the sequential order of gene activation to influence cellular states.},
}
@article {pmid41348151,
year = {2025},
author = {Cheng, Y and Gao, W and Shi, S and Han, F and Dong, H},
title = {Identification of the orange pigment in Nonomuraea gerenzanensis and development of a pigment-free mutant with high yield of A40926.},
journal = {AMB Express},
volume = {},
number = {},
pages = {},
doi = {10.1186/s13568-025-01993-4},
pmid = {41348151},
issn = {2191-0855},
support = {2024TSGC0896//Shandong Province Science and Technology-based Small and Medium-sized Enterprises Innovation Capacity Enhancement Project/ ; },
abstract = {The secondary metabolite A40926, a precursor to the glycopeptide antibiotic dalbavancin, is synthesized by the rare actinomycete Nonomuraea gerenzanensis (N. gerenzanensis) within the pharmaceutical industry. The biosynthesis of A40926 is accompanied by the production of an orange pigment, which poses significant challenges and incurs high costs in the purification process of A40926. To identify this orange pigment, a comprehensive analysis was conducted, including the examination of the biosynthetic gene cluster, potential biosynthetic pathways, purification processes, and structural identification. Additionally, the ispF gene, which encodes the enzyme 2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase and is implicated in the biosynthesis of orange pigment, was deleted using the CRISPR/Cas9 system. To enhance A40926 production in the ΔIspF mutant, the overexpression of the cyclic AMP receptor protein (Crp) was implemented to assess its regulatory impact on A40926 biosynthesis. Consequently, the orange pigment produced by N. gerenzanensis was identified as lycopene, synthesized via the methylerythritol phosphate (MEP) pathway. Although the ΔIspF mutant was unable to biosynthesize the orange pigment, its production of A40926 was adversely affected and was lower than that of the original strain. Consequently, the overexpression of the global regulator Crp significantly enhanced A40926 production, achieving a yield of 841.1 mg/L. The investigation of pigment-free mutants presented in this study offers valuable insights for effectively reducing production costs within the microbial pharmaceutical industry.},
}
@article {pmid41271111,
year = {2026},
author = {Huang, Q and Zhao, T and Su, W and Li, S and Liu, J and Ge, Z and Zhang, B and Ren, X and Zhang, X and Wei, J},
title = {Screening of monoclonal vaccine strains based on real-time live-cell imaging technology.},
journal = {Journal of virological methods},
volume = {340},
number = {},
pages = {115305},
doi = {10.1016/j.jviromet.2025.115305},
pmid = {41271111},
issn = {1879-0984},
mesh = {Animals ; *Vaccinia virus/genetics/immunology/isolation &amp; purification ; *Viral Vaccines/immunology/genetics/isolation &amp; purification ; Viral Plaque Assay/methods ; Vaccines, Attenuated/immunology/genetics ; Antibodies, Viral/blood/immunology ; Humans ; Green Fluorescent Proteins/genetics ; CRISPR-Cas Systems ; Vaccines, Synthetic/immunology/genetics ; Antibodies, Neutralizing/blood ; },
abstract = {The plaque purification is a critical step in the screening of traditional live-attenuated vaccines and recombinant viral vaccines, aiming to acquire vaccine clones with homogeneous characteristics and desirable immunogenicity to address outbreaks of emerging diseases such as monkeypox, chikungunya fever, and dengue fever. The traditional plaque purification process to screen out a vaccine strain with genetically consistent stability from a mixed pool of viral clones generally requires laborsome work. We utilized live-cell imaging technique enabling us to isolate monoclonal vaccine strains to simplify and improve the efficiency of this process. Here, we genetically engineered the vaccinia virus TianTan (VTT) using CRISPR/Cas9 system to generate recombinant VTT viruses (VTT-WS01-EGFP) that expressed enhanced green fluorescent protein (EGFP). Initially, we performed 9 rounds of plaque purification using traditional plaque assay, yielding 50 candidate clones. The Incucyte Live-Cell Imaging and Analysis system was subsequently performed to conduct a rigorous, high-resolution screening of these candidates in a more automated, sensitive and high-throughput way. Through this screening process, we ultimately obtained 31 pure viral clones that were free of parental strain contamination, followed by the analysis of plaque formation, fluorescent plaque size, and plaque morphology, and 11 candidate clones were selected for immunological evaluation. Furthermore, we found that clone 49 induced a relatively high titer of anti-VTT neutralizing antibodies and elicited the production of cross-reactive IgG against monkeypox virus antigens, thereby validating its potential as a candidate strain as a monkeypox virus vaccine. Taken together, our data demonstrates that live-cell imaging technique significantly accelerates the screening process for the isolation of monoclonal viral clones as recombinant viral vaccines, and holds considerable potential in attenuated strain selection as well as investigations into biological characteristics of viruses, including viral replication.},
}
@article {pmid41260396,
year = {2026},
author = {Zhong, Z and Li, G and Liang, G and Ren, T and Teng, C and Xiong, J and Ji, G and Zheng, M and Pan, Y and Qin, Y and Ouyang, K and Yin, Y and Chen, Y and Huang, W and Wei, Z},
title = {Establishment of a nucleic acid detection method for foot-and-mouth disease virus serotype O utilizing RPA-CRISPR/Cas12a technology.},
journal = {Journal of virological methods},
volume = {340},
number = {},
pages = {115304},
doi = {10.1016/j.jviromet.2025.115304},
pmid = {41260396},
issn = {1879-0984},
mesh = {*Foot-and-Mouth Disease Virus/genetics/isolation &amp; purification/classification ; Sensitivity and Specificity ; *Nucleic Acid Amplification Techniques/methods ; *CRISPR-Cas Systems ; *Foot-and-Mouth Disease/diagnosis/virology ; Animals ; Serogroup ; Recombinases/metabolism/genetics ; DNA Primers/genetics ; RNA, Viral/genetics ; *Molecular Diagnostic Techniques/methods ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {This study aimed to develop a rapid and visually interpretable nucleic acid detection assay for Foot-and-Mouth Disease Virus serotype O (FMDV-O) by integrating recombinase polymerase amplification (RPA) with CRISPR/Cas12a technology. Specific RPA primers and CRISPR RNA (crRNA) sequences were designed and optimized based on the conserved 3D gene region of FMDV-O. An assay combining RPA pre-amplification with Cas12a-mediated cleavage was subsequently established. The sensitivity and specificity of the RPA-CRISPR/Cas12a method were systematically evaluated, and its diagnostic utility was further assessed using clinical samples. The results demonstrated that the primer set RPA-F1/R1 paired with crRNA1 constituted the optimal combination, with an ideal reaction system comprising 50 nM Cas12a protein and 200 nM crRNA. This system exhibited a detection limit of 2.60 × 10[2] copies/μL for target plasmid DNA following a 20-minute incubation at 37°C. Specificity analysis confirmed positive detection exclusively for FMDV-O plasmids, with no cross-reactivity observed with other tested pathogens. When applied to clinical samples, the proposed method demonstrated a superior detection rate relative to conventional PCR. In conclusion, a novel diagnostic platform for FMDV-O was successfully developed based on RPA-CRISPR/Cas12a. This method is characterized by its rapidity, operational simplicity, high sensitivity, and excellent specificity, holding significant promise for application in clinical diagnostics, epidemiological surveillance, and field-based testing.},
}
@article {pmid41231539,
year = {2025},
author = {Jia, N and Zhou, YJ and Gao, J},
title = {Engineering recombination machinery facilitates the construction of yeast cell factories.},
journal = {FEMS yeast research},
volume = {25},
number = {},
pages = {},
doi = {10.1093/femsyr/foaf066},
pmid = {41231539},
issn = {1567-1364},
support = {22478382//National Natural Science Foundation of China/ ; E411040705//Energy Revolution S&amp;T Program of Yulin Innovation Institute of Clean Energy/ ; },
mesh = {*Metabolic Engineering/methods ; *Gene Editing/methods ; *DNA End-Joining Repair ; *Saccharomyces cerevisiae/genetics/metabolism ; *Homologous Recombination ; *Recombination, Genetic ; CRISPR-Cas Systems ; DNA Breaks, Double-Stranded ; },
abstract = {Advances in genome editing have been promoted by programmable nucleases like CRISPR-Cas9, which triggers endogenous DNA repair mechanisms by inducing double-strand break (DSB). Cellular responses to DSBs are governed by competing repair pathways: error-prone non-homologous end joining (NHEJ) and high-fidelity homologous recombination (HR). This review systematically compares the molecular mechanisms and key regulators of NHEJ and HR, with a focus on recent breakthroughs in recombination engineering in non-conventional yeasts. These advances address challenges in precise genome editing, enabling robust metabolic engineering of yeast cell factories for sustainable bioproduction.},
}
@article {pmid41347244,
year = {2025},
author = {Daraghmeh, DN and AbuIriban, RW and Nawawreh, N and Abuamro, AM and Alassar, MM and Daraghma, SN and Alhajahmed, NM and Thandar, Y},
title = {Advancements in alternative approaches to address antimicrobial resistance in bacterial pneumonia: a comprehensive review.},
journal = {Frontiers in microbiology},
volume = {16},
number = {},
pages = {1704931},
pmid = {41347244},
issn = {1664-302X},
abstract = {PURPOSE: This review explores both current and emerging alternative treatment approaches to combat AMR specifically in the context of bacterial pneumonia, highlighting therapies that extend beyond conventional antibiotics.<br><br>METHODS: PubMed, Embase, and Google Scholar were searched for full-text, English-language articles, with emphasis on publications from 2020 to 2025. Earlier seminal studies were also included when necessary to provide historical, mechanistic, or conceptual context. The review focuses was on alternative strategies that have shown effectiveness in preclinical or clinical settings to combat AMR in relation to bacterial pneumonia.<br><br>RESULTS: Emerging strategies to tackle AMR in bacterial pneumonia involve several innovative approaches including stem cells, bacteriophage therapy, metal based nanoparticles (e.g., silver, copper, and gold). The adjunctive use of probiotics and herbal medicine has demonstrated potential in enhancing clinical outcomes and modulating host immunity. Moreover, gene editing technologies like CRISPR-CAS and various vaccination programs are being investigated for their roles in prevention and resistance management. While these methods show promise, many are still in the early stages of development and encounter challenges related to standardization, safety, and regulatory approval.<br><br>CONCLUSION: Alternative therapies present exciting possibilities for addressing AMR in bacterial pneumonia. However, to effectively translate these innovations into clinical practice, we need thorough research, international collaboration, and supportive policy frameworks. By combining these strategies with antimicrobial stewardship initiatives, we can help maintain antibiotic effectiveness and enhance patient outcomes.},
}
@article {pmid41346702,
year = {2026},
author = {Patra, C and Hussein, Z and Ace, VD and Misnik, EV and Rybalko, DS and Salimova, AA and Ereshko, DS and Dubovichenko, MV and Nour, MAY and Drozd, VS and Kolpashchikov, DM},
title = {The efficacy of oligonucleotide-based gene therapeutics in gene silencing.},
journal = {Theranostics},
volume = {16},
number = {2},
pages = {599-616},
pmid = {41346702},
issn = {1838-7640},
mesh = {Humans ; *Genetic Therapy/methods ; *Gene Silencing ; *Oligonucleotides, Antisense/therapeutic use/genetics ; RNA, Small Interfering/genetics/therapeutic use ; Animals ; *Oligonucleotides/genetics/therapeutic use ; },
abstract = {Oligonucleotide-based gene therapeutics (OGTs) have emerged as a promising strategy for treating a variety of diseases, offering a tool for gene modulation at the mRNA level. Despite significant progress in OGTs development, their efficacy in both experimental and clinical settings has often fallen short of expectations. Current estimates suggest that less than 1% of transfected OGTs are released into the cytosol, significantly limiting the interaction with target RNA. Moreover, data suggests that only about 2% of the tested siRNAs achieve the expected 70% target gene knockdown in vitro. Clinically approved OGTs appear to be effective only against genetic disorders that lack effective alternative treatment, and even in these cases their therapeutic contribution remains marginal. Notably, the majority of approved OGTs, as well as those currently in clinical trials, are antisense oligonucleotides (ASOs) despite cell culture data showing that small interfering RNAs (siRNAs) exhibit greater potency. The delayed commercialization of siRNAs, despite high research interest, may be attributed to passenger stand-dependent off target effect and the immaturity of their design and modification strategies. This review critically evaluates the factors influencing therapeutic efficacy of OGTs and highlights the persistent gap between theoretical promise and clinical reality.},
}
@article {pmid41275796,
year = {2025},
author = {Gao, Y and Zhao, L and Shi, J and Wang, B},
title = {NtQPT2 plays critical roles in nicotine biosynthesis and development of tobacco plant.},
journal = {Biochemical and biophysical research communications},
volume = {793},
number = {},
pages = {153030},
doi = {10.1016/j.bbrc.2025.153030},
pmid = {41275796},
issn = {1090-2104},
mesh = {*Nicotiana/growth &amp; development/genetics/metabolism/enzymology ; *Nicotine/biosynthesis ; *Pentosyltransferases/genetics/metabolism ; Gene Expression Regulation, Plant ; *Plant Proteins/genetics/metabolism ; CRISPR-Cas Systems ; Gene Knockout Techniques ; Plants, Genetically Modified ; },
abstract = {The enzyme quinolinate phosphoribosyltransferase (QPT), encoded by a small gene family in tobacco plant, plays a critical role in the biosynthesis of nicotine, a defensive pyridine alkaloid in Nicotiana species, in addition to its vital function in the NAD(P)(H) synthesis. Previous studies have demonstrated that two NtQPT genes (NtQPT1 and NtQPT2) are present in N. tabacum genome, and it has been believed that NtQPT1 is responsible for NAD(P)(H) synthesis and thus essential for primary metabolism, while NtQPT2 is specifically involved in nicotine biosynthesis. In this study, we generated knockout tobacco lines for NtQPT1 and NtQPT2 respectively using the CRISPR/Cas9-based genome-editing technology and found that knockout of NtQPT2 caused both dramatic reduction of nicotine biosynthesis and a retardation of plant development, indicating that NtQPT2 is important not only to nicotine biosynthesis, but also to the development of tobacco plant. Like NtQPT2, NtQPT1 was also found to contribute to nicotine biosynthesis although to a much lesser extent than NtQPT2. Meanwhile, knockout of NtQPT1 did not significantly affect plant growth. Together with the observation that NtQPT2's expression is remarkably higher than that of NtQPT1 in root, leaf, stem and flower of tobacco plant, it is reasonable to infer that their functional diversification on nicotine biosynthesis and tobacco plant growth may be attributed largely to their markedly different transcript abundance.},
}
@article {pmid41274245,
year = {2025},
author = {Shimura, R and Yamamoto, K and Chang, YH and Otaki, A and Goyama, S},
title = {Development of a CRISPR/Cas9-degron system that enables in vivo specific gene depletion in leukemia models.},
journal = {Biochemical and biophysical research communications},
volume = {793},
number = {},
pages = {153002},
doi = {10.1016/j.bbrc.2025.153002},
pmid = {41274245},
issn = {1090-2104},
mesh = {*CRISPR-Cas Systems/genetics ; Animals ; Humans ; *Leukemia, Myeloid, Acute/genetics/pathology ; Histone-Lysine N-Methyltransferase/genetics ; Mice ; *Gene Editing/methods ; Cell Line, Tumor ; Disease Models, Animal ; CRISPR-Associated Protein 9 ; Degrons ; },
abstract = {The CRISPR/Cas9 system has transformed genome editing, yet precise temporal control of Cas9 activity remains challenging. We developed a Cas9-degron platform that couples degron-tagged Cas9 with a dTAG-based chemical degradation strategy. In the presence of dTAG, Cas9 is rapidly and near-completely degraded, preventing editing; upon dTAG withdrawal, Cas9 activity is restored, enabling precise temporal control. Using this system, we achieved selective in vivo gene depletion in acute myeloid leukemia (AML) models and confirmed that SETDB1, a histone H3K9 methyltransferase, is essential for the in vivo growth of both human (MOLM13) and murine (cSAM) AML cells. By maintaining SETDB1 intact prior to transplantation and depleting it afterward, we avoided culture-induced pre-selection bias inherent to sgRNA transduction and validated its critical role in AML progression within the in vivo context. The Cas9-degron retains activity and delivery efficiency comparable to conventional Cas9 in the absence of dTAG. Thus, this versatile system provides a superior alternative to conventional Cas9 and a powerful platform for in vivo CRISPR screening, gene function studies, and potentially temporally controlled gene therapy.},
}
@article {pmid41086995,
year = {2025},
author = {Jang, SH and Song, HG and Jung, J and Gee, HY},
title = {Recent preclinical and clinical advances in gene therapy for hereditary hearing loss.},
journal = {Molecules and cells},
volume = {48},
number = {12},
pages = {100285},
pmid = {41086995},
issn = {0219-1032},
mesh = {*Genetic Therapy/methods ; Humans ; *Hearing Loss/therapy/genetics ; Animals ; Gene Editing ; Genetic Vectors/genetics ; CRISPR-Cas Systems ; Dependovirus/genetics ; Gene Transfer Techniques ; Clinical Trials as Topic ; },
abstract = {Hereditary hearing loss is a genetically heterogeneous condition that affects millions of people worldwide and has limited curative treatment options. Recent advancements in gene therapy have opened promising avenues for correcting the underlying genetic defects in the inner ear. This review summarizes the key developments in vector platforms, delivery strategies, target genes, preclinical models, and clinical trials relevant to both gene supplementation and gene editing approaches, as well as future directions. Adeno-associated virus vectors have emerged as the leading platform for inner ear gene transfer, owing to their safety and efficacy. Clinical programs, such as those targeting OTOF variants, are currently underway and are supported by robust preclinical data. Additionally, genome editing technologies, including CRISPR/Cas9-mediated nonhomologous end joining, base editing, and prime editing, offer variant-specific therapeutic potential. Despite these advances, challenges remain in expanding the therapeutic window, ensuring long-term safety, and establishing ethical and regulatory frameworks for their use.},
}
@article {pmid41346247,
year = {2025},
author = {Singh, V and Mishra, M and Singla-Pareek, SL and Roy, JK and Pareek, A},
title = {Lysine Matters: Genetic and Biotechnological Innovations to Combat Protein Malnutrition.},
journal = {Plant, cell &amp; environment},
volume = {},
number = {},
pages = {},
doi = {10.1111/pce.70316},
pmid = {41346247},
issn = {1365-3040},
support = {//This study was supported by Department of Biotechnology, Ministry of Science and Technology, India./ ; },
abstract = {Lysine deficiency in staple crops like maize, rice, and wheat remains a major cause for global protein malnutrition, underscoring the urgent need for effective biofortification strategies. This review critically examines recent advances in enhancing lysine content, spanning conventional breeding and metabolic engineering to cutting-edge precision genome editing. While conventional breeding, exemplified by Quality Protein Maize, has improved lysine levels, it is often constrained by yield and quality trade-offs. Metabolic engineering strategies, including overexpression of lysine biosynthetic genes, suppression of catabolic genes, and modification of storage proteins, have achieved substantial lysine enrichment but face regulatory and consumer acceptance challenges due to their transgenic nature. The advent of CRISPR/Cas technology now enables precise, transgene-free editing of key enzymes such as DHDPS, AK, and LKR/SDH offering a powerful alternative, though concerns regarding off-target effects and pleiotropy remain. While integrating multi-omics with AI-driven predictive modelling can optimise metabolic flux for higher lysine yield, coupling next-generation genome editing with speed breeding offers a transformative route to develop high-lysine, high-yielding crops for sustainable nutritional security.},
}
@article {pmid41345283,
year = {2025},
author = {Saydam, S and Dinçer, P},
title = {Precision rewriting of muscle genetics: therapeutic horizons of base and prime editing in skeletal muscle disorders.},
journal = {Gene therapy},
volume = {},
number = {},
pages = {},
pmid = {41345283},
issn = {1476-5462},
abstract = {Base Editing (BE) and Prime Editing (PE), novel precision tools of the CRISPR/Cas toolbox, have emerged as transformative technologies that enable highly specific genetic modifications. Their compatibility with post-mitotic cell types makes them invaluable for treating genetic skeletal muscle disorders. Despite their severity and progressive nature, monogenic muscle diseases remain without definitive treatments. They are caused by diverse mutations in critical muscle proteins, for which gene editing offers a promising therapeutic avenue. However, traditional CRISPR/Cas9 applications face challenges such as genotoxicity and inefficiency in post-mitotic tissues. BE and PE technologies overcome these limitations by enabling safe and efficient modifications without causing double-strand breaks or requiring homology-directed repair. Their therapeutic potential comes from two key features: their ability to work in non-dividing cells such as myotubes and cardiomyocytes, and their capacity to target a broad range of mutations found in genetic muscle diseases. In this review, we explore mechanisms of BE and PE and summarize their current applications in monogenic skeletal muscle disorders. We discuss the challenges of in vivo application in skeletal muscle and highlight innovations to bypass them. Collectively, both systems offer flexible precision solutions with immense potential for mutation-specific and personalized gene therapy approaches for monogenic skeletal muscle disorders.},
}
@article {pmid41344769,
year = {2026},
author = {Liu, X and Zheng, Y and Chen, Z and Wang, S and Liao, H and Jia, J and Wang, G and Wang, J and Yuan, C and Guo, X and Yin, Y and Hu, Q},
title = {Rapid and visual detection of Listeria monocytogenes by combining one-pot LAMP-CRISPR/Cas12b with lateral flow assay.},
journal = {Food microbiology},
volume = {135},
number = {},
pages = {104977},
doi = {10.1016/j.fm.2025.104977},
pmid = {41344769},
issn = {1095-9998},
mesh = {*Listeria monocytogenes/isolation &amp; purification/genetics ; *Nucleic Acid Amplification Techniques/methods ; Animals ; Food Contamination/analysis ; Food Microbiology/methods ; Swine ; *CRISPR-Cas Systems ; *Molecular Diagnostic Techniques/methods ; Sensitivity and Specificity ; Limit of Detection ; },
abstract = {Listeria monocytogenes, the leading cause of fatalities worldwide among foodborne pathogens, poses serious risks to food safety and public health. Therefore, a rapid and accurate detection method is crucial for early interception and effective management. In this study, a one-pot LAMP-CRISPR/Cas12b detection system based on the lmo0753 gene was developed for rapid detection of L. monocytogenes by combining loop-mediated isothermal amplification (LAMP) with a CRISPR/Cas12b assay. Further integration of a lateral flow assay (LFA) to develop a LAMP-CRISPR/Cas12b-LFA assay enabled direct detection of the results on the strips with the naked eye. Nine L. monocytogenes strains belonging to eight serotypes tested positive with both the one-pot LAMP-CRISPR/Cas12b and LAMP-CRISPR/Cas12b-LFA assays. Two assays did not show cross-reactivity with L. innocua and eight other foodborne bacteria. The limits of detection were 10 CFU/mL for pure culture and 20 CFU/g for spiked pork samples. Moreover, the enrichment time was substantially shortened to 3 h for pork samples spiked with only L. monocytogenes F2365, and 4-5 h for pork samples spiked with mixed bacteria. In addition, with one-pot LAMP-CRISPR/Cas12b detection, 5 of 66 fresh pork samples, 1 of 20 ready-to-eat food samples, and 2 of 24 raw milk samples tested positive for L. monocytogenes, in agreement with the results obtained through a culture based standard method. Thus, this study established one-pot LAMP-CRISPR/Cas12b and LAMP-CRISPR/Cas12b-LFA assays for rapid, visual detection of L. monocytogenes in food samples.},
}
@article {pmid41341583,
year = {2025},
author = {Jiang, Z and Jia, B and Hu, N and Zhang, M and Xiao, H and Chen, G and Yu, J and Li, X and Shen, B and Feng, J and Wang, J},
title = {In Vivo engineering of transgenic mice for systemic human neutralizing antibody production against staphylococcal enterotoxin B.},
journal = {Frontiers in immunology},
volume = {16},
number = {},
pages = {1679421},
pmid = {41341583},
issn = {1664-3224},
mesh = {Animals ; Mice, Transgenic ; Humans ; Mice ; *Enterotoxins/immunology ; *Antibodies, Neutralizing/immunology/genetics/biosynthesis ; Female ; CRISPR-Cas Systems ; Genetic Engineering ; Antibodies, Monoclonal/immunology/genetics ; Glycosylation ; },
abstract = {Transgenic animal bioreactors provide a complementary strategy to traditional mammalian cell culture systems for the production of therapeutic human monoclonal antibodies (mAbs). Here we present a CRISPR/Cas9-mediated breakthrough in creating two novel genetically engineered (GE) mouse models with species-specific chromosomal integration of human anti-staphylococcal enterotoxin B (SEB) mAb genes at either the ROSA26 or Hipp11 (H11) safe-harbor loci - evolutionarily conserved genomic safe harbors (GSH). These genetically optimized animals demonstrated broad tissue capability for glycosylation-competent human antibodies, achieving exceptional secretion levels reaching 208 mg/L in serum, 43 mg/L in mammary secretions, 24 mg/L in saliva on average. The transgenic lines maintained this antibody production stability for >140 weeks without compromising animal viability, while preserving germline transmission fidelity through six successive generations. Furthermore, the highly glycosylated human antibodies derived from these genetic engineered mice exhibited high binding affinity to SEB (KD=0.108 nM for ROSA26; 0.154 nM for H11), providing comprehensive protection against SEB intoxication in vivo. This study opens avenues for utilizing transgenic animal bioreactors for large-scale production of fully human antibodies or disease-resistant livestock in the foreseeable future.},
}
@article {pmid41330077,
year = {2026},
author = {Fathpour, H and Fouladi, M and Jafarpour, F and Moradi-Hajidavaloo, R and Izadi, T and Shiralian-Esfahani, H and Kues, W and Nasr-Esfahani, MH and Hajian, M and Eghbalsaied, S},
title = {Crosstalk between myostatin and callipyge in CRISPR/Cas9-edited goat fibroblast cells.},
journal = {Research in veterinary science},
volume = {198},
number = {},
pages = {105992},
doi = {10.1016/j.rvsc.2025.105992},
pmid = {41330077},
issn = {1532-2661},
mesh = {Animals ; *Myostatin/genetics/metabolism ; *Goats/genetics ; *Fibroblasts/metabolism ; *CRISPR-Cas Systems ; Gene Editing/veterinary ; },
abstract = {Myostatin (MSTN) and Callipyge (CLPG) genes are key regulators of muscle growth. While MSTN inhibits muscle development, the CLPG mutation induces muscle hypertrophy through a specific imprinted genetic mechanism. The interaction between these genes remains of interest for improving livestock muscle traits. In this study, CRISPR/Cas9 was employed to edit MSTN and CLPG genes in goat fibroblast cells via electrotransfection. Cells were selected using puromycin antibiotic, and gene-editing efficiency was evaluated through Sanger sequencing. Gene expression changes were analyzed using RT-qPCR analysis. MSTN gene knockout resulted in significant downregulation of MSTN and CLPG, while GTL2 expression was upregulated by more than 50-fold. Additionally, myosin heavy chain genes (MYH1, MYH3, MYH4) were strongly upregulated, with MYH3 13-fold and MYH4 30-fold increase in the expression. In CLPG-edited cells, the expression of MSTN, TRIM28, and CLPG was reduced, while GTL2 was upregulated by 6-fold. MYH3 and MYH4 expression increased 4-fold in CLPG-edited cells, though the increase was less pronounced compared to MSTN-edited cells. DLK1 expression was undetectable in both non-edited control and gene-edited fibroblast cells. Our findings support the interaction between MSTN and CLPG, contributing to the regulation of muscle growth. Notably, the study also highlights the challenges associated with editing imprinted genes like CLPG and suggests that TRIM28 may play a role downstream of CLPG regulation. These results provide valuable insights into muscle development regulation, offering potential applications in livestock genetic improvement.},
}
@article {pmid41285661,
year = {2026},
author = {Gu, X and Zhang, T and Yao, H and Guo, F and Yang, C and Xu, H and He, X and Ma, Z and Zhang, X and Yu, S and An, R and Wang, F},
title = {CRISPR-Cas12a-integrated pregnancy test strip biosensors: Visual detection of telomerase and miRNA let-7a in cervical cancer diagnostics.},
journal = {Biosensors &amp; bioelectronics},
volume = {294},
number = {},
pages = {118241},
doi = {10.1016/j.bios.2025.118241},
pmid = {41285661},
issn = {1873-4235},
mesh = {Humans ; Female ; *MicroRNAs/genetics/isolation &amp; purification/analysis ; *Uterine Cervical Neoplasms/diagnosis/genetics ; *Biosensing Techniques ; *Telomerase/isolation &amp; purification/genetics/analysis ; CRISPR-Cas Systems/genetics ; HeLa Cells ; Pregnancy ; Limit of Detection ; Reagent Strips/analysis ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {Cervical cancer is a leading cause of female cancer-related mortality globally, and early screening based on reliable biomarkers is critical for improving prognosis. Telomerase (a key driver of cellular immortalization) and microRNA let-7a (a tumor suppressor with downregulated expression in cervical cancer) are well-validated diagnostic targets, but existing detection methods are hindered by complex procedures, high instrumentation costs, and reliance on specialized technical expertise-limiting their accessibility in resource-constrained settings. To address these limitations, we developed two novel CRISPR-Cas12a-integrated biosensors using commercially available pregnancy test strips (PTS) for instrument-free, visual readout. Both biosensors leverage a core signal mediator, probe 1 ("MB-ssDNA1-hCG"), which links CRISPR-Cas12a activation to visible color development on the PTS. The first Biosensor CRISPR-PTS-Telo detects telomerase activity in one-step without PCR: telomerase-generated (TTAGGG)n repeats activate Cas12a-crRNA1 complex, cleaving the probe 1 to release hCG, achieving a detection limit of 18 HeLa cells-comparable to sensitive laboratory assays. The second Biosensor CRISPR-PTS-let7a detects miRNA let-7a by first converting miRNA signals to Trigger DNA via Assister DNA and probe 2 ("MB-ssDNA2+Trigger"), activating Cas12a-crRNA2 complex, cleaving the probe 1 and inducing PTS coloration. This achieves a detection limit of 25.1 fM for let-7a. Validation with clinical samples (24 cervical tissues and 26 blood samples) confirmed their concordance with gold-standard methods (ELISA for telomerase, RT-qPCR for let-7a). These versatile tools hold significant potential as point-of-care testing (POCT) solutions to facilitate early, accessible cervical cancer screening.},
}
@article {pmid41093250,
year = {2025},
author = {Buck-Wiese, M and Liechocki, S and Erfle, H and Starkuviene, V},
title = {Comparative analysis of antibody-mediated loss-of-function versus gene knock-out and knock-down.},
journal = {SLAS discovery : advancing life sciences R &amp; D},
volume = {37},
number = {},
pages = {100283},
doi = {10.1016/j.slasd.2025.100283},
pmid = {41093250},
issn = {2472-5560},
mesh = {Humans ; CRISPR-Cas Systems/genetics ; *Gene Knockout Techniques/methods ; *Gene Knockdown Techniques/methods ; *Antibodies ; Talin/genetics/metabolism ; RNA Interference ; RNA, Small Interfering/genetics ; Gene Expression Profiling ; Cell Adhesion/genetics ; },
abstract = {In this study we compare three methods for manipulating cell function: RNA interference (RNAi), CRISPR-Cas9 gene knock-out, and antibody-mediated loss-of-function. We have focused on analyzing changes in cell-matrix adhesion via targeting two key regulators, Talin1 (TLN1) and Kindlin-2 (KD2). Adhesion-relevant phenotypic assays revealed distinct temporal onset dynamics for each method. RNAi and CRISPR-Cas9 effectively reduced target mRNA and protein levels. In contrast, antibody transfection induced phenotypic changes without altering target expression, suggesting direct intracellular antibody-target interaction. Transcriptome analysis demonstrated that antibody transfection and CRISPR-Cas9 induced fewer deregulated mRNAs than RNAi. Furthermore, transfected antibodies and sgRNAs shared 30 % and 70 % of deregulated transcripts to their negative controls, respectively. Whereas only 10 % of overlap was recorded between targeting and control siRNAs. Our findings emphasize the importance of considering method-specific temporal dynamics of on-target phenotype appearance and off-target manifestation. Additionally, they highlight intracellular delivered antibodies as a valuable alternative for validating and complementing genetic approaches.},
}
@article {pmid40392482,
year = {2025},
author = {Huang, S and Wu, J and Yang, Y and Zhu, M and Chen, L and Zhang, S and Yang, Y and Sun, X and Xie, Y},
title = {Investigate the Effect of ZFP64 on mRNA Expression of HBG Based on Bioinformatics and Experimental Validation.},
journal = {Cell biochemistry and biophysics},
volume = {83},
number = {4},
pages = {4427-4437},
pmid = {40392482},
issn = {1559-0283},
support = {2020A0505100062, 2023A1515010872, 2024A1515012233//the Natural Science Foundation of Guangdong Province/ ; 32070582//National Natural Science Foundation of China/ ; 2023HLLH01//the Joint Foundation of He Lin Academical Workstation of the Third Affiliated Hospital of Guangzhou Medical University/ ; 2023A03J0386, 2023A03J0395//Guangdong Municipal Department of Science and Technology, Municipal Schools (Institutes) Jointly Funded Project/ ; .02-408-2203-2059//Guangzhou Medical University for the First-class Professional Construction Project in 2022-Enhancement of Undergraduates' Scientific Research and Innovation Ability Project/ ; 2024SRP119//plan on enhancing scientific research in GMU/ ; },
mesh = {Humans ; *RNA, Messenger/metabolism/genetics ; *Transcription Factors/genetics/metabolism ; *Computational Biology ; K562 Cells ; *gamma-Globins/genetics/metabolism ; Protein Interaction Maps ; *DNA-Binding Proteins/genetics/metabolism ; *Gene Expression Regulation ; CRISPR-Cas Systems ; },
abstract = {γ-globin genes (HBG1 and HBG2) are usually expressed during fetal life, and almost no expression after birth. Therefore, the reactivation of HBG is a key target for the treatment of hemoglobinopathy. ZFP64 is a C2H2 type zinc finger transcription factor, which has been shown to play an important role in the maintenance of gene expression in mixed lineage leukemia, and other C2H2 type zinc finger transcription factors (such as ZFP410 and ZFP644) have been shown to regulate the expression of fetal hemoglobin (HbF) in thalassemia. This study aims to investigate the effect of ZFP64 on mRNA expression of HBG. We performed bioinformatics analyses using the Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), and protein-protein interaction (PPI) networks to identify genes and transcription factors associated with ZFP64. ZFP64 was knocked out in K562 and HUDEP-2 cell lines by CRISPR-Cas9 electroporation, and the transcription levels of ZFP64, HBB and HBG were analyzed. In undifferentiated and 7-day differentiated HUDEP-2 cells, knocking down ZFP64 resulted in a 1.5-fold and 2.5-fold increase in HBG mRNA expression, respectively (p < 0.05). These findings suggest that ZFP64 is a potential regulator of HBG expression and warrants further investigation as a therapeutic target in hemoglobinopathies.},
}
@article {pmid41341502,
year = {2025},
author = {Gao, Y and Chen, J},
title = {Fast but accurate: a systematic review and meta-analysis on diagnostic performance of MRSA detection in clinical samples by using CRISPR-based rapid molecular methods.},
journal = {Frontiers in microbiology},
volume = {16},
number = {},
pages = {1703247},
pmid = {41341502},
issn = {1664-302X},
abstract = {BACKGROUND: Methicillin-resistant Staphylococcus aureus (MRSA) poses a significant global health threat due to its multidrug resistance and association with severe infections. Conventional culture methods are time-consuming, usually requiring 48-72 h to obtain results, while conventional molecular methods such as PCR or qPCR, though faster, still require trained personnel and specialized instruments, which may delay timely clinical treatment and infection control. CRISPR-based methods have emerged as promising alternative tools for MRSA detection, but their real-world performance still requires comprehensive assessment. This meta-analysis aimed to systematically evaluate the diagnostic accuracy and timeliness of CRISPR/Cas systems for MRSA detection in clinical samples.<br><br>METHODS: A systematic search of PubMed, Embase, Web of Science, and Cochrane Library was conducted using search terms related to MRSA, CRISPR/Cas, diagnostic accuracy, and rapid detection. Studies reporting sensitivity and specificity with extractable 2&#x202f;&#xd7;&#x202f;2 contingency tables were included. Quality was assessed via QUADAS-2. Meta-disc 1.4.0 and Stata 16.0 were used for statistical analysis, including pooled sensitivity, specificity, likelihood ratios, diagnostic odds ratios (DOR) and summary receiver operating characteristic (SROC). Median detection time and subgroup analyses were also conducted.<br><br>RESULTS: Twelve studies were included. The results showed that the CRISPR-based methods showed a pooled sensitivity of 99% (95% CI: 97-100%) and specificity of 100% (95% CI: 99-100%), with a PLR of 32.68 (95% CI: 15.45-69.15), NLR of 0.03 (95% CI: 0.02-0.07), and DOR of 664.25 (95% CI: 234.59-1880.84). The median detection time across included studies was 60&#x202f;min (IQR: 41.25-98.75&#x202f;min).<br><br>CONCLUSION: CRISPR-based molecular assays demonstrated exceptional accuracy and rapid detection capability for MRSA in clinical settings, significantly outperforming conventional methods. However, potential publication bias and methodological limitations warrant cautious interpretation of these results.<br><br>PROSPERO ID: CRD420251115439.},
}
@article {pmid41340056,
year = {2025},
author = {Braun, S and Knackfuß, K and Ziesmann, T and Mlinzk, L and Goerg, A and Frankenheim, J and Walter, A and Schneider-Brachert, W and Distler, U and Fritsch, J},
title = {Loss of ADAM15 prevents necroptosis induction by partial RIPK1 degradation due to enhanced TNF-R1 surface expression and basal caspase-8 activation.},
journal = {Cell communication and signaling : CCS},
volume = {23},
number = {1},
pages = {520},
pmid = {41340056},
issn = {1478-811X},
mesh = {Humans ; *Caspase 8/metabolism ; *ADAM Proteins/metabolism/genetics/deficiency ; *Receptor-Interacting Protein Serine-Threonine Kinases/metabolism ; *Proteolysis ; *Membrane Proteins/metabolism/genetics/deficiency ; *Necroptosis ; *Receptors, Tumor Necrosis Factor, Type I/metabolism ; Enzyme Activation ; Jurkat Cells ; U937 Cells ; Signal Transduction ; },
abstract = {BACKGROUND: Cell death and survival processes must be tightly regulated to ensure proper tissue homeostasis and prevent excessive inflammation and tissue damage. Death receptors, including TNF-R1, can induce either immunogenic (necroptosis) or non-immunogenic (apoptosis) cell death and relay proliferative / cell survival signaling by activating NFκB and MAPK cascades. In a recent report, we identified the metalloproteinase ADAM15 as a possible TNF-responding enzyme, leading to the hypothesis that it regulates either cell survival or death cascades.<br><br>METHODS: CRISPR/Cas-9 was used to knock out the adam15 gene. Loss of gene expression was validated by Western blot and flow cytometry in U937 and Jurkat cells. NF&#x3ba;B, MAPK signaling, and cell death cascades were monitored by Western blot, flow cytometry, and enzyme assays. A bottom-up proteome analysis was performed to elucidate cellular processes affected by ADAM15 loss. The subcellular localization of ADAM15 was monitored by microscopy and immuno-magnetic fractionation.<br><br>RESULTS: We identified ADAM15 as a regulator of necroptosis, leaving apoptosis and cell survival signaling unaffected. Loss of ADAM15 resulted in abrogated necroptosis, as evidenced by the application of death ligands TNF, TRAIL, FasL, and TL1a, as well as the BH3 mimetic Obatoclax. We observed enhanced basal Caspase-8 activity, which was not cytotoxic, and partial RIPK1 proteolysis. The loss of ADAM15 was verified in a proteome screen, which revealed alterations in various molecular pathways, including autophagy, organelle trafficking, and sorting. We observed ADAM15 in intracellular compartments, which in part have a lysosomal protein signature. We observed enhanced surface expression of TNF-R1, proposing it as a possible ADAM15 substrate.<br><br>CONCLUSIONS: ADAM15 is a previously unknown regulator of necroptosis, likely due to its role in modulating intracellular organelle sorting processes. Its proteolytic activity and possible scaffolding capacity for recruiting adaptor molecules make it a veritable drug target. The activation or deactivation of ADAM15 may be exploited to modulate various disease conditions.},
}
@article {pmid41339642,
year = {2025},
author = {Yang, WJ and Liu, BY and Xue, L},
title = {Knockout of protein arginine methyltransferase 1 inhibited cell growth and promoted cell migration in human bronchial epithelial cells.},
journal = {Scientific reports},
volume = {15},
number = {1},
pages = {43069},
pmid = {41339642},
issn = {2045-2322},
support = {2018BFC360//Fund for Key Laboratory Construction of Hubei Province/ ; 31101047//National Natural Science Foundation of China/ ; CZQ22013//"the Fundamental Research Funds for the Central Universities", South-Central MinZu University/ ; PTZ24018//i Medical Biology International Science and Technology Cooperation Base/ ; },
mesh = {Humans ; *Cell Movement/genetics ; *Cell Proliferation/genetics ; *Protein-Arginine N-Methyltransferases/genetics/metabolism ; *Epithelial Cells/metabolism/cytology ; *Bronchi/cytology/metabolism ; Cell Line ; Apoptosis/genetics ; Gene Knockout Techniques ; *Repressor Proteins/genetics/metabolism ; Cell Cycle/genetics ; CRISPR-Cas Systems ; },
abstract = {Previous studies have demonstrated that PRMT1 was involved in the progression of multiple lung diseases. However, its specific function within the bronchial epithelium was still limited and needed further exploration. In the present study, human bronchial epithelial cell line 16HBE was chosen to elucidate the biological role of PRMT1 in lung epithelium. Cell proliferation, cell-cycle distribution, cell apoptosis, and cell motility capacity were systematically evaluated following CRISPR/Cas9-mediated knockout of PRMT1. We showed that knockout of PRMT1 in 16HBE inhibited cell proliferation, redistributed cell cycle, promoted cell apoptosis, and accelerated cell migration via a series of regulated cyclins, cyclin-dependent kinase regulators, and EMT markers. Taken together, these findings identify PRMT1 as a potential modulator of epithelial cell proliferation, survival, and motility in the human bronchial epithelium, offering new insights into its possible role in epithelial remodeling during pulmonary disorders.},
}
@article {pmid41337296,
year = {2025},
author = {Cimolato, C and Letrari, S and Chiacchiera, AF and Del Favero, S and Schenato, L and Pasotti, L and Bellato, M},
title = {Modeling of Phage-Mediated CRISPRi System to Inhibit Antibiotic Resistances in Bacteria.},
journal = {Annual International Conference of the IEEE Engineering in Medicine and Biology Society. IEEE Engineering in Medicine and Biology Society. Annual International Conference},
volume = {2025},
number = {},
pages = {1-7},
doi = {10.1109/EMBC58623.2025.11253443},
pmid = {41337296},
issn = {2694-0604},
mesh = {*Bacteriophages/genetics ; *CRISPR-Cas Systems/genetics ; *Bacteria/genetics/drug effects/virology ; *Drug Resistance, Bacterial/genetics ; Anti-Bacterial Agents/pharmacology ; Humans ; },
abstract = {Antimicrobial resistance (AMR) poses a critical threat to global health, rendering traditional antibiotics increasingly ineffective and amplifying the urgency for innovative solutions. Among promising alternatives, synthetic biology emerges as a powerful tool to combat AMR. This work proposes an innovative strategy based on engineering bacteriophages to deliver CRISPR interference (CRISPRi) systems into antibiotic-resistant pathogens to precisely silence target resistance genes. A comprehensive mathematical model is developed and simulated to capture the dynamics of phage-mediated CRISPRi delivery. By explicitly incorporating mutations that affect CRISPRi functionality, the study evaluates system performance and its potential for long-term therapeutic efficacy. This model serves as a critical framework for optimizing future CRISPRi-based interventions and advancing synthetic biology-driven approaches to tackle AMR.Clinical relevance- This paper provides a quantitative modeling framework to evaluate key parameters affecting engineered phage therapy efficiency, supporting rational design and phage posology optimization.},
}
@article {pmid41336948,
year = {2025},
author = {Yu, ES and Jang, H and Kwon, J and Jeong, H and Park, J and Kang, T and Jeong, KH},
title = {On-chip Nanoplasmonic RT-RPA and CRISPR/Cas12a Assay for Point-of-care Molecular Diagnostics.},
journal = {Annual International Conference of the IEEE Engineering in Medicine and Biology Society. IEEE Engineering in Medicine and Biology Society. Annual International Conference},
volume = {2025},
number = {},
pages = {1-4},
doi = {10.1109/EMBC58623.2025.11253682},
pmid = {41336948},
issn = {2694-0604},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; SARS-CoV-2/genetics ; *COVID-19/diagnosis ; *Point-of-Care Systems ; *Nucleic Acid Amplification Techniques/methods/instrumentation ; *Lab-On-A-Chip Devices ; *Molecular Diagnostic Techniques ; *Pathology, Molecular/methods ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {Rapid and accurate nucleic acid detection at point-of-care (POC) is essential for advancing effective disease diagnosis and management. Here, we report a handheld nanoplasmonic all-in-one setup for on-chip recombinase polymerase amplification (RPA) and real-time fluorescence detection by CRISPR/Cas12a reaction. The all-in-one setup consists of AuNIs-based nanoplasmonic cavity (AuNIs-NC), a disposable plastic-on-polymer (PoP) cartridge, and fluorescence microlens array (FMLA) camera. The AuNIs-NC allows uniform and efficient photothermal heating under white LED illumination due to strong broadband light absorption and internal reflection by randomly distributed AuNIs and thin Al film. This setup allows the RPA and CRISPR/Cas 12a reactions in a single chamber of PoP cartridge, with fluorescence signals monitored by a FMLA camera. The experimental result demonstrates rapid SARS-CoV-2 E gene plasmid DNA detection within 20 min, achieving a detection sensitivity of 10 copies/ul. Testing with 16 clinical samples shows a linear trend with RT-qPCR, indicating the platform's reliable sensitivity and specificity. This compact platform offers affordable and reliable molecular diagnosis, facilitating rapid and scalable POC testing for a range of infectious diseases.Clinical Relevance- This on-chip real-time RT-RPA and CRISPR/Cas12a assay provides rapid and precise molecular diagnostics at POC using fully integrated plasmonic system.},
}
@article {pmid41332531,
year = {2025},
author = {Zhang, P and Xue, B and Xie, Y and Li, K and Yang, H and Sun, P and Zhang, L},
title = {OSM-11 modulates salinity-stress tolerance in Caenorhabditis elegans.},
journal = {bioRxiv : the preprint server for biology},
volume = {},
number = {},
pages = {},
doi = {10.1101/2025.11.20.689412},
pmid = {41332531},
issn = {2692-8205},
abstract = {Most terrestrial animals exhibit narrow salinity tolerance compared to their marine counterparts. Previous studies identified osm-11 (which encodes a Notch co-ligand) mutations as a driver of hyper-saline tolerance in Caenorhabditis elegans , but mechanistic insights remained unclear. This study employs RNA sequencing and CRISPR/Cas-9 genome editing to demonstrate that osm-11 mutations enhance salinity stress resistance through up-regulation of fatty acid metabolism (acdh-12 , acs-17) and cytochrome P450 pathways (ugt-15), while suppressing calcium signaling. Furthermore, we demonstrated that acdh-12 mutation impairs salinity-stress tolerance by activating ferroptosis and mitophagy, accompanied by down-regulated oxidative phosphorylation and up-regulated autophagic pathways. Morphological observations show that mitochondrial fragmentation contributes to wild-type nematode mortality under high salinity, while enlarged lipid droplets in wild-types correlate with reduced β-oxidation gene expression (dhs-28 , daf-22), whose knockout disrupts tolerance in mutants. These findings unravel the multi-pathway regulatory network of osm-11 -mediated salinity tolerance, providing mechanistic insights for developing protective strategies against environmental salinity stressors impacting animal survival.},
}
@article {pmid41331925,
year = {2025},
author = {Matsumoto, D and Kubota, K and Sato, Y and Kato-Inui, T and Nigorikawa, K and Miyaoka, Y and Nomura, W},
title = {Screening strategy to identify Cas9 variants with higher HDR activity based on diphtheria toxin.},
journal = {Journal of biomedical science},
volume = {32},
number = {1},
pages = {102},
pmid = {41331925},
issn = {1423-0127},
support = {JP23K13844//KAKENHI/ ; JP24K09445//KAKENHI/ ; JP20H03442//KAKENHI/ ; JP20K21253//KAKENHI/ ; JP22H02201//KAKENHI/ ; JP23K23468//KAKENHI/ ; },
mesh = {Humans ; *Diphtheria Toxin/genetics ; *CRISPR-Associated Protein 9/genetics ; *Gene Editing/methods ; *Recombinational DNA Repair/genetics ; *CRISPR-Cas Systems ; HEK293 Cells ; Mutation ; },
abstract = {BACKGROUND: In gene therapy via genome editing, it is essential to precisely repair disease-associated gene sequences without introducing random mutations. However, achieving highly accurate genome editing remains challenging owing to the low efficiency of homology-directed repair (HDR)-mediated gene repair, which relies on template DNA. Therefore, if Cas9 mutants capable of enhancing HDR can be identified, they could enable more precise gene therapies.<br><br>METHOD: In this research project, we developed a screening system that uses the acquisition of diphtheria toxin resistance as an indicator of HDR efficiency in human cells and EGFP disruption as an indicator of off-target effect.<br><br>RESULTS: By screening a library of SpCas9 variants with random mutations introduced into its nuclease domain, we identified a novel SpCas9 mutant with higher HDR efficiency than wild-type Cas9.<br><br>CONCLUSION: We explored the possibility of obtaining Cas9 mutants with high HDR efficiency via this screening system.},
}
@article {pmid41331675,
year = {2025},
author = {Wang, M and Zhang, Y and Bi, C and Li, M},
title = {CRISPR-Cas9-induced double-strand breaks disrupt maintenance of epigenetic information.},
journal = {Genome biology},
volume = {26},
number = {1},
pages = {411},
pmid = {41331675},
issn = {1474-760X},
support = {BAS/1/1080-01-01//KAUST Office of Sponsored Research/ ; 5932//KAUST Center of Excellence for Smart Health/ ; },
mesh = {Humans ; *DNA Breaks, Double-Stranded ; *CRISPR-Cas Systems ; DNA Methylation ; *Epigenesis, Genetic ; Gene Editing ; MutL Protein Homolog 1/genetics ; Human Embryonic Stem Cells/metabolism ; Genomic Imprinting ; DNA Repair ; },
abstract = {BACKGROUND: CRISPR-Cas9 genome editing enables precise genetic modifications by introducing targeted DNA double-strand breaks (DSBs). While Cas9-induced DSBs are known to cause unintended on-target mutations, their impact on the epigenetic landscape remains unexplored.<br><br>RESULTS: Here, we investigate how Cas9-induced DSBs affect DNA methylation patterns in human embryonic stem cells (hESCs). We induce DSBs at differentially methylated regions of imprinted genomic loci and perform high-coverage, long-read native DNA sequencing to simultaneously obtain genetic variant and base-resolution methylation data in a haplotype-resolved manner. Our findings reveal that DSBs cause significant changes in DNA methylation at target sites through mechanisms including homologous recombination, large structural variations, or defective methylation maintenance during DNA repair. Notably, these epigenetic changes can occur either together with or independently of genetic alterations. Beyond imprinted loci, Cas9-induced DSBs significantly disrupt DNA methylation patterns of the MLH1 epimutation alleles in colorectal cancer cells, and hypermethylated heterochromatin loci in hESCs. Clonal analysis indicates that the aberrant methylation changes are stable during in vitro passaging. Intriguingly, significant changes in DNA methylation levels are also detected around endogenous deletions in unedited genomic regions, suggesting that methylation alterations are not unique to Cas9 nuclease activity but represent a general outcome of DSB repair in human cells.<br><br>CONCLUSIONS: This study underscores the importance of assessing and mitigating unintended epigenetic consequences in genome editing applications, as such changes can profoundly affect gene regulation and cellular function.},
}
@article {pmid41277755,
year = {2025},
author = {Wang, Z and Zhang, D and Wu, Y and Ye, X and Qian, Y and Chen, F},
title = {A capillary SERS sensor based on CRISPR/Cas13a and DS Au-AgNRs for detecting miRNA-221 in serum of hepatocellular carcinoma patients.},
journal = {Analytical methods : advancing methods and applications},
volume = {17},
number = {47},
pages = {9627-9637},
doi = {10.1039/d5ay01517k},
pmid = {41277755},
issn = {1759-9679},
mesh = {*MicroRNAs/blood/genetics ; Humans ; *Carcinoma, Hepatocellular/blood/diagnosis/genetics ; *Liver Neoplasms/blood/diagnosis/genetics ; *Spectrum Analysis, Raman/methods ; Gold/chemistry ; Silver/chemistry ; CRISPR-Cas Systems ; Nanotubes/chemistry ; Biomarkers, Tumor/blood ; Limit of Detection ; },
abstract = {The low early diagnosis rate of hepatocellular carcinoma (HCC) severely impacts patient prognosis, making the development of highly sensitive and specific early diagnostic technologies crucial. MicroRNA-221 (miR-221), an aberrantly overexpressed biomarker in HCC, holds significant diagnostic potential. This paper constructed a capillary surface-enhanced Raman scattering (SERS) sensing platform utilizing CRISPR/Cas13a trans-cleavage and double-shell gold-silver nanorods (DS Au-AgNRs) to detect serum miR-221 in HCC patients. DS Au-AgNRs were synthesized and assembled onto aminated capillaries, followed by conjugation of Cy5-labeled single-stranded DNA (ssDNA) to the DS Au-AgNR surface via Au-S bonds. In the presence of miR-221, activated CRISPR/Cas13a trans-cleavage cleaves the ssDNA, releasing Cy5 from the sensor surface and diminishing the SERS signal, enabling miR-221 quantification. The synthesized DS Au-AgNRs exhibit uniform morphology and size, are uniformly distributed on the capillary, and form numerous "hotspots", thereby significantly enhancing the SERS signal. According to the characteristic peak of Cy5 at 1074 cm[-1], a linear relationship is established between the log concentration of miR-221 and the measured SERS intensity (y = -3527.97 × -35369.60, R[2] = 0.97767), with a LOD as low as 4.17 × 10[-17] M. The sensor demonstrated high specificity and high sensitivity, and its capacity to detect miR-221 expression aligned with qRT-PCR results when analyzing serum samples, confirming that hepatocellular carcinoma patients exhibited significantly higher miR-221 levels compared to healthy individuals. The capillary SERS sensor thus provides an accurate and convenient approach for early HCC diagnosis.},
}
@article {pmid41252908,
year = {2026},
author = {Zhou, F and Zhao, X and Wang, Y and Huang, Y},
title = {Cooperation of CRISPR/Cas12a and exonuclease III-assisted cascade cycling amplification for ultrasensitive electrochemical detection of ciprofloxacin.},
journal = {Talanta},
volume = {299},
number = {},
pages = {129131},
doi = {10.1016/j.talanta.2025.129131},
pmid = {41252908},
issn = {1873-3573},
mesh = {*Ciprofloxacin/analysis ; *Exodeoxyribonucleases/metabolism/chemistry ; *Biosensing Techniques/methods ; *Electrochemical Techniques/methods ; *CRISPR-Cas Systems ; Limit of Detection ; *Nucleic Acid Amplification Techniques ; *Anti-Bacterial Agents/analysis ; Food Contamination/analysis ; *CRISPR-Associated Proteins/metabolism ; DNA Probes/chemistry/genetics ; Bacterial Proteins ; Endodeoxyribonucleases ; },
abstract = {Antibiotic residues have been a serious public health concern worldwide, while sensitive and reliable detection of antibiotic residues is significant to control antibiotic contamination, ensure food safety, and safeguard human health. Herein, an ultrasensitive electrochemical biosensor is engineered for the detection of ciprofloxacin (CIP) based on the cooperation of CRISPR/Cas12a and exonuclease III (Exo III)-assisted cascade cycling amplification. The presence of CIP induces the conformational change of DNA probes and further triggers Exo III to catalyze the cascade cycling amplification, enabling propagation and ongoing accumulation of DNA fragments which act as the target strands to activate the trans-cleavage activity of CRISPR/Cas12a. Consequently, the activated CRISPR/Cas12a initiates its trans-cleavage activity to swiftly cleave the signal probes on the surface of electrode, bringing about remarkable change of electrochemical signal and eventually realizing the ultrasensitive detection of CIP. The exceptional enzymatic cycle amplification of Exo III incorporated with the superior trans-cleavage activity of CRISPR/Cas12a synergistically facilitates considerable improvement of analytical performance, resulting in a limit of detection as low as 0.022 ng mL[-1]. Benefiting from the effective amplification capacity, high fidelity and programmability of the designed detection system, the biosensor shows good precision and specificity along with robust stability for CIP detection. Moreover, the proposed electrochemical biosensor dispensing with complicated probe construction is label-free and convenient-operated, which contributes to the credible application for CIP detection in real food samples with satisfactory results, indicating promising practicability in food safety monitoring.},
}
@article {pmid41223903,
year = {2025},
author = {Alsultan, A and Karim, SM and Al-Saadi, M and Alsallami, D and Ben Said, M and Belkahia, H},
title = {Rapid and sensitive detection of Theileria equi using a novel integrated RPACRISPR/Cas13a lateral flow assay.},
journal = {Journal of equine veterinary science},
volume = {155},
number = {},
pages = {105732},
doi = {10.1016/j.jevs.2025.105732},
pmid = {41223903},
issn = {0737-0806},
mesh = {Horses ; Animals ; *Theileria/isolation &amp; purification ; *Theileriasis/diagnosis/parasitology ; *Horse Diseases/diagnosis/parasitology ; Sensitivity and Specificity ; *Nucleic Acid Amplification Techniques/veterinary/methods ; CRISPR-Cas Systems ; },
abstract = {BACKGROUND: Equine piroplasmosis (EP), caused by the intracellular protozoa Theileria equi, Babesia caballi, and Theileria haneyi, represents a major health and economic threat to the equine industry worldwide. Existing diagnostic methods, including PCR, serology, and microscopy, are constrained by their dependence on specialized equipment, lengthy protocols, and the requirement for skilled personnel.<br><br>AIM: This study aimed to develop a rapid, accurate, and field-deployable molecular diagnostic assay for T. equi.<br><br>METHODS: A nucleic acid-based diagnostic platform combining recombinase polymerase amplification (RPA) with CRISPR/Cas13-mediated detection and lateral flow device (LFD) readout was developed. The assay targets a conserved region of the erythrocyte merozoite antigen 1 (EMA-1) gene of T. equi. Validation was performed using 22 blood samples collected from horses, as well as specificity controls including B. caballi- and Anaplasma phagocytophilum-infected samples, synthetic EMA-1 DNA, and non-template controls. All assay steps were conducted at room temperature.<br><br>RESULTS: The integrated RPA-CRISPR/Cas13-LFD assay generated clear visual results within 50 minutes. It demonstrated complete specificity with no false positives across all tested samples. The method effectively differentiated horses infected with T. equi, including both clinically affected and asymptomatic individuals, from healthy, uninfected animals, confirming its high accuracy and reliability.<br><br>CONCLUSION: The developed assay provides a rapid, precise, and equipment-free diagnostic platform suitable for both field and clinical environments. Although the current protocol relies on DNA extraction, future optimization will aim to enable direct detection from unprocessed blood samples, thereby further simplifying point-of-care diagnostics for equine piroplasmosis.},
}
@article {pmid41221894,
year = {2025},
author = {Wei, R and Wang, S and Pan, Y and Pan, W and Li, N and Tang, B},
title = {CRISPR-coupled triple cascade amplification for simultaneous lateral flow detection of Mycoplasma pneumoniae and H1N1.},
journal = {Chemical communications (Cambridge, England)},
volume = {61},
number = {97},
pages = {19241-19244},
doi = {10.1039/d5cc04993h},
pmid = {41221894},
issn = {1364-548X},
mesh = {*Mycoplasma pneumoniae/isolation &amp; purification/genetics ; *Influenza A Virus, H1N1 Subtype/isolation &amp; purification/genetics ; Humans ; *Nucleic Acid Amplification Techniques/methods ; *CRISPR-Cas Systems ; Saliva/microbiology/virology ; Limit of Detection ; },
abstract = {We developed a CRISPR-coupled triple cascade system integrating recombinase polymerase amplification (RPA), CRISPR/Cas12a, and catalytic hairpin assembly (CHA) for simultaneous lateral flow detection of Mycoplasma pneumoniae and H1N1 in saliva samples, achieving a LOD of 10 aM for H1N1 RNA and 25 aM for MP DNA on a single LFA.},
}
@article {pmid41202787,
year = {2026},
author = {Xiao, X and Zhong, Y and Xie, H and Liao, H and Wang, H and Liu, H and Liang, F and Chen, J and Zhong, H and Chen, Z and Yu, L},
title = {A reverse transcription-free CRISPR/Cas12a biosensor for ultrasensitive detection of SARS-CoV-2 variants.},
journal = {Talanta},
volume = {299},
number = {},
pages = {129072},
doi = {10.1016/j.talanta.2025.129072},
pmid = {41202787},
issn = {1873-3573},
mesh = {*SARS-CoV-2/genetics/isolation &amp; purification ; *Biosensing Techniques/methods ; *CRISPR-Cas Systems/genetics ; Humans ; *COVID-19/diagnosis/virology ; Nucleic Acid Amplification Techniques/methods ; Limit of Detection ; Mutation ; RNA, Viral/genetics ; Reverse Transcription ; CRISPR-Associated Proteins/genetics ; Bacterial Proteins ; Endodeoxyribonucleases ; },
abstract = {In this work, we developed a reverse transcription-free self-primer isothermal exponential amplification reaction (RTF-SP-EXPAR) combined with a CRISPR/Cas12a biosensor for the simultaneous detection of single- and double-stranded amplification products of SARS-CoV-2 mutant variants. SP-EXPAR can recognize RNA targets directly, simultaneously generate double-stranded DNA products containing PAM sequences, and single-stranded DNA products without PAM sequences. Both types of SP-EXPAR products can be recognized by the crRNA and produce fluorescent signals, thereby enhancing detection sensitivity. This RTF-SP-EXPAR-CRISPR/Cas12a biosensor enables the detection of SARS-CoV-2 mutations within 1 h, achieving a detection limit of 7.49 aM and a dynamic range of 10 aM to 10 pM. This method shows high specificity in differentiating mutant variants from wild-type sequences. For the detection of 106 clinical samples, this RTF-SP-EXPAR-CRISPR/Cas12a assay demonstrates 100 % sensitivity and 100 % specificity compared with DNA sequencing results. These findings highlight our proposed assay's strong applicability for the application of RNA samples without reverse transcription.},
}
@article {pmid41175865,
year = {2025},
author = {Ahrens-Nicklas, RC and Musunuru, K},
title = {How to create personalized gene editing platforms: Next steps toward interventional genetics.},
journal = {American journal of human genetics},
volume = {112},
number = {12},
pages = {2826-2829},
doi = {10.1016/j.ajhg.2025.10.006},
pmid = {41175865},
issn = {1537-6605},
mesh = {Humans ; *Gene Editing/methods/trends ; *Precision Medicine/methods/trends ; *Genetic Therapy/methods/trends ; CRISPR-Cas Systems/genetics ; },
abstract = {How do we go from a single individual receiving a personalized gene-editing therapy to a future of "interventional genetics" in which such therapies are the standard of care? First and foremost: regulatory innovation.},
}
@article {pmid41135356,
year = {2026},
author = {Wang, W and Zheng, Y and Zhang, L and Bao, Y and Liu, J and Zang, J and Qu, Y and Zhang, K and Han, R and Ren, H and Zhu, L and Cao, Y and Wang, B and Shen, Q and Sun, W},
title = {A multiplex RPA-CRISPR/Cas12a platform for rapid and accurate toxinotyping of Clostridium perfringens.},
journal = {Talanta},
volume = {298},
number = {Pt B},
pages = {128998},
doi = {10.1016/j.talanta.2025.128998},
pmid = {41135356},
issn = {1873-3573},
mesh = {*Clostridium perfringens/genetics/isolation &amp; purification ; *CRISPR-Cas Systems ; *Bacterial Toxins/genetics/analysis ; *Nucleic Acid Amplification Techniques/methods ; *Recombinases/metabolism ; Humans ; Limit of Detection ; Bacterial Proteins/genetics ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {Clostridium perfringens (C. perfringens) is a leading cause of foodborne disease worldwide, requiring rapid and accurate toxinotyping for effective outbreak control and surveillance. Herein, we developed C. perfringens-multiplex RPA-CRISPR/Cas12a, an integrated detection platform combing multiplex Recombinase Polymerase Amplification (RPA) with Clustered Regularly Interspaced Short Palindromic Repeats-associated protein 12a (CRISPR/Cas12a)-mediated detection for comprehensive toxinotyping. The system simultaneously identifies six key toxin genes (cpa, cpb, etx, iap, cpe, netB) in two reaction tubes, enabling discrimination of all seven C. perfringens toxinotypes (A-G). The C. perfringens-multiplex RPA-CRISPR/Cas12a assay platform exhibited exceptional analytical performance, achieving a detection limit of ≤10 copies/μL for across all targets while maintaining absolute specificity against the human genomic DNA and 5 common foodborne pathogens. In validation testing with 12 naturally contaminated food samples, the C. perfringens-multiplex RPA-CRISPR/Cas12a assay platform demonstrated superior performance to commercial qPCR kits, accurately identifying eight Type A (cpa-gene-positive) and four Type F (cpa-gene and cpe-gene co-positive) strains. When coupled with a portable detection device, the platform completed the entire diagnostic workflow within 50 min while maintaining laboratory-level accuracy under field conditions. The rapid, cost-effective, and equipment-free system is particularly suited for decentralized toxin surveillance in resource-limited settings. By integrating high sensitivity, multiplex capability, and field applicability, this system significantly advances Point-of-care Testing (POCT) capabilities for food safety monitoring, supporting global food safety initiatives.},
}
@article {pmid41109522,
year = {2025},
author = {Garcia, AFS and Farinati, S and Draga, S and Riommi, D and Gabelli, G and Vannozzi, A and Barcaccia, G and Palumbo, F},
title = {Establishing a cutting-edge protoplast technology platform for applying new genomic techniques in Cichorium spp.},
journal = {New biotechnology},
volume = {90},
number = {},
pages = {206-222},
doi = {10.1016/j.nbt.2025.10.008},
pmid = {41109522},
issn = {1876-4347},
mesh = {*Protoplasts/metabolism ; *Gene Editing/methods ; *Genomics/methods ; CRISPR-Cas Systems ; *Cichorium intybus/genetics ; },
abstract = {Genome editing technologies, especially those based on the CRISPR/Cas9 system, have revolutionized crop breeding by enabling precise genetic modifications. Specifically, delivering preassembled ribonucleoprotein (RNP) complexes-consisting of the Cas9 endonuclease coupled to specific single guide RNAs (sgRNAs)-into protoplasts offers an effective DNA-free method that prevents the integration of foreign genetic material. Despite the availability of detailed protocols, establishing a standardized and efficient in vitro regeneration procedure-from protoplast isolation to whole plant regeneration-remains challenging due to significant variability in regeneration efficiency across different varieties and biotypes. Therefore, optimizing each step is essential to maximize the recovery of successful edited plants. In this study, we developed an efficient protocol for regenerating whole plants from protoplasts isolated from 12 representative Italian varieties of chicory and endive. We focused on leaf chicory and endive biotypes with high horticultural value, including Radicchio types, which are important targets for quality improvement. Our optimized platform supports protoplast isolation, PEG-mediated transfection, and plant regeneration, demonstrating promising potential for future genome editing applications. Notably, the high responsiveness of protoplasts to PEG-mediated transfection suggests that coupling this method with our regeneration procedure could facilitate the use of advanced biotechnological strategies. The combination of high transient transformation efficiency, versatile encapsulation techniques, and successful plant regeneration establishes chicory and endive as promising candidates for DNA-free genome editing via protoplasts, providing a technically precise approach with reduced environmental and economic impacts compared to conventional breeding methods.},
}
@article {pmid41075867,
year = {2025},
author = {Zhang, D and Liu, H and Zhong, Y},
title = {Monoclonal antibodies production in microbial systems: Current status, challenges and perspectives.},
journal = {New biotechnology},
volume = {90},
number = {},
pages = {163-173},
doi = {10.1016/j.nbt.2025.10.005},
pmid = {41075867},
issn = {1876-4347},
mesh = {*Antibodies, Monoclonal/biosynthesis/genetics ; *Metabolic Engineering ; Humans ; CRISPR-Cas Systems ; Animals ; Gene Editing ; Synthetic Biology ; },
abstract = {Monoclonal antibodies (mAbs) serve as indispensable tools in diagnostics, clinical therapeutics, and biomedical research. However, their large-scale production faces significant challenges due to the high costs and lengthy timelines associated with conventional mammalian cell-based expression systems. Microbial expression platforms have emerged as a transformative alternative, offering cost-effectiveness, rapid cultivation cycles, and superior genetic tractability for industrial-scale monoclonal antibodies production. Recent advances in Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9-mediated gene editing enable precise metabolic engineering of host strains to enhance protein folding, secretion efficiency, and translational accuracy. Synthetic biology approaches facilities the reconstruction of mammalian glycosylation pathways in microbial systems, yielding monoclonal antibodies with near-native structural integrity. Furthermore, AI (artificial intelligence)-driven optimization of expression vectors, promoter systems, and culture conditions, combined with high-throughput screening of engineered strains, significantly accelerates the identification of high-yield production clones. This review comprehensively examines current progress in microbial expression systems, strain engineering strategies, and fermentation optimization for enhanced monoclonal antibodies production, while critically discussing existing limitations and potential solutions to advance the field.},
}
@article {pmid41072226,
year = {2026},
author = {Li, L and Li, M and Wang, S and Dong, Y},
title = {Development of a CRISPR/Cas12a-assisted fluorescent aptasensor for simultaneous detection of zearalenone and ochratoxin A.},
journal = {Talanta},
volume = {298},
number = {Pt B},
pages = {128937},
doi = {10.1016/j.talanta.2025.128937},
pmid = {41072226},
issn = {1873-3573},
mesh = {*Ochratoxins/analysis ; *Zearalenone/analysis ; *Aptamers, Nucleotide/chemistry/genetics/metabolism ; *Biosensing Techniques/methods ; *CRISPR-Cas Systems ; Limit of Detection ; Food Contamination/analysis ; Spectrometry, Fluorescence ; Fluorescence ; *Fluorescent Dyes/chemistry ; },
abstract = {Mycotoxins, such as zearalenone (ZEN) and ochratoxin A (OTA), represent significant hazards to both human and animal health, necessitating strict monitoring and regulation of mycotoxin levels in food, feed, and environment. In this study, a simple and efficient CRISPR/Cas12a-assisted fluorescent aptasensor is presented for the simultaneous detection of ZEN and OTA. Utilizing a Bifunctional aptamer (B-APT), this biosensor achieves dual recognition of the both targets, subsequently converting their concentrations into observable fluorescent signals through the Cas12a/crRNA cis-cleavage activity. Rational modifications of the complementary strands specific to the two targets enable distinct emission wavelengths under the same excitation light, facilitating simultaneous and independent quantitative determination of ZEN and OTA. Under optimized conditions, the CRISPR/Cas12a-aptasensor demonstrates robust detection capabilities for individual ZEN and OTA targets, as well as their mixture, yielding consistent standard curves. This methodology exhibits reliable detection of ZEN and OTA concentrations spanning from 0.25 nM to 256 nM and 1 nM to 1024 nM, with respective limit of detection (LOD) values of 190 pM and 931 pM. Furthermore, this method showcases exceptional selectivity and considerable recovery rates (89.17 %-109.88 % for ZEN and 101.19 %-106.51 % for OTA) in corn oil samples, underscoring its efficacy as an advanced tool for ZEN and OTA detection and offering valuable insights into the simultaneous detection of diverse targets.},
}
@article {pmid40684770,
year = {2025},
author = {Ali, A and Roy, B and Schott, MB and Grove, BD},
title = {AKAP12 Variant 1 Knockout Enhances Vascular Endothelial Cell Motility.},
journal = {Journal of vascular research},
volume = {62},
number = {6},
pages = {312-329},
pmid = {40684770},
issn = {1423-0135},
support = {P20 GM104360/GM/NIGMS NIH HHS/United States ; P20 GM113123/GM/NIGMS NIH HHS/United States ; P30 GM103329/GM/NIGMS NIH HHS/United States ; U54 GM128729/GM/NIGMS NIH HHS/United States ; },
mesh = {*A Kinase Anchor Proteins/genetics/metabolism ; *Cell Movement ; Humans ; *Cell Cycle Proteins/genetics/metabolism ; *Endothelial Cells/metabolism ; Cells, Cultured ; *Human Umbilical Vein Endothelial Cells/metabolism ; Intercellular Junctions/metabolism/genetics ; Gene Knockout Techniques ; CRISPR-Cas Systems ; },
abstract = {INTRODUCTION: Previous work indicates that AKAP12 is expressed in endothelial cells as two variants and may play a role in cell motility. However, the role of each variant in cell motility is unknown; therefore, this study investigated the role of AKAP12 in endothelial cell motility with a specific focus on AKAP12 variants, AKAP12v1 and AKAP12v2.<br><br>METHODS: AKAP12 expression levels in cultured endothelial cells were determined by Western blotting and immunofluorescence microscopy. AKAP12 knockdown and AKAP12 variant knockout were done using antisense oligonucleotide and siRNA treatment and CRISPR/Cas9 knockout, respectively. The effect of AKAP12 variant knockout was further analyzed by RNA-seq.<br><br>RESULTS: AKAP12 expression was cell density-dependent, with the highest expression in subconfluent cultures and lowest in confluent cultures. AKAP12 expression was also elevated in cells at the wound edge of wounded endothelial cell monolayers. Knockdown of both variants inhibited cell migration, but CRISPR/Cas9 knockout of AKAP12v1 enhanced migration. RNA-seq revealed that loss of AKAP12v1 affected genes associated with cell migration and intercellular junctions.<br><br>CONCLUSION: We propose that AKAP12v1 and AKAP12v2 play distinct yet complementary roles in endothelial cell migration and likely work together in controlling the signaling events associated with vascular repair and development.},
}
@article {pmid41330932,
year = {2025},
author = {Van Vu, T and Thi Nguyen, N and Kim, J and Sung, YW and Chung, WS and Kim, JY},
title = {The evolving landscape of precise DNA insertion in plants.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {10428},
pmid = {41330932},
issn = {2041-1723},
support = {RS-2022-NR070609//National Research Foundation of Korea (NRF)/ ; RS-2025-02263262//National Research Foundation of Korea (NRF)/ ; RS-2021-NR060105//National Research Foundation of Korea (NRF)/ ; RS-2020-NR049590//National Research Foundation of Korea (NRF)/ ; RS-2020-NR049590//National Research Foundation of Korea (NRF)/ ; RS-2025-02263262//National Research Foundation of Korea (NRF)/ ; RS-2022-NR070609//National Research Foundation of Korea (NRF)/ ; RS-2025-02263262//National Research Foundation of Korea (NRF)/ ; RS-2021-NR060105//National Research Foundation of Korea (NRF)/ ; RS-2025-02263262//National Research Foundation of Korea (NRF)/ ; RS-2022-NR070609//National Research Foundation of Korea (NRF)/ ; RS-2020-NR049590//National Research Foundation of Korea (NRF)/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; Gene Editing/methods ; Genome, Plant ; Plants, Genetically Modified/genetics ; *DNA, Plant/genetics ; *Plants/genetics ; *Mutagenesis, Insertional/methods ; Gene Targeting/methods ; },
abstract = {Precise DNA insertion into plant genomes is central to advancing crop improvement and synthetic biology. CRISPR-Cas systems have enabled programmable DNA integration using tools such as gene targeting (GT), prime editing (PE), and recombinase- or transposase-based platforms. These tools are transitioned from theoretical concepts to practical applications, supporting applications like in-locus protein tagging, regulatory element engineering, and multi-gene stacking. Key challenges persist, such as inefficient large-fragment insertion, delivery barriers, and regulatory hurdles. This review traces the evolution from random to CRISPR-Cas-based systems, analyzes current limitations, and discusses emerging solutions paving the way for predictable DNA insertion in modern plant biotechnology.},
}
@article {pmid41330849,
year = {2025},
author = {Boob, AG and Zhang, C and Pan, Y and Zaidi, A and Whitaker, RJ and Zhao, H},
title = {Discovery, characterization, and application of chromosomal integration sites in the hyperthermophilic archaeon Sulfolobus islandicus.},
journal = {Trends in biotechnology},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.tibtech.2025.11.003},
pmid = {41330849},
issn = {1879-3096},
abstract = {Sulfolobus islandicus, an emerging archaeal model organism, offers unique advantages for metabolic engineering and synthetic biology applications owing to its ability to thrive in extreme environments. Although several genetic tools have been established for this organism, the lack of well-characterized chromosomal integration sites has limited its potential as a cellular factory. Here, we systematically identified and characterized 13 artificial CRISPR RNAs targeting eight integration sites in S. islandicus using the CRISPR-COPIES pipeline and a multi-omics-informed computational workflow. We leveraged the endogenous CRISPR-Cas system to integrate the reporter gene lacS and validated heterologous expression through a β-galactosidase assay, revealing significant positional effects. As a proof of concept, we utilized these sites to genetically manipulate lipid ether composition by overexpressing glycerol dibiphytanyl glycerol tetraether (GDGT) ring synthase B (GrsB). This study expands the genetic toolbox for S. islandicus and advances its potential as a robust platform for archaeal synthetic biology and industrial biotechnology.},
}
@article {pmid41330674,
year = {2026},
author = {Hou, L and Ruan, F and Zhao, K and Li, B},
title = {Boosting split-crRNA CRISPR/Cas12a activity by 3'-end extension of DNA activator for direct microRNA sensing.},
journal = {Analytica chimica acta},
volume = {1382},
number = {},
pages = {344841},
doi = {10.1016/j.aca.2025.344841},
pmid = {41330674},
issn = {1873-4324},
mesh = {*MicroRNAs/analysis/genetics/blood ; *CRISPR-Cas Systems ; *Biosensing Techniques/methods ; Humans ; *DNA/chemistry/metabolism/genetics ; Limit of Detection ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {BACKGROUND: The unique trans-cleavage activity of CRISPR/Cas12a has been extensively utilized in the domain of biosensing. Nevertheless, the detection of miRNAs using the traditional CRISPR/Cas12a system requires nucleic acid amplification or reverse transcription to convert miRNA into DNA, which increases reaction time and the risk of contamination.<br><br>RESULTS: This study presents a split-crRNA CRISPR/Cas12a-based biosensing for direct detection of miRNA. The target miRNA-375 was utilized as the spacer region of the crRNA, facilitating its binding to the truncated scaffold RNA, thereby resulting in the formation of a complete crRNA. More importantly, we discovered that the cleavage activity of split-crRNA CRISPR/Cas12a was significantly enhanced by extending sequences at the 3'-end of the DNA activator. Compared with the conventional split-crRNA CRISPR/Cas12a system, the split-crRNA CRISPR/Cas12a with 24-nucleotide random sequence extension at the 3'-end of the DNA activator exhibited a 6.4-fold increase in activity. The enhancement mechanism of 3'-end extension of DNA activator was discussed. This proposed split-crRNA CRISPR/Cas12a system was applied to detect miRNA-375 with a linear range of 5 pM-1&#xa0;nM, and the detection limit was estimated to be 0.6 pM (3&#x3c3;). Furthermore, this system was used to detect miRNA-375 in 10&#xa0;% diluted human serum, achieving satisfactory recovery rate (98&#xa0;%-106&#xa0;%).<br><br>SIGNIFICANCE: This finding indicates that it is feasible to enhance the activity of the split-crRNA CRISPR/Cas12a by extending the 3'-end of the DNA activator, thereby achieving highly sensitive direct detection of miRNA. It is a simple yet effective strategy for enhancing the sensitivity of direct miRNA detection.},
}
@article {pmid41330665,
year = {2026},
author = {Bao, Y and Ding, W and Zhang, L and Wang, W and Liu, J and Qu, Y and Zhu, L and Zhang, K and Zhong, G and Han, R and Shen, Q and Wang, B and Gu, X and Cao, Y and Sun, W},
title = {A novel multiplex RPA/CRISPR-Cas12a integrated biosensor for on-site detection of high-risk HPV genotypes.},
journal = {Analytica chimica acta},
volume = {1382},
number = {},
pages = {344830},
doi = {10.1016/j.aca.2025.344830},
pmid = {41330665},
issn = {1873-4324},
mesh = {*CRISPR-Cas Systems/genetics ; *Biosensing Techniques/methods ; Humans ; Genotype ; *Nucleic Acid Amplification Techniques/methods ; *Papillomaviridae/genetics/isolation &amp; purification ; *Recombinases/metabolism ; Papillomavirus Infections/virology/diagnosis ; DNA, Viral/genetics ; },
abstract = {Accurate genotyping of high-risk human papillomavirus (HR-HPV) at the point of care is critical for global cervical cancer elimination, but its application remains limited by the need for complex equipment and specialized procedures, particularly in resource-limited regions. Here, we develop H-MRC12a-an integrated platform based on degenerate primers and type-specific crRNAs that combines multiplex recombinase polymerase amplification (RPA) with CRISPR-Cas12a trans-cleavage activity-for ultrasensitive detection of eight key HR-HPV genotypes (16, 18, 31, 33, 52, 53, 58, 66). By introducing a degenerate primer strategy coupled with type-specific crRNAs, the system overcomes interference from primer dimer formation and achieves single-copy sensitivity within 50 min under isothermal conditions (37 °C). Clinical validation demonstrated 100 % concordance with qPCR and identified three additional low viral-load positives (Ct > 35) that were missed by conventional methods. Crucially, the platform enables visual readout under UV light and eliminates the need for specialized instruments. This 'broad-spectrum capture and precision typing' paradigm establishes a versatile framework for multiplexed pathogen detection, advancing accessible molecular diagnostics for global health equity.},
}
@article {pmid41288387,
year = {2025},
author = {Kim, B and Yuk, M and Park, M and Ryu, H and Park, J and Yu, H and Park, S and Hong, JT and Lim, KH and Han, SB and Song, N and Park, H},
title = {CRISPR editing of miR-33 restores ApoE lipidation and amyloid-β metabolism in ApoE4 sporadic Alzheimer's disease.},
journal = {Brain : a journal of neurology},
volume = {148},
number = {12},
pages = {4400-4415},
doi = {10.1093/brain/awaf244},
pmid = {41288387},
issn = {1460-2156},
support = {//National Research Foundation of Korea/ ; 2021R1C1C1006551//Korean government/ ; //Bio &amp; Medical Technology Development Program/ ; //National Research Foundation/ ; RS-2024-00440787//Korean government/ ; },
mesh = {*MicroRNAs/genetics/metabolism ; *Alzheimer Disease/genetics/metabolism/pathology ; Animals ; Humans ; Mice ; *Gene Editing/methods ; *Apolipoprotein E4/genetics/metabolism ; *Lipid Metabolism/genetics ; *Amyloid beta-Peptides/metabolism ; Male ; CRISPR-Cas Systems ; Female ; Astrocytes/metabolism ; Mice, Transgenic ; *Apolipoproteins E/metabolism/genetics ; Aged ; Disease Models, Animal ; },
abstract = {Sporadic Alzheimer's disease (sAD) is marked by dysregulated lipid metabolism, prominently involving apolipoprotein E (ApoE). MicroRNA-33 (miR-33) has emerged as a key regulator of lipid homeostasis, yet its role in sAD remains unclear. This study investigated miR-33 dysregulation in APOE ε4 allele (ApoE4)-associated sAD and explored its therapeutic potential using clustered regulatory interspaced short palindromic repeats (CRISPR)-mediated gene editing. Elevated miR-33 expression was observed in both AD patients, particularly those with ApoE4-associated sAD, and in the ApoE4 mouse model, implicating its role in AD pathology. Using CRISPR/Cas9, we modulated miR-33 expression in astrocytes to regulate ApoE lipidation and ameliorate AD-related pathology. Our results show that targeted miR-33 regulation in astrocytes via CRISPR/Cas9 restores ApoE lipidation and mitigates AD pathology in both in vitro and in vivo AD mice. Additionally, applying this gene therapy approach in ApoE4 sAD patient cell lines highlights its translational potential for therapeutic intervention. In conclusion, our findings elucidate miR-33's role in AD pathogenesis and underscore the therapeutic promise of CRISPR-mediated miR-33 targeting for restoring lipid homeostasis and ameliorating AD pathology. This study provides valuable insights into developing miRNA-based gene therapy strategies for treating sAD.},
}
@article {pmid41284724,
year = {2025},
author = {da Silva, GLA and Damasceno, JD and Black, JA and Lapsley, C and McCulloch, R and Tosi, LRO},
title = {ATR, a DNA damage kinase, modulates DNA replication timing in Leishmania major.},
journal = {PLoS genetics},
volume = {21},
number = {11},
pages = {e1011899},
doi = {10.1371/journal.pgen.1011899},
pmid = {41284724},
issn = {1553-7404},
mesh = {*Leishmania major/genetics ; *Ataxia Telangiectasia Mutated Proteins/genetics/metabolism ; DNA Damage/genetics ; *DNA Replication/genetics ; CRISPR-Cas Systems ; *DNA Replication Timing/genetics ; *Protozoan Proteins/genetics/metabolism ; },
abstract = {All cells possess mechanisms to maintain and replicate their genomes, whose integrity and transmission are constantly challenged by DNA damage and replication impediments. In eukaryotes, the protein kinase Ataxia-Telangiectasia and Rad3-related (ATR), a member of the phosphatidylinositol 3-kinase-like family, acts as a master regulator of the eukaryotic response to DNA injuries, ensuring DNA replication completion and genome stability. Here we aimed to investigate the functional relevance of the ATR homolog in the DNA metabolism of Leishmania major, a protozoan parasite with a remarkably plastic genome. CRISPR/cas9 genome editing was used to generate a Myc-tagged ATR cell line (mycATR), and a Myc-tagged C-terminal knockout of ATR (mycATRΔC-/-). We show that the nuclear localisation of ATR depends upon its C-terminus. Moreover, its deletion results in single-stranded DNA accumulation, impaired cell cycle control, increased levels of DNA damage, and delayed DNA replication re-start after replication stress. In addition, we show that ATR plays a key role in maintaining L. major's unusual DNA replication program, where larger chromosomes duplicate later than smaller chromosomes. Our data reveals loss of the ATR C-terminus promotes the accumulation of DNA replication signal around replicative stress fragile sites, which are enriched in larger chromosomes. Finally, we show that these alterations to the DNA replication program promote chromosome instability. In summary, our work shows that ATR acts to modulate DNA replication timing, limiting the plasticity of the Leishmania genome.},
}
@article {pmid41233591,
year = {2025},
author = {Li, L and Gao, J and Yi, D and Sheft, AP and Schimenti, JC and Ding, X},
title = {A primordial germ cell-like-cell platform enables CRISPRi screen for epigenetic fertility modifiers.},
journal = {EMBO reports},
volume = {26},
number = {23},
pages = {6044-6078},
pmid = {41233591},
issn = {1469-3178},
support = {R01 HD082568/HD/NICHD NIH HHS/United States ; 1P50HD096723//HHS | NIH | Eunice Kennedy Shriver National Institute of Child Health and Human Development (NICHD)/ ; CO29155//New York State Stem Cell Science (NYSTEM)/ ; C30293GG//New York State Stem Cell Science (NYSTEM)/ ; R01 HD082568/HD/NICHD NIH HHS/United States ; },
mesh = {Animals ; Mice ; *Germ Cells/metabolism/cytology ; *Epigenesis, Genetic ; Male ; Positive Regulatory Domain I-Binding Factor 1/genetics/metabolism ; Cell Differentiation/genetics ; Nanog Homeobox Protein/genetics/metabolism ; *Fertility/genetics ; Transcription Factor AP-2/genetics/metabolism ; Transcription Factors/genetics/metabolism ; *CRISPR-Cas Systems ; Female ; Repressor Proteins/genetics/metabolism ; RNA-Binding Proteins/genetics ; DNA-Binding Proteins/genetics/metabolism ; },
abstract = {Primordial germ cells (PGCs) are the precursors of gametes, and the ability to derive PGC-like cells (PGCLCs) from pluripotent stem cells has transformed germline research. A key limitation remains producing PGCLCs in sufficient numbers for large-scale applications. Here, we show that overexpression of Nanog plus three PGC master regulators - Prdm1, Prdm14, and Tfap2c - in mouse epiblast-like cells and formative embryonic stem cells yields abundant and highly enriched PGCLCs without costly recombinant cytokines. Nanog enhances the PGC regulatory network, suppresses somatic differentiation, and stabilizes PGCLC fate. Transcriptomically, these PGCLCs are developmentally more advanced than cytokine-induced counterparts and can be sustained long-term or differentiated into spermatogonia-like cells. Using this platform, we conduct a CRISPRi screen of 701 epigenetic genes to identify those needed for PGCLC formation. Downregulation of Ncor2, a histone deacetylase (HDAC) recruiter, has the greatest impact. Additionally, the HDAC inhibitors valproic acid and sodium butyrate suppress PGCLC formation and sperm counts of in utero-exposed animals. This work establishes a scalable system for functional screening of genes that influence germline development.},
}
@article {pmid41214358,
year = {2025},
author = {Liu, XY and Li, YF and Zhou, JZ and Huang, JQ and Wang, LJ and Yang, L and Li, CY and Fang, X and Chen, XY},
title = {Gamma-selinene synthase catalyzes the first step of dihydroagarofuran sesquiterpene alkaloid biosynthesis in Tripterygium.},
journal = {Science China. Life sciences},
volume = {68},
number = {12},
pages = {3696-3710},
pmid = {41214358},
issn = {1869-1889},
mesh = {*Tripterygium/genetics/enzymology/metabolism ; *Sesquiterpenes/metabolism/chemistry ; *Alkaloids/biosynthesis ; *Plant Proteins/metabolism/genetics ; Biosynthetic Pathways ; Phylogeny ; CRISPR-Cas Systems ; },
abstract = {Tripterygium wilfordii and T. hypoglaucum, which belong to family Celastraceae, are perennial vine shrubs with medicinal uses in treating rheumatoid arthritis, cancer, and male contraception. Among the bioactive ingredients, the macrocyclic dihydroagarofuran sesquiterpene alkaloids (DASAs), also serving as chemotaxonomic markers of Celastraceae, are well-known for cytotoxicity and insecticidal properties. Despite intensive phytochemical investigations over the last half-century, the DASAs biosynthetic pathway remains unsolved. Here, we mined multi-omics data of Tripterygium species and identified 14 sesquiterpene synthase genes. Detailed characterization revealed that a group of enzymes (TwTPS5, TwTPS16, ThTPS5) catalyzed the key step committed to the biosynthesis of DASAs in Tripterygium, as evidenced by CRISPR/Cas9 knockout of TwTPS5 in T. wilfordii. Our biochemical assay showed that these enzymes converted FPP to gamma-selinene. Homologues in other Celastraceae species (EaTPS3, CaTPS5, and CaTPS6) also produced gamma-selinene, suggesting functional conservation in this DASAs-producing lineage. Notably, knocking out TwTPS5 resulted in a reduction of DASAs and enhanced accumulation of pharmaceutically valuable compounds, generating a safer and innovative medicinal plant resource for future applications.},
}
@article {pmid41139756,
year = {2025},
author = {Kang, CM and Jeong, RD},
title = {Rapid and visual detection of Lentinula edodes spherical virus using CRISPR/Cas12a-based RT-RPA.},
journal = {Virus genes},
volume = {61},
number = {6},
pages = {724-732},
pmid = {41139756},
issn = {1572-994X},
mesh = {*Shiitake Mushrooms/virology/genetics ; *CRISPR-Cas Systems ; *Fungal Viruses/genetics/isolation &amp; purification ; Republic of Korea ; *Nucleic Acid Amplification Techniques/methods ; Sensitivity and Specificity ; },
abstract = {Lentinula edodes (shiitake) is a globally important edible mushroom, but viral infections can hinder mycelial growth, reduce yield, and degrade quality, leading to economic losses. This study examined viral infections in L. edodes cultivated in South Korea. We developed a CRISPR/Cas12a-based reverse transcription recombinase polymerase amplification (RT-RPA) assay for the rapid and accurate detection of L. edodes spherical virus (LeSV), the predominant mycovirus-infecting L. edodes in Korea. The optimized RT-RPA-CRISPR/Cas12a assay can be completed within 20 min at 34 °C and demonstrated 100 times higher sensitivity than RT-PCR. Field sample verification further confirmed its superior detection rate over RT-PCR. The RT-RPA-CRISPR/Cas12a method described herein has the potential to facilitate early diagnosis of LeSV infections and support disease management in L. edodes cultivation.},
}
@article {pmid41016102,
year = {2026},
author = {Guo, X and Zhang, J and Han, K and Li, Y and Wang, D},
title = {Aptamer-based PSA biosensing technology: From nanomaterials to CRISPR-diagnostics technology.},
journal = {Talanta},
volume = {298},
number = {Pt A},
pages = {128871},
doi = {10.1016/j.talanta.2025.128871},
pmid = {41016102},
issn = {1873-3573},
mesh = {*Prostate-Specific Antigen/analysis/blood ; Humans ; *Biosensing Techniques/methods ; *Aptamers, Nucleotide/chemistry ; *Nanostructures/chemistry ; *Prostatic Neoplasms/diagnosis ; Male ; *CRISPR-Cas Systems ; },
abstract = {Prostate cancer is a leading cause of cancer-related mortality in men, making early detection crucial for improving patient outcomes. Prostate-specific antigen (PSA) is a key biomarker for prostate cancer. Traditional PSA detection methods, such as ELISA and CLIA, are widely used but have limitations including complex sample preparation and long assay times. Aptamer-based biosensors offer a promising alternative due to their high affinity, specificity, and ease of customization. Recent advancements in aptamer-based PSA biosensors, including the use of novel nanomaterials and dual-modal detection technologies, have significantly enhanced sensitivity and reliability. The integration of CRISPR-Dx technology has further improved the accuracy of PSA detection in complex biological samples. These innovations hold great potential for developing portable point-of-care testing (POCT) devices for early screening and personalized treatment. This review examines the latest developments in aptamer-based biosensing technology for PSA detection from 2019 to 2025, focusing on nanomaterial innovation, CRISPR-Dx integration, and dual-modal sensing. It aims to provide a comprehensive overview of the strengths and limitations of current technologies and to outline future directions for more efficient and clinically valuable PSA detection methods.},
}
@article {pmid40985613,
year = {2025},
author = {Chen, S and Chen, X and Peng, Y and Li, Q and Zhou, J and Li, J and Du, G and Chen, J and Zhang, G},
title = {CRISPR-DNA Polymerase Assisted Targeted Mutagenesis for Regulable Laboratory Evolution.},
journal = {Advanced science (Weinheim, Baden-Wurttemberg, Germany)},
volume = {12},
number = {45},
pages = {e11448},
doi = {10.1002/advs.202511448},
pmid = {40985613},
issn = {2198-3844},
support = {2024YFA0918300//National Key Research and Development Program of China/ ; 32172153//National Natural Science Foundation of China/ ; BK20202002//Natural Science Foundation of Jiangsu Province/ ; },
mesh = {*DNA-Directed DNA Polymerase/genetics/metabolism ; *CRISPR-Cas Systems/genetics ; *Mutagenesis/genetics ; Escherichia coli/genetics ; *Directed Molecular Evolution/methods ; Mutation/genetics ; },
abstract = {Targeted hypermutation tools are useful for engineering proteins and pathways, and exploring the evolutionary landscapes. However, existing targeted hypermutation tools for genomic loci mostly exhibit restricted mutation windows and limited mutational types. Here, by integrating mutagenic, high-processivity bacteriophage T5 or T7 DNA polymerases (DNAPs) with CRISPR-Cas9, the study develops an in vivo mutagenesis system that enables all possible types of nucleotide substitutions and an expanded mutation window of up to 2 kilobases, achieving a maximum mutation rate 1.1 × 10[6]-fold higher than wild-type Escherichia coli. Through MS2-mediated recruitment of T5 or T7 DNAP for co-localization with nickase nCas9, off-target rate is reduced by up to 96.8% without compromising on-target rate. Further benefiting from the dTnpB-based transcriptional repression system, the mutagenesis process can be properly regulated during continuous evolution. Finally, the CRISPR-TDNAP-assisted targeted mutagenesis for regulable laboratory evolution (CTRLE) confers cellular triple-antibiotic resistance in 8 days, and enhances the efficiency of the twin-arginine translocation pathway by over threefold in 6 days. Furthermore, CTRLE proves effective in Bacillus subtilis and Kluyveromyces lactis, yielding targeted mutation rates 1.2 × 10[5]-fold and 5 × 10[7]-fold higher than host backgrounds, respectively. Collectively, CTRLE provides an efficient and universal way to accelerate the continuous evolution of different microbial cells.},
}
@article {pmid40961712,
year = {2026},
author = {Zheng, Y and Li, Y and Zhu, J and Yi, J and Li, T and Tang, H},
title = {Synergistic UCNP/CRISPR-Cas12a enhanced near-infrared photoelectrochemical biosensor for quantitative Kanamycin detection.},
journal = {Talanta},
volume = {298},
number = {Pt A},
pages = {128857},
doi = {10.1016/j.talanta.2025.128857},
pmid = {40961712},
issn = {1873-3573},
mesh = {*Kanamycin/analysis ; *Biosensing Techniques/methods ; *Electrochemical Techniques/methods ; *CRISPR-Cas Systems ; Milk/chemistry ; Zinc Oxide/chemistry ; *Food Contamination/analysis ; Animals ; *Anti-Bacterial Agents/analysis ; Infrared Rays ; Aptamers, Nucleotide/chemistry ; Limit of Detection ; Sulfides/chemistry ; *Nanoparticles/chemistry ; Cadmium Compounds/chemistry ; *Bacterial Proteins/metabolism/genetics ; Electrodes ; Ytterbium/chemistry ; Photochemical Processes ; Yttrium/chemistry ; Endodeoxyribonucleases ; Zinc Compounds ; CRISPR-Associated Proteins ; },
abstract = {Antibiotic residues in food pose significant health risks, and sensitive on-site detection remains challenging in complex matrices. We report a near-infrared photoelectrochemical (NIR-PEC) biosensor with CRISPR-Cas12a signal amplification for sensitive detection of kanamycin in food. A ZnO/CdS heterojunction photoanode boosts visible-light charge separation, producing a photocurrent of 4.6 mA-96 % higher than ZnO alone. Upconversion nanoparticles (NaYF4:Yb[3+], Er[3+]) convert 980 nm light into 542 nm emission to drive the heterojunction under NIR illumination. In the presence of kanamycin, activator DNA is displaced from an aptamer, triggering Cas12a to cleave ssDNA probes on the electrode and reduce photocurrent. The sensor shows a linear response from 10 to 1000 nM, a detection limit of 0.284 nM and less than 2.8 % cross-reactivity with related compounds. Average recovery in spiked milk samples averages 97 % (RSD = 2.2 %). This platform offers high specificity and sensitivity for antibiotic residue analysis in complex food matrices.},
}
@article {pmid40956287,
year = {2025},
author = {Shen, Y and Lu, B and Ma, B and Ding, X},
title = {Enhanced One-Pot Dual-CRISPR-Based Assay Lyophilized on a 3D-Printed Disc for Field-Deployable Multiplex Bacteria Detection.},
journal = {Advanced science (Weinheim, Baden-Wurttemberg, Germany)},
volume = {12},
number = {45},
pages = {e09355},
doi = {10.1002/advs.202509355},
pmid = {40956287},
issn = {2198-3844},
support = {2023YFD2402800//National Key Research and Development Program of China for Young Scientists/ ; 2022YFF1102600//National Key Research and Development Program of China for Young Scientists/ ; 82373629//National Natural Science Foundation of China/ ; BE2023725//Jiangsu Provincial Key Research and Development Program/ ; 4060692202/023//Medical Foundation of Southeast University/ ; },
mesh = {*Printing, Three-Dimensional ; Freeze Drying/methods ; *Nucleic Acid Amplification Techniques/methods ; *Bacteria/genetics/isolation &amp; purification ; *CRISPR-Cas Systems/genetics ; },
abstract = {One-pot CRISPR-based detection combining recombinase polymerase amplification (RPA) enables rapid and accurate nucleic acid testing but faces challenges in performance, multiplexing, and field-ready lyophilization. Here, an enhanced one-pot, helicase-assisted RPA (hRPA)-combined, dual-CRISPR/uAsCas12a (EOD-CRISPR) assay is described which can be lyophilized on a 3D-printed microfluidic disc to achieve field-deployable multiplex bacteria detection. In EOD-CRISPR reactions, the reaction speed, sensitivity, and fluorescence signal are significantly enhanced due to the synergistic effect of bovine serum albumin, hRPA, and uAsCas12a nuclease. The 3D-printed disc features four central chambers encircled by eight outer chambers, permitting detecting four targets simultaneously. For stable lyophilization on disc chambers, glassfiber membranes are inserted as substrates to adsorb the EOD-CRISPR reagents containing a protectant of 5% trehalose and 1% glycine. Toward point-of-need testing, EOD-CRISPR-lyophilized discs are applied to build an onsite detection platform. Through detecting synthetic food samples contaminated by four foodborne bacteria (i.e., Bacillus cereus, Salmonella enterica, Staphylococcus aureus, and Escherichia coli O157:H7), the onsite detection platform is validated and the sensitivity (80%-88.9%) and specificity (92.3%-100%) are comparable to those of standard PCR methods. Therefore, the field-deployable multiplex EOD-CRISPR assays holds great potentials for onsite bacteria detection and beyond.},
}
@article {pmid40946480,
year = {2026},
author = {Zhang, Q and Liu, Z and Sheng, E and Liu, Z and Zhao, R and Li, M},
title = {Glucosemeter-based aptasensor with an efficient and green system driven by CRISPR/Cas12a for ZEN detection.},
journal = {Talanta},
volume = {298},
number = {Pt A},
pages = {128844},
doi = {10.1016/j.talanta.2025.128844},
pmid = {40946480},
issn = {1873-3573},
mesh = {*CRISPR-Cas Systems ; *Biosensing Techniques/methods ; *Zearalenone/analysis ; *Aptamers, Nucleotide/chemistry/genetics/metabolism ; Limit of Detection ; *CRISPR-Associated Proteins/metabolism/genetics ; Bacterial Proteins ; Endodeoxyribonucleases ; },
abstract = {To address the challenge of readily available and cost-effective monitoring, a novel aptasensor platform was proposed relying on the clustered regularly interspaced short palindromic repeats/associated protein 12a (CRISPR/Cas12a)-driven and glucosemeter-assisted system for zearalenone (ZEN). The magnetic bead (MB)-based probes of MB@Apt-cDNA and MB@invertase were prepared to provide specific recognition and efficient conversion. When ZEN presented, the released cDNA activated the cutting function of CRISPR/Cas12a for ssDNA, releasing numerous invertase to convert the environmentally friendly sucrose. Then, the generated glucose signals were positively related to ZEN levels, which quantified by a glucosemeter. The proposed aptasensor displayed an excellent sensitivity, achieving the limit of detection (LOD) and detection range of 0.218 ng/mL and 0.218-109.89 ng/mL, respectively. The platform also exhibited high specificity (with cross-reactivity values below 6.81 % for other analytes) and high accuracy for ZEN detection. The satisfactory reliability and practicability had been demonstrated through its application in real agricultural byproduct samples. The area under the curve was 0.979, with an optimal operating point being (0.125, 1.000). This study can provide an alternative pathway for improving the detection performance and accessibility to public in limited situation, suggesting the efficient CRISPR/Cas12a and green glucosemeter detection strategies for other pollutant monitoring.},
}
@article {pmid40939440,
year = {2026},
author = {Xiang, Q and Zhou, W and Li, D},
title = {Dumbbell-shaped DNA topology drives self-sustaining CRISPR/Cas12a exponential amplification for ultrasensitive monitoring of DNA methyltransferase activity.},
journal = {Talanta},
volume = {298},
number = {Pt A},
pages = {128841},
doi = {10.1016/j.talanta.2025.128841},
pmid = {40939440},
issn = {1873-3573},
mesh = {*CRISPR-Cas Systems ; *DNA/chemistry/metabolism ; DNA Methylation ; Nucleic Acid Conformation ; Humans ; *Enzyme Assays/methods ; *Nucleic Acid Amplification Techniques/methods ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {DNA methylation is an essential epigenetic mechanism, and abnormal methylation has been linked to the onset and progression of many diseases, representing a potential threat to health. Monitoring DNA methyltransferase (MTase) activity is essential for understanding DNA methylation regulation and developing MTase-targeted inhibitors. To address this challenge, we developed a dumbbell-shaped DNA topology that drives self-sustaining (autocatalytic) CRISPR/Cas12a system for exponential signal amplification, enabling ultrasensitive fluorescent detection of DNA MTase activity. In this strategy, a DNA dumbbell topological structure (DDTS) is designed, in which two double-strand DNA (dsDNA) loops effectively block the activity of CRISPR/Cas12a. Upon Dam MTase presence, DpnI endonuclease cleaves the methylated recognition sites in the DNA probe, disrupting the DDTS topology to generate linear dsDNA activators. These activators restore the trans-cleavage of CRISPR/Cas12a, which further cleaves the single-stranded DNA (ssDNA) domain in DDTS probes to produce additional activators, creating an exponential amplification loop through autocatalysis. The system achieves a detection limit of 6.37 × 10[-4] U/mL for Dam MTase, with a linear range of 1 × 10[-3] to 15 U/mL, and shows excellent selectivity over other MTases and nucleases. It also enables inhibitor screening, with the half-maximal inhibitory concentration (IC50) value of 1.84 μM for 5-fluorouracil. Therefore, the method has great potential for application in the early diagnosis of diseases and drug discovery.},
}
@article {pmid40898619,
year = {2025},
author = {Noh, B and Gopalappa, R and Lin, H and Gee, HY and Choi, JY and Kim, HH and Jung, J},
title = {Engineered virus-like particles for in vivo gene editing ameliorate hearing loss in murine DFNA2 model.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {33},
number = {12},
pages = {6449-6462},
doi = {10.1016/j.ymthe.2025.08.049},
pmid = {40898619},
issn = {1525-0024},
mesh = {Animals ; Mice ; *Gene Editing/methods ; Disease Models, Animal ; *Genetic Therapy/methods ; *Hearing Loss/therapy/genetics ; CRISPR-Cas Systems ; Hair Cells, Auditory, Outer/metabolism ; Genetic Vectors/genetics ; Gene Transfer Techniques ; KCNQ Potassium Channels/genetics ; *Virion/genetics ; CRISPR-Associated Protein 9/genetics ; },
abstract = {Although gene editing therapy is applicable to human diseases, its efficiency and safety require further investigation. Further, non-virus-mediated gene editor delivery is challenging in the inner ear. Here, engineered virus-like particles (eVLPs) were used for inner ear delivery of SpCas9 and single-guided RNA to delete the Kcnq4 dominant-negative mutant allele, which causes progressive hearing loss in a non-syndromic hearing loss murine model. eVLP-delivered SpCas9 was administered to the inner ears of Kcnq4[W277S/+] mice to target the Kcnq4-expressing outer hair cells (OHCs). Hearing loss was significantly alleviated 7 weeks after eVLP administration. OHC survival improved significantly, and OHC-innervating neurite (connected to type II spiral ganglion neuronal body) loss was ameliorated. Finally, OHC membrane potential was hyperpolarized with eVLP gene editor treatment in Kcnq4-mutant mice, indicating that their OHCs were healthier and more stable than those of uninjected mice. Our findings suggest that eVLPs are feasible inner ear gene editor deliverers to treat hearing loss.},
}
@article {pmid40885667,
year = {2025},
author = {Liu, W and Duan, W and Peng, Z and Liao, Y and Wang, X and Liu, R and Jing, Q and Jiang, H and Fan, Y and Ge, L and Huang, L and Xing, Y},
title = {Highly efficient prime editors for mammalian genome editing based on porcine retrovirus reverse transcriptase.},
journal = {Trends in biotechnology},
volume = {43},
number = {12},
pages = {3253-3278},
doi = {10.1016/j.tibtech.2025.07.029},
pmid = {40885667},
issn = {1879-3096},
mesh = {Animals ; *Gene Editing/methods ; Swine ; *RNA-Directed DNA Polymerase/genetics/metabolism ; Humans ; *Endogenous Retroviruses/genetics/enzymology ; CRISPR-Cas Systems ; Swine, Miniature ; HEK293 Cells ; },
abstract = {Prime editing is a versatile and precise genome-editing tool. Most prime editors (PEs) rely on reverse transcriptase (RT) derived from Moloney murine leukemia virus (MMLV). Here, we established a PE, pvPE, using a RT derived from a porcine endogenous retrovirus (PERV) from a Bama mini-pig. Through various optimization strategies, including RT engineering, structural modifications, and La protein fusion, we gradually upgraded to pvPE-V4. This version achieved 24.38-101.69-fold higher efficiency compared with pvPE-V1 and up to 2.39-fold higher efficiency than another upgraded PE, PE7, with significantly fewer unintended edits across multiple mammalian cell lines. We further show that nocodazole (Noc) significantly enhanced pvPE efficiency by 2.25-fold on average. Using our pvPE system, we efficiently modified three genes simultaneously in porcine fibroblasts and subsequently generated cloned pigs that could serve as valuable models for Alzheimer's disease (AD) in humans. Our results highlight the broad application prospects of pvPE systems in mammalian genome editing.},
}
@article {pmid40878354,
year = {2025},
author = {Shan, S and Pisias, MT and Zhang, Z and Mavrodiev, EV and Gitzendanner, MA and Hauser, BA and Grover, CE and Barbazuk, WB and Soltis, PS and Yang, B and Soltis, DE},
title = {Development of an efficient CRISPR-mediated genome editing platform in the diploid-polyploid model system Tragopogon (Asteraceae).},
journal = {Journal of experimental botany},
volume = {76},
number = {22},
pages = {6700-6713},
doi = {10.1093/jxb/eraf380},
pmid = {40878354},
issn = {1460-2431},
support = {IOS-1923234//National Science Foundation/ ; DEB-2043478//National Science Foundation/ ; },
mesh = {*Gene Editing/methods ; *Polyploidy ; *CRISPR-Cas Systems ; *Tragopogon/genetics ; *Genome, Plant ; Diploidy ; Alcohol Oxidoreductases/genetics/metabolism ; },
abstract = {Polyploidy or whole-genome duplication (WGD) is a significant evolutionary force. However, the mechanisms governing polyploid genome evolution remain unclear, limited largely by a lack of functional analysis tools in organisms that best exemplify the earliest stages of WGD. Tragopogon (Asteraceae) includes an evolutionary model system for studying the immediate consequences of polyploidy. In this study, we significantly improved the transformation system and obtained genome-edited T. porrifolius (2x) and T. mirus (4x) primary generation (T0) individuals. Using CRISPR/Cas9, we knocked out the dihydroflavonol 4-reductase (DFR) gene, which controls anthocyanin synthesis, in both species. All transgenic allotetraploid T. mirus individuals had at least one mutant DFR allele, and 71.4% had all four DFR alleles edited. The resulting mutants lacked anthocyanin, and these mutations were inherited in the T1 generation. This study demonstrates a highly efficient CRISPR platform, producing genome-edited Tragopogon individuals that have completed the life cycle. The approaches used and challenges faced in building the CRISPR system in Tragopogon provide a framework for building similar systems in other non-genetic models. Genome editing in Tragopogon paves the way for novel functional biology studies of polyploid genome evolution and the consequences of WGD on complex traits, holding enormous potential for both basic and applied research.},
}
@article {pmid40744808,
year = {2025},
author = {Luo, G and Trinh, MDL and Falkenberg, MKD and Chiurazzi, MJ and Najafi, J and Nørrevang, AF and Correia, PMP and Palmgren, M},
title = {Unlocking in vitro transformation of recalcitrant plants.},
journal = {Trends in plant science},
volume = {30},
number = {12},
pages = {1306-1321},
doi = {10.1016/j.tplants.2025.07.001},
pmid = {40744808},
issn = {1878-4372},
mesh = {*Gene Editing/methods ; *Transformation, Genetic ; Plants, Genetically Modified/genetics ; CRISPR-Cas Systems ; *Plants/genetics ; },
abstract = {Genome editing offers powerful opportunities for crop improvement by enabling precise and targeted mutagenesis. Tools such as CRISPR-associated protein 9 and single-guide RNAs can be introduced into plant cells via in vitro transformation, which, despite the rise of in planta methods, remains an important method because it is highly effective when successful. However, transformation-induced stress is a critical and underexplored barrier to successful in vitro transformation, especially in recalcitrant plants. In this opinion article, we discuss in vitro methods for transforming recalcitrant plants, the challenges encountered, and potential solutions through the conceptual lens of stress biology. Reducing cellular stress, transiently weakening the immune response and optimizing regeneration protocols may be essential for expanding the transformation capacity across a broader range of plant species.},
}
@article {pmid41328758,
year = {2026},
author = {Plewnia, A and Hoenig, BD and Lötters, S and Heine, C and Erens, J and Böning, P and Bending, GD and Krehenwinkel, H and Williams, MA},
title = {The Emergence of a CRISPR-Cas Revolution in Ecology: Applications, Challenges, and an Ecologist's Overview of the Toolbox.},
journal = {Molecular ecology resources},
volume = {26},
number = {1},
pages = {e70086},
pmid = {41328758},
issn = {1755-0998},
support = {//University of Warwick/ ; NE/S010270/1//Natural Environment Research Council/ ; },
mesh = {*CRISPR-Cas Systems ; *Ecology/methods ; *Gene Editing/methods ; },
abstract = {CRISPR-Cas (Clustered Regularly Interspaced Short Palindromic Repeats-CRISPR-associated nucleases) systems allow researchers to detect, capture, and even alter parts of an organism's genome. However, while the use of CRISPR-Cas has revolutionised many fields in the life sciences, its full potential remains underutilised in ecology and biodiversity research. Here we outline the emerging applications of CRISPR-Cas in ecological contexts, focusing on three main areas: nucleic acid detection, CRISPR-enhanced sequencing, and genome editing. CRISPR-based nucleic acid detection of environmental DNA samples is already reshaping species monitoring, providing highly sensitive and non-invasive tools for both scientists and the public alike, with reduced costs and minimal experience required. Further, CRISPR-enhanced sequencing, including Cas-mediated target enrichment, enables efficient recovery of ecologically relevant loci and supports diverse applications such as amplification-free metagenomics. Finally, while genome editing on wild species remains largely theoretical in ecology, these tools are already being used in controlled settings to study adaptation and resilience in the face of ongoing global stressors. Together, the applications of CRISPR-Cas are paving the way for more affordable, accessible, and impactful applications for species conservation, and promise to improve our ability to tackle the ongoing global biodiversity crisis.},
}
@article {pmid41328592,
year = {2025},
author = {Geng, Y and Jiang, C and Zhang, H and Yang, H and Peng, Y and Chen, Y and Hu, C and Liu, H and Li, S and Chen, H and Xie, S and Guo, A},
title = {Genome-scale CRISPR screen identifies host factors associated with bovine parainfluenza virus 3 infection.},
journal = {Virulence},
volume = {16},
number = {1},
pages = {2589554},
doi = {10.1080/21505594.2025.2589554},
pmid = {41328592},
issn = {2150-5608},
mesh = {Animals ; Cattle ; *Parainfluenza Virus 3, Bovine/physiology/genetics ; CRISPR-Cas Systems ; Virus Replication ; *Host-Pathogen Interactions/genetics ; Cell Line ; *Respirovirus Infections/virology/veterinary/genetics ; Wnt-5a Protein/genetics/metabolism ; *Cattle Diseases/virology ; Virus Internalization ; Gene Knockout Techniques ; },
abstract = {Bovine parainfluenza virus type 3 (BPIV-3) is a major pathogen associated with the bovine respiratory disease complex. However, the limited understanding of host factors crucial for BPIV-3 replication has hindered the development of effective preventive and therapeutic strategies. To tackle this critical issue, we constructed a bovine genome-wide CRISPR/Cas9 knockout library in Madin-Darby bovine kidney cells, which was then used to systematically identify and characterize the host genes essential for BPIV-3a replication. Subsequently, 10 genes were validated using both RT-qPCR and viral titration assays. Furthermore, through gene knockout or knockdown and rescue experiments, we identified three key genes required for BPIV-3a replication: Wnt family member 5A (WNT5A), solute carrier family 16 member 13 (SLC16A13), and selenoprotein N (SELENON). However, their effects on viral adhesion and internalization varied. WNT5A was involved in both processes, SLC16A13 participated solely in internalization, while SELENON had no significant impact on either. Beyond BPIV-3a, these three genes were also found to be essential for the infection of BPIV-3c and Bovine enterovirus. In conclusion, this study offers novel insights into the molecular mechanisms governing the replication and pathogenesis of BPIV-3a, BPIV-3c, and bovine enterovirus within host cells, thereby providing a foundation for identifying potential targets in the development of novel antiviral strategies.},
}
@article {pmid41328409,
year = {2026},
author = {Zhang, H and Song, Y and Liu, W and Zheng, X and An, X and Li, C and Chen, W and Wang, H and Zhang, Y},
title = {Defect-complementation homologous recombination: A novel strategy for precise genome engineering of virulent phages.},
journal = {Synthetic and systems biotechnology},
volume = {12},
number = {},
pages = {59-70},
pmid = {41328409},
issn = {2405-805X},
abstract = {Engineered bacteriophages (phages) have been developed to overcome the limitations of natural phage therapies and serve as precision-targeted agents against drug-resistant bacterial infections. However, their application has been constrained by the low efficiency of existing genome-editing tools, largely because of the absence of effective selection markers. This study proposed a novel strategy, termed defect-complementation homologous recombination (DCHR), for precise phage genome editing. In this approach, CRISPR-Cas9 cleaves a donor plasmid in host cells to release a linear donor template carrying homology arms, an essential phage gene used as a selection marker, and two lox sites. The donor template undergoes homologous recombination with the genome of essential gene-deficient phage, thereby enabling targeted genome modifications. Using DCHR, we successfully generated large genomic deletions (1.48-kb gp0.4-0.7 and 1.02-kb gp4.3-4.7), achieved gene insertion (3.08-kb lacZ), and introduced a single-base substitution (TGA to TAA) in the stop codon of gp9 within the same T7 phage genome, all with 100 % accuracy. The significant advantages of DCHR are as follows: (i) High-efficiency screening: Only progeny phages derived from successful homologous recombination retain viability and replicative capacity, thereby greatly simplifying recombinant isolation. (ii) Editing flexibility: Unlike CRISPR-Cas systems, DCHR cannot be constrained by protospacer adjacent motif dependence and allows modifications across diverse genomic loci. (iii) High recombination efficiency: DCHR can achieve a recombinant phage titer of 3.1 × 10[5] PFU mL[-1] (plaque-forming units per mL) without relying on exogenous homologous recombination systems. In summary, DCHR demonstrates potential as a precise and efficient general genome-editing tool that facilitates design of engineered phages and advances functional genomic studies.},
}
@article {pmid41328347,
year = {2026},
author = {Li, Q and Bao, Q and Zhao, S and Wu, F and Li, Y and Wang, K and Li, W and Gao, H},
title = {Advancements in CRISPR-based therapies for ocular pathologies: from disease mechanisms to intervention strategies.},
journal = {Theranostics},
volume = {16},
number = {1},
pages = {156-192},
pmid = {41328347},
issn = {1838-7640},
mesh = {Humans ; *Genetic Therapy/methods ; *Eye Diseases/therapy/genetics ; *Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; Animals ; },
abstract = {Eye diseases caused by genetic mutations affect over 2.2 billion people worldwide. The development of CRISPR technology has opened exciting possibilities for how we diagnose and treat these conditions. However, designing effective CRISPR systems, managing potential risks, and considering the ethical questions around gene therapy in clinical practice are major challenges. To move forward successfully, it's important to evaluate how practical CRISPR-based treatments are for eye diseases from a clinical perspective, while also understanding how CRISPR systems work. In this review, we start by covering the basic principles behind CRISPR technology and explore its different types. Next, we look at various ways CRISPR is being used in eye research and treatments, from early studies to new clinical approaches. Lastly, we address the regulatory environment and ethical issues involved, discussing existing rules, safety concerns, and guidelines for genetic modifications in medical settings. Our goal is to share new insights into innovative treatments for eye diseases and to support the safe use of CRISPR in clinical eye care. This review aims to be a helpful resource for researchers, doctors, and regulators working on CRISPR-based therapies.},
}
@article {pmid41326076,
year = {2026},
author = {Niu, RC and Zeng, QH and Wang, WJ and Hu, J and Liu, TX and Zhang, SZ},
title = {Multi-omics analyses identify the modulators COX6A1 and NAL as regulators of silk cocoon formation in Plutella xylostella.},
journal = {Pesticide biochemistry and physiology},
volume = {216},
number = {Pt 1},
pages = {106764},
doi = {10.1016/j.pestbp.2025.106764},
pmid = {41326076},
issn = {1095-9939},
mesh = {Animals ; *Moths/genetics/metabolism/growth &amp; development ; *Silk/biosynthesis/genetics/metabolism ; *Insect Proteins/genetics/metabolism ; Larva/metabolism/genetics/growth &amp; development ; Proteomics ; CRISPR-Cas Systems ; *Electron Transport Complex IV/genetics/metabolism ; Transcriptome ; Multiomics ; },
abstract = {The diamondback moth (DBM) is a major global pest of cruciferous crops. Silk production, essential for DBM larval locomotion and pupal attachment, is governed by fibroin heavy chain (FibH), fibroin light chain (FibL), and fibrohexamerin (P25). However, the regulatory mechanisms and downstream key genes involved in silk production in DBM remain poorly understood. To address this, we integrated transcriptomic and proteomic data from CRISPR/Cas9 generated PxFibH, PxFibL, and PxP25 mutants to investigate the impact of silk gene deletions in the silk gland and identify modulators of silk formation. In the transcriptomic analysis, we identified 1994, 913, and 1266 differentially expressed genes (DEGs) in the three mutant strains, respectively. GO and KEGG enrichment analysis revealed significant involvement in pathways such as oxidation-reduction process, transmembrane transport, enzyme activity, and extracellular matrix (ECM) receptor interaction. At the proteomic level, 604, 210, and 266 differentially expressed proteins (DEPs) were identified in the three mutants, respectively. GO and KEGG enrichment analysis of these DEPs consistently highlighted energy metabolism, hydrolase activity, and catalytic activity pathways. Integrated multi-omics analyses identified three conserved regulator genes: cytochrome c oxidase subunit 6A1 (COX6A1), N-acetylneuraminate lyase (NAL), and protein phosphatase 1 regulatory subunit 14B (PPP1R14B). CRISPR/Cas9 knockout of PxCOX6A1 resulted in incomplete cocoon formation, along with increased larval mortality, prolonged development, and reduced oviposition. PxNAL knockout was lethal, while heterozygotes exhibited decreased cocoon formation, pupal weight, and fecundity. This study reveals FibH/FibL/P25-dependent metabolic networks regulating silk production and identifies COX6A1 and NAL as novel targets for environmentally sustainable pest control strategies.},
}
@article {pmid41325825,
year = {2025},
author = {Zhang, Y and Deng, S},
title = {Geminivirus vectors: From gene silencing to synthetic biology.},
journal = {Biotechnology advances},
volume = {87},
number = {},
pages = {108771},
doi = {10.1016/j.biotechadv.2025.108771},
pmid = {41325825},
issn = {1873-1899},
abstract = {Geminiviruses, the largest plant DNA virus family, cause devastating diseases in crops worldwide. These viruses possess distinctive features, such as the stem-loop structure and replication protein (Rep), which enable the creation of functional geminiviral replicons (GVRs) in plants. Over three decades, geminiviruses have been developed into vectors for virus-induced gene silencing (VIGS), high-level protein expression, and genome editing. This review introduces the genomic structure, Rep protein domains and functions, as well as the historical applications of geminiviruses, then highlights their prominent roles in VIGS and synthetic biology. As VIGS vectors, bipartite geminiviruses utilize AV1 gene replacement, while monopartite species rely on satellite DNAs to insert target sequences, enabling gene silencing in diverse plants. In synthetic biology, GVRs facilitate high-level protein expression through autonomous replication and enhance CRISPR/Cas genome editing efficiency in crops. Additionally, gene regulatory elements, including tissue-specific promoters and gene expression enhancement sequences from geminiviral genomes or satellite DNA expand their utility in genetic engineering. Finally, this review provides an outlook on the future development of geminivirus vectors. GVRs can work as plasmid-like DNAs for supporting diverse and creative designs in plant synthetic biology. The stem-loop structure and Rep are not unique to geminiviruses, a fact that suggests potential cross-kingdom applications of GVRs beyond plants. Vast viral resources enable further acceleration of GVR applications through resource mining and optimization. Moreover, attenuated or engineered geminiviral strains hold promise as "plant vaccines" via cross-protection. Collectively, geminivirus vectors bridge fundamental viral research with practical innovations in crop improvement, biomanufacturing, and synthetic biology.},
}
@article {pmid41261131,
year = {2025},
author = {Pierce, SE and Erwood, S and Oye, K and An, M and Krasnow, N and Zhang, E and Raguram, A and Seelig, D and Osborn, MJ and Liu, DR},
title = {Prime editing-installed suppressor tRNAs for disease-agnostic genome editing.},
journal = {Nature},
volume = {648},
number = {8092},
pages = {191-202},
pmid = {41261131},
issn = {1476-4687},
mesh = {Humans ; *Gene Editing/methods ; Animals ; *RNA, Transfer/genetics/metabolism ; Mice ; Codon, Nonsense/genetics ; Cystic Fibrosis/genetics/therapy ; Codon, Terminator/genetics ; Female ; Male ; CRISPR-Cas Systems/genetics ; Disease Models, Animal ; Cystic Fibrosis Transmembrane Conductance Regulator/genetics ; HEK293 Cells ; },
abstract = {Precise genome-editing technologies such as base editing[1,2] and prime editing[3] can correct most pathogenic gene variants, but their widespread clinical application is impeded by the need to develop new therapeutic agents for each mutation. For diseases that are caused by premature stop codons, suppressor tRNAs (sup-tRNAs) offer a more general strategy. Existing approaches to use sup-tRNAs therapeutically, however, require lifelong administration[4,5] or show modest potency, necessitating potentially toxic overexpression. Here we present prime editing-mediated readthrough of premature termination codons (PERT), a strategy to rescue nonsense mutations in a disease-agnostic manner by using prime editing to permanently convert a dispensable endogenous tRNA into an optimized sup-tRNA. Iterative screening of thousands of variants of all 418 human tRNAs identified tRNAs with the strongest sup-tRNA potential. We optimized prime editing agents to install an engineered sup-tRNA at a single genomic locus without overexpression and observed efficient readthrough of premature termination codons and protein rescue in human cell models of Batten disease, Tay-Sachs disease and cystic fibrosis. In vivo delivery of a single prime editor that converts an endogenous mouse tRNA into a sup-tRNA extensively rescued disease pathology in a model of Hurler syndrome. PERT did not induce detected readthrough of natural stop codons or cause significant transcriptomic or proteomic changes. Our findings suggest the potential of disease-agnostic therapeutic genome-editing approaches that require only a single composition of matter to treat diverse genetic diseases.},
}
@article {pmid41201289,
year = {2025},
author = {Liu, T and Su, C and Yan, X and Zhang, Y and Wang, Y and Duan, K},
title = {Creating Hypoallergenic and Low Antinutritional Soybeans through CRISPR/Cas12a-Mediated Multiplex Gene Editing.},
journal = {Journal of agricultural and food chemistry},
volume = {73},
number = {48},
pages = {30922-30932},
doi = {10.1021/acs.jafc.5c08935},
pmid = {41201289},
issn = {1520-5118},
mesh = {*Glycine max/genetics/immunology/chemistry/metabolism ; Animals ; *Gene Editing ; *CRISPR-Cas Systems ; Mice ; *Soybean Proteins/genetics/immunology ; *Plants, Genetically Modified/genetics/immunology/metabolism/chemistry ; *Allergens/immunology/genetics ; Humans ; Food Hypersensitivity/immunology ; Female ; },
abstract = {Soybean protein is a valuable plant-based nutrition source for food. However, allergens and antinutritional factors like Gly Bd 30k (P34), Kunitz trypsin inhibitor (KTI), and soybean agglutinin (LE) pose risks. In this study, we employed the CRISPR/Cas12a system to generate hypoallergenic and low antinutritional soybean lines without agronomic traits penalties. The edited lines exhibited markedly reduced levels and enzymatic activity of trypsin inhibitors, as well as soybean agglutinin content. Feeding mice with these edited soybeans, deficient in P34, KTI, and LE proteins, significantly alleviated allergenic responses. This research provides a promising resource of hypoallergenic and low antinutritional soybeans for applications in food production, animal feed, and immunotherapy.},
}
@article {pmid40715744,
year = {2025},
author = {Zhang, K and Yuan, X and Lu, S and Shu, Y and Wang, C and Cen, J and Wu, B and Hui, L},
title = {Expansion of human hepatocytes and their application in three-dimensional culture and genetic manipulation.},
journal = {Nature protocols},
volume = {20},
number = {12},
pages = {3722-3754},
pmid = {40715744},
issn = {1750-2799},
support = {92168202//National Natural Science Foundation of China (National Science Foundation of China)/ ; 32370793//National Natural Science Foundation of China (National Science Foundation of China)/ ; 32221002//National Natural Science Foundation of China (National Science Foundation of China)/ ; },
mesh = {Humans ; *Hepatocytes/cytology/metabolism ; CRISPR-Cas Systems ; Gene Editing/methods ; *Cell Culture Techniques, Three Dimensional/methods ; Lentivirus/genetics ; *Cell Culture Techniques/methods ; },
abstract = {Hepatocytes are one of the most important cell types in the liver, carrying out key functions. They are essential for hepatocyte-based therapy, disease modeling and drug development. However, the availability of primary human hepatocytes (PHHs) is limited by a shortage of donors. It is therefore of great value to expand PHHs in large quantities. Here we provide a detailed protocol for the large-scale expansion of PHHs (proliferating human hepatocytes, ProliHHs) derived from healthy donors and patients with inherited liver diseases, which can be rematured in a three-dimensional culture system. Moreover, we provide a protocol for the genetic manipulation of ProliHHs, including lentivirus transduction and CRISPR-Cas9-mediated knockout and knock-in. The protocol described here will help to realize the full potential of ProliHH-based therapy, organoid-based liver disease modeling and drug screening. The protocol to expand PHHs takes ~1-2 months, the protocol to establish the 3D-cultured ProliHHs takes ~8 d and the protocol to perform gene editing takes ~3 d. Personnel with basic scientific training can conduct these protocols.},
}
@article {pmid40715743,
year = {2025},
author = {Guruprasad, P and Ramasubramanian, R and Nason, S and Carturan, A and Liu, S and Paruzzo, L and Hornet, V and Plesset, J and Patel, RP and Bhoj, V and Beatty, GL and Ruella, M},
title = {Manufacturing of CRISPR-edited primary mouse CAR T cells for cancer immunotherapy.},
journal = {Nature protocols},
volume = {20},
number = {12},
pages = {3629-3654},
pmid = {40715743},
issn = {1750-2799},
support = {R00CA212302//U.S. Department of Health &amp; Human Services | National Institutes of Health (NIH)/ ; R01-37-CA262362-03//U.S. Department of Health &amp; Human Services | National Institutes of Health (NIH)/ ; },
mesh = {Animals ; Mice ; *Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; *Receptors, Chimeric Antigen/genetics/immunology ; *T-Lymphocytes/immunology/metabolism ; *Neoplasms/therapy/immunology ; *Immunotherapy, Adoptive/methods ; Humans ; Tumor Microenvironment/immunology ; },
abstract = {Editing chimeric antigen receptor (CAR) T cells by using CRISPR-Cas9 has become a routine strategy to improve their antitumor function or safety profile. Xenograft tumor models in immunodeficient mice are often used to evaluate the function of CRISPR-edited human CAR T cells. These models, however, lack functional immune systems and thus fail to recapitulate barriers such as the immunosuppressive tumor microenvironment (TME) that CAR T cells will encounter in patients. Thus, genetically modifying mouse CAR T cells for use in immune-intact models is an attractive approach to explore the impact of a given gene deletion on CAR T cells within a natural TME. Here, we describe a protocol to perform CRISPR-Cas9 editing in primary mouse T cells, thereby enabling studies of gene-edited CAR T within the TME and in the presence of a functional immune system. This protocol is integrated into a standard mouse CAR T manufacturing workflow, a process that typically spans ~5-6 days. The first stage of this protocol involves isolating mouse T cells, electroporating them with a ribonucleoprotein complex and activating them by using magnetic bead stimulation. The second stage involves transducing the CAR gene and expanding these cells, and the third stage focuses on validating knockout efficiency and the functionality of gene-edited mouse CAR T cells. This procedure requires a proficiency in aseptic cell culture techniques and a basic understanding of T cell biology. We anticipate that efficient and reliable genetic modification of mouse T cells will have wide-ranging applications for cancer immunotherapies and related fields.},
}
@article {pmid40640356,
year = {2025},
author = {Ma, Y and Xiong, Y and Xu, J and Xu, H and Fu, Z and Zhao, GR and Wu, Y and Yuan, YJ},
title = {Assembly and delivery of large DNA via chromosome elimination in yeast.},
journal = {Nature protocols},
volume = {20},
number = {12},
pages = {3755-3782},
pmid = {40640356},
issn = {1750-2799},
support = {[32471483]//National Natural Science Foundation of China (National Science Foundation of China)/ ; [23JCYBJC00220]//Natural Science Foundation of Tianjin City (Natural Science Foundation of Tianjin)/ ; },
mesh = {*Saccharomyces cerevisiae/genetics ; CRISPR-Cas Systems ; *Chromosomes, Fungal/genetics ; Haploidy ; *Gene Editing/methods ; RNA, Guide, CRISPR-Cas Systems/genetics ; *DNA/genetics ; *DNA, Fungal/genetics ; },
abstract = {Manipulation of large-scale genetic information provides a powerful approach to deciphering and engineering complex biological functions. However, the manipulation of large DNA, such as assembly and delivery, remains complex and difficult. Here we describe the experimental design strategy and protocol for a chromosome elimination-mediated large DNA assembly and delivery method (HAnDy), which enables efficient Mb-scale DNA assembly and delivery in yeast conveniently. This protocol is divided into three parts: (1) CRISPR-Cas9-mediated elimination of chromosome, which includes design and integration of a synthetic single-guide RNA (sgRNA) site near the centromere, activation of chromosome elimination by mating, and verification of the chromosome elimination. It can be used to eliminate multiple chromosomes, achieving haploidization in yeast. (2) Haploidization-mediated DNA assembly, which includes the design and construction of initial assembly strains, DNA assembly by programmed haploidization and verification of the assembled clones. (3) Haploidization-mediated DNA delivery, which includes the design and construction of inducible haploidization strains, DNA delivery by programmed haploidization and verification of the delivered clones. Users can utilize this protocol entirely or selectively depending on their needs. With the use of this protocol, it takes 10 d to achieve chromosome elimination and 7-11 d to achieve a standard cycle of haploidization-mediated DNA assembly or delivery. This protocol provides an efficient approach that is useful for the elimination, assembly and delivery of large DNA in yeast, requiring basic molecular biology skills.},
}
@article {pmid40541508,
year = {2025},
author = {Woodward, MJ and Dallaire, A and Paszkowski, U and Kokkoris, V},
title = {Is genetic manipulation of arbuscular mycorrhizal fungi possible?.},
journal = {Trends in microbiology},
volume = {33},
number = {12},
pages = {1331-1343},
doi = {10.1016/j.tim.2025.06.002},
pmid = {40541508},
issn = {1878-4380},
mesh = {*Mycorrhizae/genetics ; CRISPR-Cas Systems ; Transformation, Genetic ; Gene Editing/methods ; Protoplasts ; },
abstract = {Unlike many fungi, arbuscular mycorrhizal (AM) fungi have proven recalcitrant to genetic manipulation due to their obligate biotrophic lifestyle and multinucleate, coenocytic cellular structure. In this review, we examine past attempts at AM fungal transformation, we identify key biological and technical barriers and explore recent advances to overcome them. We focus on techniques never before applied in AM fungi, including CRISPR/Cas9, microinjection, and protoplast-based transformation, and we explore how they provide viable strategies for achieving this elusive goal. We conclude by outlining guidelines for future research, distinguishing between established approaches that are readily applicable to AM fungi and others that first require addressing key outstanding questions in AM fungal cell biology and genetics to ensure success.},
}
@article {pmid41325824,
year = {2025},
author = {Uddin, N and Ullah, MW and Zhu, D and Li, X and Yang, S and Xin, X},
title = {Engineering lignin pathway and plant cell wall modification and genome editing for advanced renewable bioenergy and material applications.},
journal = {Biotechnology advances},
volume = {},
number = {},
pages = {108772},
doi = {10.1016/j.biotechadv.2025.108772},
pmid = {41325824},
issn = {1873-1899},
abstract = {Lignin biosynthesis and plant cell wall engineering are central to plant structural integrity and biomass utility. Recent advances in molecular and synthetic biology have opened opportunities to tailor lignin contents, composition, and polymer structure for renewable bioenergy and sustainable biomaterial applications. This review provides an integrative perspective on biosynthesis, regulation, and engineering of lignin. It summarizes the current progress in understanding the genetic, transcriptional, epigenetic, and metabolic networks that control lignin formation, with a focus on emerging tools such as CRISPR/Cas genome editing, synthetic promoters, and metabolic rewiring. Beyond cataloguing current knowledge, it critically analyzes the trade-offs involved in lignin modification for biomaterials, addressing unresolved challenges such as monolignol transport, metabolic flux control, and species-specific regulatory divergence. Engineered lignin and modified plant cell walls hold significant potential for biorefineries, advanced polymers, pharmaceuticals, and carbon sequestration, yet their translation from the laboratory to the field remains limited. Engineered lignin offers real-world applications across diverse industries, including bioenergy, bioplastics, carbon fiber composites, pharmaceuticals, and sustainable construction materials, thereby reinforcing its pivotal role in advancing a circular bioeconomy. The review further proposes future research directions that integrate multi-omics, single-cell technologies, machine learning, and field-based validation to enable precision lignin engineering. Strategic advances in this field will support next-generation bioenergy systems, advanced biomaterials, and the transition to a circular bioeconomy.},
}
@article {pmid41325351,
year = {2025},
author = {Jansen van Vuuren, J and Matthews, MC and Robène, I and Rozsasi, S and Campa, M and Burger, J and Viljoen, A and Mostert, D},
title = {Combined Recombinase Polymerase Amplification CRISPR/Cas12a Assay for Detecting Fusarium oxysporum f. sp. cubense Tropical Race 4.},
journal = {Journal of visualized experiments : JoVE},
volume = {},
number = {225},
pages = {},
doi = {10.3791/68841},
pmid = {41325351},
issn = {1940-087X},
mesh = {*Fusarium/genetics/isolation &amp; purification ; *CRISPR-Cas Systems ; *Recombinases/genetics/chemistry/metabolism ; *Nucleic Acid Amplification Techniques/methods ; Plant Diseases/microbiology ; DNA, Fungal/genetics/analysis ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {Regular and accurate surveillance stands central to the efficient management of plant diseases. It can indicate which course of action is most appropriate, and whether prevention, eradication, or no action is required. Surveillance based on symptomology in host plants alone is often not reliable due to similarities in the symptoms caused by biotic and abiotic stresses. Laboratory-based molecular methods such as polymerase chain reaction (PCR) and quantitative (q)PCR are the most commonly and reliably used for plant pathogen detection, but rely on expensive equipment and skilled operators. Here, we describe a protocol combining a simplified DNA extraction, recombinase polymerase amplification (RPA), and clustered regularly interspaced short palindromic repeats (CRISPR)/Cas12a (RPA-Cas12a) for the detection of the invasive pathogen, Fusarium oxysporum f. sp. cubense tropical race 4 (Foc TR4). The technique provides a simple single-tube detection alternative that is analytically robust with improved specificity compared to available molecular detection assays and negates the need for expensive and sophisticated laboratory equipment.},
}
@article {pmid41324747,
year = {2025},
author = {Priyanka, SS and Iqbal, G and Nidarshan, NC and Kumari, K and Vanjre, S and Rasal, K and Sonwane, A and Brahmane, M and Goswami, M},
title = {Avenues of genome editing for color trait improvement in ornamental fishes: current status and future perspectives.},
journal = {Transgenic research},
volume = {34},
number = {1},
pages = {51},
pmid = {41324747},
issn = {1573-9368},
mesh = {Animals ; *Gene Editing/methods ; *Fishes/genetics/growth &amp; development ; *Pigmentation/genetics ; CRISPR-Cas Systems/genetics ; Animals, Genetically Modified/genetics/growth &amp; development ; },
abstract = {Ornamental fish industries are growing sectors contributing significantly to livelihood, trade and export, driven by the worldwide demand for colourful and unique species. Pigmentation is the focal point of the visual appeal of ornamental fish, market value, and species-specific interaction. The pigment cells are called chromatophores, derived from neural crest cells and controlled by sophisticated genetic mechanisms, conferring these fish with distinctive colours and patterns. Historically, selective breeding and dietary pigment supplementation have been applied to enhance colouration. Such traditional practices, however, are prone to disadvantages such as slow development, genetic ambiguity, and unforeseen consequences. With the invention of genome editing, such as CRISPR-Cas9, researchers now have a sensitive and powerful tool to control pigmentation traits at the genetic level. Central pigmentation gene manipulation, such as Tyr, Mc1r, and Slc45a2, can allow researchers to introduce stable and uniform color changes. Such enhancements confer unparalleled control of fish colour, a promising avenue in the ornamental fish industry. This review discusses the genetic nature of fish pigmentation. It reports recent advances in CRISPR-based modifications and describes their possible applications and implications for future ornamental fish breeding.},
}
@article {pmid41324601,
year = {2025},
author = {Bhattacharya, S and Goyal, K and Satpati, P},
title = {Thermodynamics of PAM Recognition by Cas9 of Streptococcus pyogenes.},
journal = {Journal of chemical information and modeling},
volume = {},
number = {},
pages = {},
doi = {10.1021/acs.jcim.5c01934},
pmid = {41324601},
issn = {1549-960X},
abstract = {The CRISPR/Cas9 system from Streptococcus pyogenes (SpCas9) requires a canonical 5'-NGG-3' PAM sequence in target DNA for effective genome editing. Base-specific interactions between the guanines (second and third position) and arginine dyad (R1333 and R1335) ensured specificity. We evaluated the PAM recognition strength of SpCas9 by using alchemical free energy calculations, revealing the energetics that influence genome editing accuracy. SpCas9 does not discriminate at the first position of the NGG sequence, but it penalizes mutations in the second and third positions. SpCas9 imposes a higher penalty for guanine mutation in the third PAM position compared to the second due to the greater conformational rigidity of R1335 in relation to R1333. Conformational rigidity of R1335 prevents side-chain readjustment for new protein-DNA interactions in noncanonical PAMs. A guanine-to-cytosine substitution in either the second or third position of canonical PAM disrupts direct protein-PAM interactions and leads to solvent exposure. This happens due to strong electrostatic repulsion between the arginine dyad's guanidinium groups and the amine group of cytosine. Interestingly, the strength of SpCas9 in disfavoring a single cytosine substitution (by >10 kcal/mol) is comparable to that of disfavoring double base substitutions in the NGG sequence. The ability of SpCas9 to differentiate between noncanonical and canonical PAMs (ΔΔG) is directly related to the number of direct interactions between SpCas9 and the PAM sequence, as well as the degree of solvent exposure. Loss of direct interactions and increased solvent exposure enhance ΔΔG. The calculated ΔΔG adequately explains the observed differences in DNA cleavage activity of SpCas9 across various DNA substrates with different PAM sequences. This study connects thermodynamics, structures, and activity to elucidate PAM selectivity in SpCas9 and may also apply to other CRISPR/Cas systems, offering valuable insights for the rational design of Cas9 variants with modified PAM specificities.},
}
@article {pmid41323291,
year = {2025},
author = {Li, Y and Hall-Ponselè, AM},
title = {Plant Cell Strain Improvement Through Engineering Biology for Industrial Plant Cell Culture.},
journal = {Engineering biology},
volume = {9},
number = {1},
pages = {e70002},
pmid = {41323291},
issn = {2398-6182},
abstract = {Plant cell culture (PCC) presents a promising and sustainable alternative to traditional agricultural methods for producing specialty bioactive compounds. However, its widespread industrial application has been hindered by challenges such as low yields, cell line instability and inconsistent product quality. engineering biology (EB) offers a powerful toolkit to overcome these limitations by systematically improving plant cell lines. This review focuses on the application of EB principles to enhance PCC for the production of high-value bioactives from an industry-oriented perspective. We explore three core pillars of the EB toolkit: (1) Multiomics and in silico design, which leverage comprehensive data integration and predictive modelling for rational target identification; (2) gene manipulation and pathway bioengineering, encompassing precise genome editing (e.g., CRISPR/Cas), synthetic gene circuits and directed evolution for targeted metabolic reprogramming and (3) biosensors for high-throughput screening and real-time monitoring, enabling rapid testing and optimisation of engineered cell lines. The synergistic integration of these tools within the iterative design-build-test-learn (DBTL) cycle is highlighted as a key strategy for accelerating strain improvement. Ultimately, the convergence of these EB approaches is transforming PCC into a robust platform for producing pharmaceuticals, functional foods and green chemicals, contributing to a biobased economy with a minimal ecological footprint.},
}
@article {pmid41321825,
year = {2025},
author = {Senthilraja, G and Sandhya, M and Priyadharshini, E and Anand, T and Kavitha, M and Tharmalingam, N},
title = {Targeting effector proteins of plant pathogens as a strategy for durable plant disease resistance.},
journal = {Frontiers in microbiology},
volume = {16},
number = {},
pages = {1681047},
pmid = {41321825},
issn = {1664-302X},
}
@article {pmid41317788,
year = {2025},
author = {Brogan, DJ and Lin, CP and Benetta, ED and Wang, T and Chen, F and Li, H and Lin, C and Komives, EA and Akbari, OS},
title = {Synthetic type III-E CRISPR-Cas effectors for programmable RNA-targeting.},
journal = {Journal of molecular biology},
volume = {},
number = {},
pages = {169566},
doi = {10.1016/j.jmb.2025.169566},
pmid = {41317788},
issn = {1089-8638},
abstract = {The recent discovery of the type III-E class of CRISPR-Cas effectors has reshaped our fundamental understanding of CRISPR-Cas evolution and classification. Type III-E effectors are composed of several Cas7-like domains and a single Cas11-like domain naturally fused together to create a single polypeptide capable of targeting and degrading RNA. Here we identified a novel type III-E-like effector composed of three Cas7 domains and a Cas1 domain which was not active but could be engineered into an active chimeric RNA-targeting Cas effector by domain additions and swaps from other type III-E effectors. The results reveal that various domains in type III-E effectors can be swapped for the equivalent domain from a different type III-E effector. Remarkably, the Cas1 domain located at the C-terminus of Cas7-1 could be swapped in place of the Cas11 domain located between the Cas7.1 and the Cas7.2 domains of DiCas7-11. The results reveal a new modality for engineering type III-E effectors from the blueprints found in nature.},
}
@article {pmid41315365,
year = {2025},
author = {Wang, Y and Liao, Y and Sun, Y and Mitra, B and Guo, R and Piedras, BI and White, S and Tang, HY and Asara, JM and Tempera, I and Lieberman, PM and Gewurz, BE},
title = {The CTLH ubiquitin ligase substrates ZMYND19 and MKLN1 negatively regulate mTORC1 at the lysosomal membrane.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {10731},
pmid = {41315365},
issn = {2041-1723},
support = {R01 AI164709/AI/NIAID NIH HHS/United States ; PF-24-1250090-01-IBCD//American Cancer Society (American Cancer Society, Inc.)/ ; P01CA269043//U.S. Department of Health &amp; Human Services | NIH | NCI | Division of Cancer Epidemiology and Genetics, National Cancer Institute (National Cancer Institute Division of Cancer Epidemiology and Genetics)/ ; P01 CA269043/CA/NCI NIH HHS/United States ; R01AI164709//Division of Intramural Research, National Institute of Allergy and Infectious Diseases (Division of Intramural Research of the NIAID)/ ; PF-23-1144614-01-IBCD//American Cancer Society (American Cancer Society, Inc.)/ ; PF-24-1194768-01-TBE//American Cancer Society (American Cancer Society, Inc.)/ ; PF-24-1308318-01-TBE//American Cancer Society (American Cancer Society, Inc.)/ ; R01 DE033907/DE/NIDCR NIH HHS/United States ; R00 DE031016/DE/NIDCR NIH HHS/United States ; },
mesh = {Humans ; *Lysosomes/metabolism ; *Mechanistic Target of Rapamycin Complex 1/metabolism/genetics ; *Ubiquitin-Protein Ligases/metabolism/genetics ; HEK293 Cells ; Cell Line, Tumor ; CRISPR-Cas Systems ; *Intracellular Membranes/metabolism ; Signal Transduction ; Phosphatidylinositol 3-Kinases/metabolism ; Cell Proliferation ; },
abstract = {Most Epstein-Barr virus-associated gastric carcinoma (EBVaGC) harbor non-silent mutations that activate phosphoinositide 3 kinase (PI3K) to drive downstream metabolic signaling. To gain insights into PI3K/mTOR pathway dysregulation in this context, we perform a human genome-wide CRISPR/Cas9 screen for hits that synergistically blocked EBVaGC proliferation together with the PI3K antagonist alpelisib. Multiple subunits of carboxy terminal to LisH (CTLH) E3 ligase, including the catalytic MAEA subunit, are among top screen hits. CTLH negatively regulates gluconeogenesis in yeast, but not in higher organisms. The CTLH substrates MKLN1 and ZMYND19, which highly accumulated upon MAEA knockout, associate with one another and with lysosome outer membranes to inhibit mTORC1. Rather than perturbing mTORC1 lysosomal recruitment, ZMYND19 and MKLN1 block the interaction between mTORC1 and Rheb and also with mTORC1 substrates S6 and 4E-BP1. Thus, CTLH enables cells to rapidly tune mTORC1 activity at the lysosomal membrane via the ubiquitin/proteasome pathway.},
}
@article {pmid41261109,
year = {2025},
author = {Zhang, T and Zheng, Y and Sheng, M and Wang, Q and Jin, Z and Li, Z and Chen, W and Huang, J and Yang, X},
title = {Electrochemical Biosensing Platform Based on MOF Carrier Signal Probes and CRISPR/Cas12a Cleavage for Sensitive Detection of MicroRNAs.},
journal = {Analytical chemistry},
volume = {97},
number = {47},
pages = {26265-26273},
doi = {10.1021/acs.analchem.5c05821},
pmid = {41261109},
issn = {1520-6882},
mesh = {*MicroRNAs/analysis ; *Metal-Organic Frameworks/chemistry ; *Biosensing Techniques/methods ; *Electrochemical Techniques/methods ; Humans ; *CRISPR-Cas Systems ; DNA, Single-Stranded/chemistry ; *CRISPR-Associated Proteins/metabolism ; *Endodeoxyribonucleases/metabolism ; Methylene Blue/chemistry ; Limit of Detection ; Bacterial Proteins ; },
abstract = {Dysregulation of microRNA (miRNA) expression is associated with a variety of human diseases, including cancers, and the development of highly sensitive miRNA detection is important for the early diagnosis of cancer. In this study, we developed an electrochemical biosensing platform for miRNA detection through the integration of a DNA-gated metal-organic framework (MOF) signal probe, duplex-specific nuclease (DSN)-assisted signal amplification, and a CRISPR/Cas12a system. The zirconium-based MOF UiO-66-NH2 was engineered as a nanocarrier for methylene blue (MB) entrapment, and single-stranded DNA (ssDNA) was capped on the MOF surface as a "gatekeeper" that allowed the controlled release of MB molecules, resulting in a DNA-gated MOF electrochemical signal probe. In the presence of target miRNA, target-initiated DSN cyclic amplification and digestion of the CRISPR/Cas12a initiator DNA sequence blocked the collateral cleavage activity of Cas12a toward ssDNA, thus preventing the release of MB and generating a high electrochemical signal. Conversely, the CRISPR/Cas12a system would be activated to cleave ssDNA in the absence of the target, leading to the release of a signal molecule and a low response. With the usage of the proposed biosensing strategy, sensitive detection of miRNA let-7a, a biomarker associated with nonsmall cell lung cancer, has been achieved. Therefore, this work expands the application scope of a MOF as a nanocarrier to prepare electrochemical signal probes and provides a valuable biosensing method for clinical diagnosis.},
}
@article {pmid41254269,
year = {2025},
author = {Hernández-Huertas, L and Moreno-Sánchez, I and Crespo-Cuadrado, J and Vargas-Baco, A and da Silva Pescador, G and Zhang, Y and Wen, Z and Florens, L and Santos-Pereira, JM and Bazzini, AA and Moreno-Mateos, MA},
title = {CRISPR-RfxCas13d screening uncovers Bckdk as a post-translational regulator of maternal-to-zygotic transition in teleosts.},
journal = {The EMBO journal},
volume = {44},
number = {23},
pages = {7021-7059},
pmid = {41254269},
issn = {1460-2075},
support = {RyC-2017-23041//MEC | Agencia Estatal de Investigaci&#xf3;n (AEI)/ ; PGC2018-097260-B-I00//MEC | Agencia Estatal de Investigaci&#xf3;n (AEI)/ ; PID2021-127535NB-I00//MEC | Agencia Estatal de Investigaci&#xf3;n (AEI)/ ; MDM-2016-0687//MEC | Agencia Estatal de Investigaci&#xf3;n (AEI)/ ; CEX2020-001088-M//MEC | Agencia Estatal de Investigaci&#xf3;n (AEI)/ ; CNS2022-135564//MEC | Agencia Estatal de Investigaci&#xf3;n (AEI)/ ; PRE2019-087721//Ministerio de Ciencia e Innovaci&#xf3;n (MCIN)/ ; PREDOC_01569//Junta de Andaluc&#xed;a (Andalusian Board)/ ; EMC21_00188//Junta de Andaluc&#xed;a (Andalusian Board)/ ; P20_00866//Junta de Andaluc&#xed;a (Andalusian Board)/ ; UPO-1380590//Junta de Andaluc&#xed;a (Andalusian Board)/ ; Margarita Salas Postdoctoral contract//EC | NextGenerationEU (NGEU)/ ; PRTR-C17.I1//EC | NextGenerationEU (NGEU)/ ; NIHR01GM136849//HHS | National Institutes of Health (NIH)/ ; NIH R21OD034161//HHS | National Institutes of Health (NIH)/ ; },
mesh = {Animals ; *Zebrafish/genetics/embryology/metabolism ; *Zygote/metabolism ; *Zebrafish Proteins/genetics/metabolism ; *Protein Processing, Post-Translational ; Gene Expression Regulation, Developmental ; CRISPR-Cas Systems ; Female ; Phosphorylation ; },
abstract = {The maternal-to-zygotic transition (MZT) is a reprograming process encompassing zygotic genome activation (ZGA) and the clearance of maternally-provided mRNAs. While some factors regulating MZT have been identified, there are thousands of maternal RNAs whose function has not been ascribed yet. Here, we have performed a proof-of-principle CRISPR-RfxCas13d maternal screen, in which we targeted mRNAs encoding kinases and phosphatases or proteins regulating them in zebrafish. This screen identified branched-chain ketoacid dehydrogenase kinase, Bckdk, as a novel post-translational regulator of MZT. Bckdk mRNA knockdown caused epiboly defects, ZGA deregulation, H3K27ac reduction and a partial impairment of miR-430 processing. Phospho-proteomic analysis revealed that Phf10/Baf45a, a chromatin remodeling factor, is less phosphorylated upon Bckdk depletion. Further, phf10 mRNA knockdown also altered ZGA, and expression of a phospho-mimetic mutant of Phf10 rescued the developmental defects observed after bckdk mRNA depletion, as well as restored H3K27ac levels. Altogether, our results demonstrate the competence of CRISPR-RfxCas13d screenings to uncover new regulators of early vertebrate development and shed light on the post-translational control of MZT mediated by protein phosphorylation.},
}
@article {pmid41219509,
year = {2025},
author = {Johnson, KA and Goswami, HN and Catchpole, RJ and Ahmadizadeh, F and Zhao, P and Wells, L and Li, H and Terns, MP},
title = {A phage-encoded anti-CRISPR protein co-opts host enolase to prevent type III CRISPR immunity.},
journal = {Nature microbiology},
volume = {10},
number = {12},
pages = {3162-3175},
pmid = {41219509},
issn = {2058-5276},
support = {R35GM118160//Foundation for the National Institutes of Health (Foundation for the National Institutes of Health, Inc.)/ ; GR000347//Foundation for the National Institutes of Health (Foundation for the National Institutes of Health, Inc.)/ ; 2400220//National Science Foundation (NSF)/ ; },
mesh = {*Phosphopyruvate Hydratase/metabolism/genetics ; *Streptococcus thermophilus/virology/immunology/genetics ; *Viral Proteins/metabolism/genetics/chemistry ; *CRISPR-Cas Systems ; *Streptococcus Phages/genetics/metabolism ; *Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) systems provide powerful adaptive immunity against phage infection. In response, phages use anti-CRISPR (Acr) proteins to evade CRISPR immunity. The few type III Acrs identified so far show conditional effectiveness in countering type III immunity or rely on unknown or poorly understood inhibitory mechanisms. Here we report the discovery of AcrIIIA2, a type III-A Acr encoded by Streptococcus thermophilus phages. Biochemical and structural analyses reveal that phage AcrIIIA2 co-opts host enolase, a highly abundant glycolysis enzyme, to form a ternary complex with the S. thermophilus type III-A (Csm) CRISPR ribonucleoprotein complex, obstructing its immune responses. The enolase-chaperoned AcrIIIA2 blocks the initial step of phage RNA binding, thereby preventing downstream type III anti-phage immune responses. Enolase participates in the anti-immune response by serving as an essential structural scaffold, stabilizing Acr-CRISPR interactions. These findings uncover a new anti-defence strategy that exploits a well-conserved host factor to block CRISPR immunity.},
}
@article {pmid41205968,
year = {2025},
author = {Zhang, Z and Chen, H and Tang, K and Xu, J and Jiang, L and Ning, Y},
title = {Triple-helix molecular switch-based aptasensor integrating exonuclease I-assisted target recycling and CRISPR-Cas13a-mediated signal amplification for fluorescence detection of adenosine triphosphate.},
journal = {International journal of biological macromolecules},
volume = {333},
number = {Pt 1},
pages = {148753},
doi = {10.1016/j.ijbiomac.2025.148753},
pmid = {41205968},
issn = {1879-0003},
mesh = {*Adenosine Triphosphate/analysis ; *Exodeoxyribonucleases/metabolism ; *Aptamers, Nucleotide/chemistry ; *Biosensing Techniques/methods ; *CRISPR-Cas Systems ; Methicillin-Resistant Staphylococcus aureus ; Graphite/chemistry ; Fluorescence ; Limit of Detection ; Spectrometry, Fluorescence ; },
abstract = {It is essential to develop new methods for detecting adenosine triphosphate (ATP), as it plays crucial roles in the regulating numerous biological processes and its levels are significantly correlated with various diseases. This study developed a fluorescent aptasensor for ATP detection utilizing a triple-helix molecular switch (THMS) based on exonuclease I (Exo I)-assisted target recycling and CRISPR-Cas13a-mediated signal amplification. Without the target, all carboxyfluorescein (fluorescence-active molecule; FAM)-labeled probes remain close to the graphene oxide (GO) substrate, resulting in the fluorescence quenching of FAM. In contrast, the target initiates THMS opening, which triggers the simultaneous target recycling and signal amplification. The iterative reactions lead to FAM accumulation, and the fluorescence can be assayed at excitation/emission wavelengths of 480/514 nm, respectively. The target was detected at concentrations from 1 fM to 1 nM with a detection limit of 0.54 fM. The aptasensor showed satisfactory performance for evaluating ATP in methicillin-resistant Staphylococcus aureus and studying drug action mechanisms, highlighting its potential for clinical diagnostics and screening antibacterial agents.},
}
@article {pmid41174223,
year = {2025},
author = {Liao, Y and Yan, J and Kong, IY and Li, Z and Ding, W and Clark, S and Maestri, D and Yoshida, T and Giulino-Roth, L and Gewurz, BE},
title = {Lysine-specific histone demethylase complex restricts Epstein-Barr virus lytic reactivation.},
journal = {Nature microbiology},
volume = {10},
number = {12},
pages = {3290-3304},
pmid = {41174223},
issn = {2058-5276},
support = {R01 AI164709/AI/NIAID NIH HHS/United States ; U01CA275301//Foundation for the National Institutes of Health (Foundation for the National Institutes of Health, Inc.)/ ; R01DE033907//Foundation for the National Institutes of Health (Foundation for the National Institutes of Health, Inc.)/ ; R01 DE033907/DE/NIDCR NIH HHS/United States ; U01 CA275301/CA/NCI NIH HHS/United States ; P01CA269043//Foundation for the National Institutes of Health (Foundation for the National Institutes of Health, Inc.)/ ; R01CA228700//Foundation for the National Institutes of Health (Foundation for the National Institutes of Health, Inc.)/ ; R01 CA228700/CA/NCI NIH HHS/United States ; R01AI164709//Foundation for the National Institutes of Health (Foundation for the National Institutes of Health, Inc.)/ ; P01 CA269043/CA/NCI NIH HHS/United States ; },
mesh = {*Herpesvirus 4, Human/physiology/genetics ; Humans ; *Histone Demethylases/metabolism/genetics ; *Virus Activation ; Animals ; Virus Latency ; Mice ; Epstein-Barr Virus Infections/virology ; Co-Repressor Proteins/metabolism/genetics ; Histones/metabolism ; Lysine/metabolism ; Trans-Activators/metabolism/genetics ; Cell Line, Tumor ; Methylation ; Burkitt Lymphoma/virology ; CRISPR-Cas Systems ; Host-Pathogen Interactions ; Nerve Tissue Proteins ; },
abstract = {Epstein-Barr virus (EBV) infects >95% of adults and contributes to several human cancers. EBV can remain latent where viral lytic genes are silenced, precluding the use of antiviral agents such as ganciclovir. Little is known about the host factors involved in EBV latency. Here we performed a human genome-wide CRISPR-Cas9 screen in Burkitt lymphoma B cells, which identified lysine-specific histone demethylase 1 (LSD1) and its corepressors REST corepressor 1 (CoREST) and zinc finger protein 217 (ZNF217) as critical for EBV latency. Gene knockout or LSD1 inhibition triggered EBV reactivation, and the latter sensitized cells to ganciclovir cytotoxicity, including in murine tumour xenografts. Mechanistically, ZNF217 recruits LSD1 and CoREST to form a complex that binds a specific DNA motif associated with regions implicated in EBV reactivation. It removes histone 3 lysine 4 (H3K4) methylation marks and restricts host DNA looping. Alternatively, the H3K4 lysine methyltransferase 2D supports EBV lytic reactivation. Our results highlight H3K4 methylation as a major EBV lytic switch regulator and therapeutic target.},
}
@article {pmid40992203,
year = {2025},
author = {Xu, Y and Wang, L and Jiang, J and Zhao, G and Wang, Z},
title = {Knockdown of argininosuccinate lyase influences the growth of Mycolicibacterium smegmatis in vitro and in vivo.},
journal = {Tuberculosis (Edinburgh, Scotland)},
volume = {155},
number = {},
pages = {102693},
doi = {10.1016/j.tube.2025.102693},
pmid = {40992203},
issn = {1873-281X},
mesh = {Animals ; *Mycobacterium smegmatis/growth &amp; development/drug effects/enzymology/genetics ; *Argininosuccinate Lyase/genetics/metabolism ; Gene Knockdown Techniques ; *Mycobacterium Infections, Nontuberculous/microbiology/genetics/drug therapy/enzymology ; Disease Models, Animal ; *Bacterial Proteins/genetics/metabolism ; Arginine/pharmacology/metabolism ; Liver/microbiology ; Antitubercular Agents/pharmacology ; CRISPR-Cas Systems ; Kidney/microbiology ; },
abstract = {The rising prevalence of drug-resistant tuberculosis (DR-TB), coupled with stagnation in the development of novel therapeutics, underscores the urgent need for new drug targets and innovative anti-tuberculosis agents. In this study, we demonstrate that CRISPR interference-mediated knockdown of argH, a nitrogen metabolism-associated gene encoding argininosuccinate lyase, significantly impairs the growth of Mycolicibacterium smegmatis (formerly Mycobacterium smegmatis). This growth defect was alleviated in a concentration-dependent manner by arginine supplementation. In a goldfish infection model, argH knockdown led to a marked reduction in bacterial burden within both liver and kidney tissues. Notably, bacitracin and 5-fluorouracil exhibited synergistic effects when combined with argH knockdown. Metabolomic profiling revealed significant perturbations in multiple amino acids, as well as in succinyl-CoA and lactate levels, suggesting that suppression of argH impairs M. smegmatis proliferation by disrupting amino acid homeostasis and interfering with aerobic respiration.},
}
@article {pmid41315225,
year = {2025},
author = {Wen, HP and Yu, C and Bi, S and Jiang, LH and Wang, ZG and Yao, Z and Pang, DW and Liu, SL},
title = {Programmable targeted RNA degradation via dCas13d-directed chaperone-mediated autophagy (dCasCMA).},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {10738},
pmid = {41315225},
issn = {2041-1723},
support = {22293032//National Natural Science Foundation of China (National Science Foundation of China)/ ; 22374138//National Natural Science Foundation of China (National Science Foundation of China)/ ; 21977054//National Natural Science Foundation of China (National Science Foundation of China)/ ; 24JCZDJC01240//Natural Science Foundation of Tianjin City (Natural Science Foundation of Tianjin)/ ; 23JCYBJC01880//Natural Science Foundation of Tianjin City (Natural Science Foundation of Tianjin)/ ; },
mesh = {Humans ; Animals ; *Chaperone-Mediated Autophagy/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics/metabolism ; *RNA Stability/genetics ; *CRISPR-Cas Systems/genetics ; Mice ; HEK293 Cells ; Autophagy ; },
abstract = {RNA-targeted degradation technologies offer significant promise for treating diseases by selectively disrupting gene expression. However, a robust method to specifically, efficiently, and programmability degrade targeted RNAs in mammalian cells is still in demand. Here, we present a versatile platform, dCas13d-directed chaperone-mediated autophagy (dCasCMA), which integrates the precise targeting capabilities of dCas13/CRISPR with the degradation efficiency of chaperone-mediated autophagy (CMA) to achieve efficient degradation of specific RNAs. By combining dCas13d with a CMA-targeting motif and customizable guide RNA (gRNA), the platform allows for accurate targeting of both exogenous and endogenous RNAs in cells. Moreover, the incorporation of multiplexed gRNA expression arrays enables the simultaneous degradation of multiple RNA targets during viral pathogenesis in live cells and in vivo. Our findings emphasize the platform's modular design, which enables flexible combinations of dCCTM components with user-defined gRNA sequences. This versatility positions it as a promising tool for developing innovative therapies for various diseases.},
}
@article {pmid41315077,
year = {2025},
author = {Loedige, KW and White, AL and McMurrough, TA and Stead, BE and Edgell, DR},
title = {A buffer-tuning strategy to profile domain-specific activity of chimeric I-TevI/CRISPR gene editors in vitro.},
journal = {Scientific reports},
volume = {15},
number = {1},
pages = {42742},
pmid = {41315077},
issn = {2045-2322},
support = {RGPIN-2022-05459//Natural Sciences and Engineering Research Council of Canada/ ; RGPIN-2022-05459//Natural Sciences and Engineering Research Council of Canada/ ; ALLRP 571374 - 21//Mitacs/ ; ALLRP 571374 - 21//Mitacs/ ; },
mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems ; Buffers ; RNA, Guide, CRISPR-Cas Systems/genetics ; Protein Domains ; DNA Cleavage ; *Endodeoxyribonucleases/metabolism/genetics ; Humans ; DNA/metabolism ; },
abstract = {Protein-DNA interactions can be manipulated in vitro by changing buffer conditions. Here, we develop a methodology to map the cleavage preferences of chimeric gene editors that are fusions of the I-TevI nuclease domain to CRIPSR nucleases by manipulating in vitro salt concentrations. We found that DNA cleavage by the I-TevI (Tev) nuclease domain at CNNNG sites was de-coupled from the gRNA-targeted site in low salt buffers. For TevCas12a, this non-targeted cleavage activity was enriched at Tev CNNNG cleavage motifs optimally positioned within a 30-bp window upstream of a Cas12a TTTV PAM site. Non-targeted cleavage did not require Cas12a nuclease activity or specific Cas12a gRNA targeting. Similar non-targeted products were observed in low salt buffer conditions for TevSaCas9, Tev-meganuclease and Tev-zinc finger editors. Cas12a and SaCas9 activity at gRNA-directed sites and sites with multiple mismatches were also sensitive to buffer salt concentration. Oxford Nanopore sequencing revealed a remarkably similar Tev CNNNG cleavage preference at different salt concentrations and in different fusion contexts, emphasizing the robustness and specificity of Tev activity. More generally, our work highlights the sensitivity of gene editors to in vitro reaction conditions and how these conditions can be leveraged to functionally dissect the activity of individual domains of chimeric gene editors.},
}
@article {pmid41314912,
year = {2025},
author = {Chen, L and Ouyang, W and Hu, Y and Peng, L and Chen, P and Guo, W and Yang, H and Xu, J and Pan, M and Xu, D and Wang, X and Zhang, C and Chen, S and Hao, Q and Yuan, S and Huang, Y and Shan, Z and Yang, Z and Xia, R and Hewezi, T and Chen, H and Tran, LP and Zhou, X and Cao, D},
title = {Creating artificial miR2118a/b to boost yield and broad-spectrum resistance in soybean via CRISPR/Cas9-targeted mutation.},
journal = {Trends in biotechnology},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.tibtech.2025.10.022},
pmid = {41314912},
issn = {1879-3096},
abstract = {While regulatory functions of mature miRNAs are well established, the functions of miRNAs* and their potential for genetic engineering in crop improvement remain underexplored. Here, we used clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas9) to generate artificial miR2118a/b (amiR2118a/b) by editing miR2118a/b-5p and obtained several amir2118a/b mutants in soybean (Glycine max). miR2118a/b-5p modifications altered the secondary structure of precursor amiR2118a/b (pre-amiR2118a/b) and reduced mature miR2118a/b levels. These amir2118a/b mutants retained the ability to initiate biogenesis of phased small interfering RNAs (phasiRNAs), albeit with a reduced abundance compared with wild-type (WT) plants. Furthermore, these mutants upregulated the expression of genes related to growth and defense under normal and Pseudomonas syringae pv. glycinea (Psg)-infected conditions, respectively. Notably, two transgene-free amir2118 mutants exhibited enhanced resistance to Psg, soybean cyst nematode (SCN), and root-knot nematode (RKN), and achieved increased yield under pathogen-free field conditions. This study provides a strategy to generate artificial miRNAs (amiRNAs) for crop improvement through the CRISPR/Cas system by mutating miRNAs* in crops.},
}
@article {pmid41314751,
year = {2025},
author = {Fatima, M and Tariq, I and Tariq, A and Talib, S and Fatima, M and Shehzadi, M and Aqib, AI},
title = {Pharmacogenomics and CRISPR-based therapies.},
journal = {Progress in brain research},
volume = {297},
number = {},
pages = {319-343},
doi = {10.1016/bs.pbr.2025.08.009},
pmid = {41314751},
issn = {1875-7855},
mesh = {Humans ; *Pharmacogenetics/methods ; *Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; *Precision Medicine/methods ; *Genetic Therapy/methods ; Animals ; },
abstract = {Pharmacogenomics and CRISPR-based treatments are two areas of precision medicine that are advancing together. Pharmacogenomics involves studying how differences in someone's genes can change the effect of medications on them. Pharmacogenomics helps reduce adverse reactions to drugs and improve healing by choosing and measuring drugs according to a patient's genetic information. Additionally, CRISPR-Cas systems now serve as leading genome editing tools that allow precise alterations at given points of the genome. CRISPR technology's use in pharmacogenomics creates new opportunities for modifying gene expression, fixing harmful mutations, and creating innovative treatment approaches. A more proactive approach to illness treatment is supported by this synergy, in which genetic factors serve as both direct targets for intervention and a basis for medication selection. This chapter examines the theoretical and practical frameworks that link CRISPR-based treatments with pharmacogenomics, emphasizing recent uses in pharmacoresistance, cancer, and monogenic diseases. To guarantee safe and fair deployment, it also covers the ethical, legal, and technical issues that need to be resolved. When combined, these technologies hold the potential to revolutionize medicine by facilitating individualized and curative drugs.},
}
@article {pmid41310509,
year = {2025},
author = {Ekrami, A and Taheri, B and Daneshfar, S and Moradi, M and Ghorbani, A and Akhash, N and Jafarzadeh, Z and Farshadzadeh, Z and Saki, M},
title = {Occurrence of CRISPR-Cas genes and lack of association with antibiotic resistance in Shigella isolates collected from patients with diarrhea in Ahvaz, southwest Iran.},
journal = {BMC infectious diseases},
volume = {25},
number = {1},
pages = {1666},
pmid = {41310509},
issn = {1471-2334},
abstract = {BACKGROUND: So far, few studies have examined the association between CRISPR-Cas and antibiotic resistance in Shigella isolates. Hence, this study sought to address this issue in Shigella species isolated from stool samples of patients with diarrhea in Ahvaz, southwest Iran.<br><br>METHODS: In this cross-sectional study, stool samples were collected from 103 children (3&#x2013;14 years) with diarrhea admitted to Abuzar Hospital affiliated to the Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran. All samples were cultured on the xylose lysine desoxycholate agar and hektoen enteric agar and incubated at 37&#xa0;&#xb0;C for 24&#xa0;h. Primary identification of the Shigella species was performed by biochemical tests and polymerase chain reaction (PCR). Antibiotic resistance rates were evaluated by the Kirby-Bauer disc diffusion. The prevalence of 12 CRISPR-Cas genes was investigated by PCR.<br><br>RESULTS: Overall, 83 Shigella isolates were identified by standard biochemical tests. Finally, 72 Shigella isolates including 45 S. flexneri (46.8%) and 27 (53.2%) S. sonnei were confirmed by PCR. The most effective antibiotics were trimethoprim/sulfamethoxazole (n&#x2009;=&#x2009;48, 66.7%), imipenem (n&#x2009;=&#x2009;46, 63.9%), and ceftazidime (n&#x2009;=&#x2009;43, 59.7%), respectively. All isolates harbored at least one of the CRISPR-Cas genes. Occurrence of CRISPR-Cas genes was as follows: CRa (100.0%), CRb (100.0%), CRc (100.0%), CSe2 (100.0%), CRf (95.8%), CSe1_Cas3 (87.5%), Cas2_Cas (84.7%), Cas (81.9%), CRd (80.6%), Cas7 (76.4%), Cas6e_Cas5 (72.2%), and CRe (48.6%). There was no significant association between the occurrence of CRISPR-Cas elements and resistance to any antibiotic in Shigella isolates (P-value&#x2009;&#x2265;&#x2009;0.9999), except for Cas gene with ceftriaxone and cefepime.<br><br>CONCLUSION: This study revealed high resistance rates of various antibiotics in Shigella isolates. However, there was no significant association between the existences of CRISPR-Cas genes with antibiotic resistance. Further investigation with higher sample size is needed to confirm this observation.<br><br>SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s12879-025-12100-0.},
}
@article {pmid41310261,
year = {2025},
author = {Spaans, GW and van der Berg, JP and Bouwman, LMS and Kleter, GA},
title = {Advancements in genomic crop techniques and considerations for regulation and food safety.},
journal = {Transgenic research},
volume = {34},
number = {1},
pages = {49},
pmid = {41310261},
issn = {1573-9368},
mesh = {*Crops, Agricultural/genetics/growth &amp; development ; *Plants, Genetically Modified/genetics/growth &amp; development ; *Food Safety ; Gene Editing ; Plant Breeding ; *Genomics/methods ; Food, Genetically Modified ; Mutagenesis ; Humans ; Genome, Plant ; },
abstract = {Advancements in genomic crop techniques have led to the development of new genetic technologies, such as base- and prime editing, but improvements have been made to existing conventional techniques as well. Fields in which these advancements occur include targeted mutagenesis, conventional random mutagenesis, and developments with null segregants, e.g., crops from which transgenic elements have been crossed out. In this review, we describe the developments in these three fields and provide considerations concerning regulatory and safety aspects. Because of differences in legislation of modern biotechnology between countries or regions, regulatory challenges are to be expected given the ongoing developments in genomic crop techniques. Moreover, the nature of the mutations induced with these newly developed techniques is not different from those induced with conventional techniques, making the modified crop plants indistinguishable from non-modified counterparts of the same crop species. Thus, enforcement of regulations cannot solely rely on technical analytical methods. Also, potential off-target or unintended effects in the primary mutants remain underexplored. Yet, these do not raise safety concerns owing to the experience with the crop breeding practice of iterative cycles for desirable traits selection, as well as the segregation and discard of unwanted phenotypes. Given that regulation will always change after innovation and developments within the sector advance rapidly, we advocate that both authorities and the breeding sector pro-actively implement a food safety culture. Such a safety culture will help developers of genomic technologies in crops to identify potential food safety issues at an early stage of development of future products.},
}
@article {pmid41309901,
year = {2025},
author = {Moroi, K and Yamamoto, T and Kurita, T},
title = {Double-strand break-free and transgene-free genome editing in the microalga Nannochloropsis oceanica using removable vectors containing the CRISPR base editing system.},
journal = {Scientific reports},
volume = {15},
number = {1},
pages = {42431},
pmid = {41309901},
issn = {2045-2322},
mesh = {*Gene Editing/methods ; *Microalgae/genetics ; *CRISPR-Cas Systems ; *Genetic Vectors/genetics ; Plasmids/genetics ; DNA Breaks, Double-Stranded ; Transgenes ; *Stramenopiles/genetics ; },
abstract = {The accumulation of lipids by algae makes them attractive for carbon-neutral fuel production; however, the industrial-scale production of algal lipids has yet to be achieved. Currently, researchers are trying to improve the lipid productivity of algal strains using genome editing for molecular breeding with CRISPR-Cas9, which allows the efficient alteration of genomic information. However, CRISPR-based gene modification via double-strand breaks sometimes induces unintended large deletions that are toxic to host cells. Here, we applied the cytidine base editor combined with an episomal vector backbone containing a centromere and autonomous replication sequence to the microalga Nannochloropsis oceanica. The cytosine base editor introduces cytidine-to-thymidine base substitutions using deaminase without double-strand breaks, and an episomal vector enables plasmid removal after base substitution. We succeeded in inducing cytidine-to-thymidine substitution at the six target sites of five endogenous genes. The base substitution activity ranged from 29.2% to 47.6% on cytidine bases at the 16th to 19th positions from the protospacer adjacent motifs. The removal of base editor plasmids was also detected, which is essential for constructing transgene-free strains. Our results provide insights into the applicability of further technologies in the genetic modification of microalgae.},
}
@article {pmid41309688,
year = {2025},
author = {Zhang, F and Peng, Y and Fan, D and Song, G and Gao, X and Tian, Y},
title = {Engineering a CRISPR-associated IscB system for developing miniature genome-editing tools in human cells and mouse embryos.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {10693},
pmid = {41309688},
issn = {2041-1723},
mesh = {Animals ; *Gene Editing/methods ; Humans ; Mice ; *CRISPR-Cas Systems/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; Embryo, Mammalian/metabolism ; HEK293 Cells ; Clustered Regularly Interspaced Short Palindromic Repeats ; CRISPR-Associated Proteins/genetics/metabolism ; },
abstract = {IscB, as the putative ancestor of Cas9, possesses a compact size, making it suitable for in vivo delivery. OgeuIscB is the first IscB protein known to function in eukaryotic cells but requires a complex TAM (NWRRNA). Here, we characterize a CRISPR-associated IscB system, named DelIscB, which recognizes a flexible TAM (NAC). Through systematically engineering its protein and sgRNA, we obtain enDelIscB with an average 48.9-fold increase in activity. By fusing enDelIscB with T5 exonuclease (T5E), we find that enDelIscB-T5E displays robust efficiency comparable to that of enIscB-T5E in human cells. Moreover, by fusing cytosine or adenosine deaminase with enDelIscB nickase, we establish efficient miniature base editors (ICBE and IABE). Finally, we efficiently generate mouse models by microinjecting mRNA/sgRNA of enDelIscB and enDelIscB-T5E into mouse embryos. Collectively, our work presents a set of enDelIscB-based miniature genome-editing tools with great potential for diverse applications in vivo.},
}
@article {pmid41242303,
year = {2025},
author = {Dong, M and Zhang, J and Long, L and Wang, H and Zhang, A and Zeng, W and Li, J},
title = {Establishment of a liver-specific Albumin-Cre recombinase transgenic golden hamster model using gRosa26-Targeted dual-fluorescent reporter system for hepatocyte-specific genetic manipulation.},
journal = {Biochemical and biophysical research communications},
volume = {792},
number = {},
pages = {152912},
doi = {10.1016/j.bbrc.2025.152912},
pmid = {41242303},
issn = {1090-2104},
mesh = {Animals ; *Integrases/genetics/metabolism ; *Mesocricetus/genetics ; *Hepatocytes/metabolism ; Cricetinae ; *Genes, Reporter ; Animals, Genetically Modified ; *Liver/metabolism ; *Albumins/genetics/metabolism ; CRISPR-Cas Systems ; Gene Editing/methods ; *RNA, Untranslated/genetics ; Male ; Mice ; Organ Specificity ; },
abstract = {BACKGROUND AND AIMS: Despite its superior physiological relevance to human liver metabolism and diseases, the absence of reliable tissue-specific gene manipulation systems has considerably limited genetic research in the Syrian golden hamster (Mesocricetus auratus). The Cre/loxP-based Rosa26-targeted dual-fluorescent reporter system (mT/mG) is widely utilized in murine models for accurate lineage tracing and conditional gene editing. However, such powerful genetic tools are not available for golden hamsters. Herein, we established a functional Rosa26-targeted mT/mG reporter system and a hepatocyte-specific albumin-Cre Recombinase (Alb-Cre) driver line in golden hamsters, providing an important platform for hepatocyte-specific gene manipulation.<br><br>METHODS: The Rosa26 homolog (gRosa26) was identified in the golden hamster genome. Then, a CRISPR/Cas9-based two-cell microinjection system was developed to precisely knock-in gRosa26. The dual-fluorescent Cre reporter (mT/mG) was inserted into gRosa26. Thereafter, a liver-specific Alb-Cre transgenic line was developed using murine Alb enhancer/promoter elements. Finally, Cre-mediated recombination efficiency was evaluated in double transgenic hamsters.<br><br>RESULTS: The gRosa26 locus exhibited >70&#xa0;% sequence conservation with mice. Golden hamsters with the mT/mG reporter exhibited ubiquitous tdTomato expression, with efficient EGFP activation after Cre-mediated recombination. Furthermore, Alb-Cre transgenic golden hamsters exhibited hepatocyte-specific Cre expression. In double transgenic hamsters, robust hepatocyte-specific tdTomato-to-EGFP recombination was observed by postnatal days 21 and 42. This validated the functionality of the gRosa26-targeted mT/mG system and Alb-driven Cre expression.<br><br>CONCLUSIONS: We reported the development of a functional Rosa26-targeted mT/mG dual-reporter system and a liver-specific Alb-Cre driver in Syrian golden hamsters for the first time. This platform facilitates precise spatiotemporal gene editing in hepatocytes and serves as an essential genetic tool for liver research in this emerging model organism.},
}
@article {pmid41237878,
year = {2025},
author = {Ashok, K and Ellango, R and Venkatesh, R and Bhargava, CN and Pavithran, S and Dhawane, Y and Manamohan, M and Jha, GK and Asokan, R},
title = {Differential expression of genes in pre-blastoderm embryos of oriental fruit fly, Bactrocera dorsalis (Hendel) (Diptera: Tephritidae) microinjected with white locus CRISPR/Cas9 ribo nucleo protein (RNP) complex.},
journal = {International journal of biological macromolecules},
volume = {333},
number = {Pt 2},
pages = {148735},
doi = {10.1016/j.ijbiomac.2025.148735},
pmid = {41237878},
issn = {1879-0003},
mesh = {Animals ; *Tephritidae/genetics/embryology ; *CRISPR-Cas Systems/genetics ; *Ribonucleoproteins/genetics ; *Gene Expression Regulation, Developmental ; Microinjections ; Gene Editing ; Gene Expression Profiling ; Insect Proteins/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; *CRISPR-Associated Protein 9/genetics ; Transcriptome ; },
abstract = {CRISPR/Cas9-based genome editing is currently revolutionizing applications in agriculture, human health, and other domains. Targeted gene editing employing CRISPR/Cas9 is achieved by delivering the various components viz. guide RNA, traCrRNA, and the Cas9 protein in DNA or RNA or RNP format. RNP enables fast, DNA-free editing without genomic integration. While innate immunity to guide RNA is studied in humans and plants, similar research in insects remains unexplored. In the present study, the RNP complex (sgRNA+Cas9) was administered via microinjection of embryos (eggs) in the pre-blastoderm (G0) stage. Transcriptome sequencing was performed using the Illumina Novoseq 6000 platform to understand the prevalence of innate immunity against the delivered guide RNA for the white locus of Bactrocera dorsalis. 33 up- and 67 down-regulated genes were found in the differential gene expression analysis from the RNA-Seq data. The correlation between RT-qPCR and RNA-Seq gene expression levels showed a strong coefficient of determination (R2 = 0.984). The genes related to stress, intron removal, and effector recognition were overexpressed. However, the genes responsible for growth and development were significantly downregulated. Thus, the present study identified the possible reasons that could play a vital role in influencing the editing outcomes employed through the RNP complex.},
}
@article {pmid41205942,
year = {2025},
author = {Feng, Z and He, X and Lin, M and Zhao, H and Chen, Y and Chen, J and Li, Z and Shen, Y and Chen, J and Yang, X and Chen, Q},
title = {Genome-wide CRISPR screen identifies ALG5, ALG6, NF2, and FUT8 as key host proteins involved in transmissible gastroenteritis virus infection.},
journal = {International journal of biological macromolecules},
volume = {333},
number = {Pt 2},
pages = {148770},
doi = {10.1016/j.ijbiomac.2025.148770},
pmid = {41205942},
issn = {1879-0003},
mesh = {Animals ; *Transmissible gastroenteritis virus/physiology ; Swine ; *Fucosyltransferases/genetics/metabolism ; *Gastroenteritis, Transmissible, of Swine/virology/genetics/metabolism ; *CRISPR-Cas Systems ; Cell Line ; Virus Internalization ; Glycosylation ; *Host-Pathogen Interactions/genetics ; Virus Replication ; Galactoside 2-alpha-L-fucosyltransferase ; },
abstract = {Transmissible gastroenteritis virus (TGEV) represents a significant threat to global swine production. In the absence of effective antiviral therapies, control relies primarily on vaccination. To identify potential therapeutic targets, we performed a genome-wide CRISPR/Cas9 screen in porcine IPEC-J2 cells, which revealed asparagine-linked glycosylation 5 (ALG5), asparagine-linked glycosylation 6 (ALG6), neurofibromin 2 (NF2), and fucosyltransferase 8 (FUT8) as essential host factors for TGEV infection. Functional characterization demonstrated that ALG5, ALG6, and NF2 knockout impaired viral adsorption and internalization through disruption of aminopeptidase N (pAPN) transcription or N-glycosylation. Consistently, tunicamycin-mediated inhibition of N-glycosylation suppressed TGEV infection. In contrast, FUT8 knockout specifically affects viral internalization and early replication by preventing the formation of double-membrane vesicles (DMVs) but does not affect pAPN expression. This role was independent of FUT8's fucosyltransferase activity, as the enzymatic inhibitor FDW028 had no effect. Mechanistically, we found that FUT8 interacts with the TGEV nonstructural proteins NSP3 and NSP4 to facilitate DMV biogenesis. Our findings delineate distinct mechanisms by which host factors support TGEV infection and provide novel insights for the development of targeted antiviral strategies.},
}
@article {pmid41135303,
year = {2026},
author = {Lin, Y and Zhou, Y and Yuan, X and Wang, X and Xu, H and Lei, F and Wang, L and Lan, Q and Zhu, B},
title = {RPA-CRISPR-Cas12a/RPA-CRISPR-Cas12a-LFD: Two detection platforms for the rapid identification of five forensic body fluids.},
journal = {Forensic science international. Genetics},
volume = {81},
number = {},
pages = {103376},
doi = {10.1016/j.fsigen.2025.103376},
pmid = {41135303},
issn = {1878-0326},
mesh = {Humans ; Female ; Semen/chemistry ; Menstruation ; RNA, Messenger/genetics/metabolism ; Saliva/chemistry ; *CRISPR-Cas Systems ; *Forensic Genetics/methods ; Male ; Vagina/metabolism ; Genetic Markers ; Cervix Mucus/chemistry ; beta-Globins/genetics ; Real-Time Polymerase Chain Reaction ; },
abstract = {Identifying tissue source types of human peripheral blood, menstrual blood, vaginal secretion, semen, and saliva left at a crime scene can offer crucial clues for case investigation. Although messenger RNA (mRNA) profiling has become a robust tool for body fluid identification, conventional methods remain constrained by time-consuming workflows and dependence on laboratory infrastructure, hindering rapid on-site forensic analysis. To address these limitations, this study designed the RPA-CRISPR-Cas12 experiment and independently developed an RPA-CRISPR-Cas12a-LFD detection system, which achieved simple, rapid (<40 min), and accurate forensic identification of five kinds of critical body fluids. Initially, we mined the Genotype-Tissue Expression database and related literatures to screen candidate mRNA markers, subsequently employed RT-qPCR to evaluate their expression levels, and applied the Boruta algorithm to optimize the number of mRNA markers, ultimately identifying five mRNA markers: HBB (peripheral blood), MMP10 (menstrual blood), DKK4 (vaginal secretion), AKAP4 (semen), and HTN3 (saliva). The developed system exhibited exceptional specificity. Furthermore, the sensitivity values of HBB, MMP10, DKK4, AKAP4, and HTN3 markers in RPA-CRISPR-Cas12 and RPA-CRISPR-Cas12a-LFD detection systems were 0.1, 0.1, 1, 0.1, 0.1 ng; and 0.1, 0.1, 1, 1 and 1 ng, respectively. Among them, the sensitivity of HBB and DKK4 markers was superior to that of previous studies. Notably, the platform successfully resolved mixed samples (excluding AKAP4 marker), and MMP10 and DKK4 markers retained detectability in aged menstrual blood and vaginal secretion samples. The proposed method provides the groundwork for further development of tissue origin identification tools of forensic body fluids, which may eventually be suitable for on-site rapid detection environments.},
}
@article {pmid41074741,
year = {2025},
author = {Karmakar, A and Hota, A and Tanga, S and Kumar, V and Das, P and S, AE and Thapa, M and Maji, B},
title = {Engineered Thermostable Chemically Responsive GlowCas9 System for Real-Time Therapeutic Monitoring Applications.},
journal = {Angewandte Chemie (International ed. in English)},
volume = {64},
number = {49},
pages = {e202511707},
doi = {10.1002/anie.202511707},
pmid = {41074741},
issn = {1521-3773},
mesh = {*Gene Editing ; *CRISPR-Cas Systems ; Humans ; *CRISPR-Associated Protein 9/genetics/metabolism/chemistry ; Animals ; Temperature ; *Protein Engineering ; Genetic Therapy ; },
abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) has revolutionized gene therapy applications due to its ease of design and efficiency, albeit accompanied by off-target effects. Spatiotemporally regulated Cas9 offers safer gene editing methods due to its lower off-target mediated genotoxicities; however, probing the Cas9 in real time for assessing the delivery efficiency, gene editing timeframe, and spatial target information remains elusive. Here, we report an engineered Cas9 system, GlowCas9, that can be probed chemically in real-time across all assay systems, including cell lysate, native gel, ex vivo, and in vivo. Importantly, we rationally engineered the Cas9 system to attain remarkable thermostability with enhanced gene editing activities. Besides its sensitive reporter and enhanced gene editing activity, the GlowCas9 system exhibits precision HDR-based gene knock-in ability in cells and significantly outperformed the WTCas9. Overall, we report a new thermostable engineered SpCas9 system for real-time theratracking applications compatible with diverse assay formats, including live ex vivo and in vivo. This GlowCas9 system will add a new dimension of non-invasive real-time tracking in gene therapy development, both ex vivo and in vivo.},
}
@article {pmid40957713,
year = {2025},
author = {Tani, M and Hatano, K and Ishizuya, Y and Oka, T and Kanaki, T and Inoguchi, S and Yoshimura, A and Horibe, Y and Liu, Y and Nesrine, S and Okuda, Y and Yamamoto, A and Uemura, T and Yamamichi, G and Hayashi, T and Yamamoto, Y and Kato, T and Kawashima, A and Yamaguchi, T and Obika, S and Yusa, K and Nonomura, N and Nimura, K},
title = {Development of a Synthetic Lethality-Based Combination Therapy Using LIG1 and PARP Inhibitors for Prostate Cancer.},
journal = {Cancer science},
volume = {116},
number = {12},
pages = {3403-3416},
doi = {10.1111/cas.70194},
pmid = {40957713},
issn = {1349-7006},
support = {19K18610//Japan Society for the Promotion of Science/ ; 21K09345//Japan Society for the Promotion of Science/ ; 24K12482//Japan Society for the Promotion of Science/ ; },
mesh = {Humans ; Male ; *Poly(ADP-ribose) Polymerase Inhibitors/pharmacology ; Animals ; Cell Line, Tumor ; Mice ; Phthalazines/pharmacology ; Piperazines/pharmacology ; *Synthetic Lethal Mutations/drug effects ; Xenograft Model Antitumor Assays ; Apoptosis/drug effects ; *DNA Ligase ATP/antagonists &amp; inhibitors/genetics/metabolism ; *Prostatic Neoplasms, Castration-Resistant/drug therapy/genetics/pathology ; *Prostatic Neoplasms/drug therapy/genetics ; DNA Damage/drug effects ; Drug Synergism ; *Antineoplastic Combined Chemotherapy Protocols/pharmacology ; Drug Resistance, Neoplasm/genetics ; CRISPR-Cas Systems ; Mice, Nude ; Tumor Suppressor Protein p53/genetics/metabolism ; },
abstract = {Despite advances in androgen receptor signaling inhibitors (ARSIs) and poly (ADP-ribose) polymerase inhibitors (PARPIs), metastatic castration-resistant prostate cancer (mCRPC) remains lethal. PARPIs clinical efficacy is limited in patients with homologous recombination repair deficiencies, such as BRCA1/2 mutations, due to resistance. Thus, identifying novel synthetic lethal interactions with PARP may expand treatment options and improve therapeutic efficacy. Here, to identify genes that influence sensitivity to the PARPI olaparib, we conducted a genome-wide CRISPR-Cas9 knockout screening of 18,010 genes in DU145, 22Rv1, and LNCaP prostate cancer cell lines. Our screening identified PARP and LIG1 as synthetic lethality-inducing factors, whereas TP53 conferred resistance to PARPIs. Simultaneous inhibition of LIG1 and PARP increased DNA damage and apoptosis. Additionally, the combination of the LIG1 inhibitor L82-G17 with olaparib exhibited synergistic effects. To the best of our knowledge, we validated this combination therapy in vivo for the first time, suppressing tumor growth in a DU145 xenograft model while minimizing toxicity in normal tissues. Immunohistochemical analysis revealed that LIG1 was overexpressed in CRPC tissues, suggesting its potential as a therapeutic target. This study established LIG1 as a novel synthetic lethality-inducing factor in prostate cancer, showing that L82-G17 enhances the efficacy of olaparib, regardless of the BRCA mutation status. These findings suggest that the combination of PARP and LIG1 inhibitors could be a novel therapeutic strategy for mCRPC.},
}
@article {pmid40745506,
year = {2025},
author = {Minh, NNT and Lee, E and Kim, SA},
title = {The cochaperone BAG3 promotes the stabilization of p53 under heat stress conditions.},
journal = {FEBS open bio},
volume = {15},
number = {12},
pages = {1945-1954},
doi = {10.1002/2211-5463.70096},
pmid = {40745506},
issn = {2211-5463},
support = {RS-2024-00358563//National Research Foundation of Korea/ ; },
mesh = {Humans ; *Tumor Suppressor Protein p53/metabolism/genetics ; *Heat-Shock Response/physiology/genetics ; *Adaptor Proteins, Signal Transducing/metabolism/genetics ; *Apoptosis Regulatory Proteins/metabolism/genetics ; HeLa Cells ; HSP70 Heat-Shock Proteins/metabolism ; *Molecular Chaperones/metabolism ; CRISPR-Cas Systems ; Gene Knockout Techniques ; },
abstract = {Bcl-2-associated athanogene 3 (BAG3) is the only member of the BAG cochaperone family that is induced by stressful stimuli such as heat shock and heavy metals. In the present study, bag3 knockout (KO) HeLa cells were generated via the CRISPR-Cas9 system, and the role of BAG3 in relation to p53 under heat stress conditions was investigated. Normally, the levels of p53 were low in both wild-type (WT) and KO cells, while heat shock increased the levels of nuclear p53 in both cell lines. However, the increased level of p53 was much greater in WT cells than in KO cells, which suggested that BAG3 played a role in controlling the level of p53 under heat stress conditions. The mRNA level of p53 did not increase in either WT or KO cells during the heat stress period, which suggested that the differences in the levels of p53 were not due to transcriptional regulation. Both treatment with the proteasome inhibitor MG132 and heat shock drastically increased p53 levels to a similar extent in WT cells. Interestingly, both BAG3 and Hsp70 rapidly translocated to the nucleus and formed a complex with p53 upon heat stress. During a 1-h recovery period from heat stress, the transcriptional activity of p53 increased up to 4-fold in WT cells, but only 1.69-fold in KO cells. These results demonstrate that Hsp70 and BAG3 are involved in the quality control of p53 under heat stress conditions and suggest a role for BAG3 as a cochaperone protein.},
}
@article {pmid40643191,
year = {2025},
author = {Zhou, R and Seth, SR and Reeves, J and Burns, AH and Hsieh, C and Horn, TW and Xue, LJ and Tsai, CJ},
title = {Populus VariantDB v3.2 facilitates CRISPR and functional genomics research.},
journal = {Tree physiology},
volume = {45},
number = {13},
pages = {143-148},
doi = {10.1093/treephys/tpaf081},
pmid = {40643191},
issn = {1758-4469},
support = {ERKP886//Office of Biological and Environmental Research of the US Department of Energy, Office of Science/ ; DE-SC0023166//Office of Biological and Environmental Research of the US Department of Energy, Office of Science/ ; DE-SC0023338//Office of Biological and Environmental Research of the US Department of Energy, Office of Science/ ; //Georgia Research Alliance Hank Haynes Forest Biotechnology Endowment/ ; },
mesh = {*Populus/genetics ; *Genomics/methods ; *Genome, Plant ; *CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; *Databases, Genetic ; Gene Editing ; },
abstract = {The success of CRISPR genome editing studies depends critically on the precision of guide RNA (gRNA) design. Sequence polymorphisms in outcrossing tree species pose design hazards that can render CRISPR genome editing ineffective. Despite recent advances in tree genome sequencing with haplotype resolution, sequence polymorphism information remains largely inaccessible to various functional genomics research efforts. The Populus VariantDB v3.2 addresses these challenges by providing a user-friendly search engine to query sequence polymorphisms of heterozygous genomes. The database accepts short sequences, such as gRNAs and primers, as input for searching against multiple poplar genomes, including hybrids, with customizable parameters. We provide examples to showcase the utilities of VariantDB in improving the precision of gRNA or primer design. The platform-agnostic nature of the probe search design makes Populus VariantDB v3.2 a versatile tool for the rapidly evolving CRISPR field and other sequence-sensitive functional genomics applications. The database schema is expandable and can accommodate additional tree genomes to broaden its user base.},
}
@article {pmid41309578,
year = {2025},
author = {Arana, S and Du, PP and Vaughan-Jackson, A and Enright, N and Spees, K and Valbuena, R and Garcia, CA and Nguyen, T and Venida, A and Seczynska, M and Bintu, L and Lehner, PJ and Prolo, LM and Bassik, MC},
title = {Reduced Cas9 transgene silencing by incorporation of intron sequences.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {10656},
pmid = {41309578},
issn = {2041-1723},
support = {R01HG011866//U.S. Department of Health &amp; Human Services | NIH | National Human Genome Research Institute (NHGRI)/ ; },
mesh = {*Introns/genetics ; *Transgenes/genetics ; *Gene Silencing ; Humans ; *CRISPR-Cas Systems/genetics ; Gene Editing/methods ; *CRISPR-Associated Protein 9/genetics/metabolism ; HEK293 Cells ; Chromatin/metabolism/genetics ; Animals ; Cell Line ; },
abstract = {Silencing remains a significant challenge for exogenous gene expression, limiting both the penetrance and expressivity of transgenes. In particular, silencing of Cas9 expression is a major technical limitation for many gene editing and CRISPR screening applications. Here, we demonstrate that including introns in Cas9 expression cassettes significantly reduces silencing across multiple cell lines. Notably, the incorporation of an intron into a CRISPRa construct results in reduced silencing, increased expression levels, and markedly enhanced activation of target genes. We investigate diverse intron sequences and discover that T-rich introns over 2 kb confer the greatest protection against silencing. In addition, we find that introns can work synergistically with chromatin opening elements to further mitigate silencing, suggesting regulatory mechanisms are acting at both the DNA and RNA level to silence exogenous genes. Our work highlights the potential of introns to optimize genetic constructs for enhanced expression and improved cellular engineering requiring constitutive expression of large transgenes.},
}
@article {pmid41260510,
year = {2025},
author = {Jiang, W and Yang, A and Ma, J and Lv, D and Liu, M and Wang, C and Chen, S and Fang, H and Chu, Y and He, Z and Li, W and Liu, Y and Zhao, Y and Zhou, Z and Long, G and Jiang, H},
title = {Genetic determinants of efficacy of antiviral drugs revealed by genome-wide CRISPR screens.},
journal = {Antiviral research},
volume = {244},
number = {},
pages = {106309},
doi = {10.1016/j.antiviral.2025.106309},
pmid = {41260510},
issn = {1872-9096},
mesh = {*Antiviral Agents/pharmacology ; Animals ; Humans ; *CRISPR-Cas Systems ; Mice ; Virus Replication/drug effects ; Cell Line, Tumor ; Ribavirin/pharmacology ; Adenosine Monophosphate/analogs &amp; derivatives/pharmacology ; Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {Nucleoside and nucleobase analog antiviral drugs are pivotal in antiviral therapy, but comprehensive methods to understand their cellular response mechanisms and genetic regulators are still lacking. Here, we show that Eμ-Myc; Arf[-/-] mouse lymphoma cells, which are highly apoptosis-prone, enabled genome-wide CRISPR-Cas9 screening on such drugs to identify genes that modulate their efficacy. Using retroviral sgRNA libraries and MAGeCK analysis, we uncovered key regulators of drug transport, activation, and inactivation for these drugs. For ribavirin, adenosine kinase (ADK) and adenylsuccinate synthase (ADSS) were critical for nucleotide metabolism and bioactivation. Remdesivir uptake and activation depended on the transporter SLC29A3 and phosphoamidase HINT1, whereas favipiravir resistance was linked to NT5C2-mediated dephosphorylation. Viral replication assays in Huh7 cells validated that knockout of SLC29A3, HINT1, or NT5C2 significantly altered antiviral efficacy. This study delineates the genetic network governing nucleotide analog response, providing mechanistic insights and potential biomarkers for personalized antiviral therapy.},
}
@article {pmid40905205,
year = {2025},
author = {Bataa, D and Kajiura, H and Sawada-Choi, RLS and Yamashita, Y and Ishimizu, T and Misaki, R and Takeda, A and Fujiyama, K},
title = {CRISPR/Cas9-Mediated Knockouts of the ALG3 and GNTI in N. benthamiana and Their Application to Pharmaceutical Production.},
journal = {Plant biotechnology journal},
volume = {23},
number = {12},
pages = {5894-5916},
doi = {10.1111/pbi.70326},
pmid = {40905205},
issn = {1467-7652},
support = {22K06143//Japan Society for the Promotion of Science/ ; 22K19186//Japan Society for the Promotion of Science/ ; },
mesh = {*Nicotiana/genetics/metabolism ; *CRISPR-Cas Systems/genetics ; Glycosylation ; Polysaccharides/metabolism ; Gene Knockout Techniques ; Plants, Genetically Modified/metabolism ; *Plant Proteins/genetics/metabolism ; Gene Editing ; },
abstract = {N-Glycosylation critically influences the efficacy, safety and pharmacokinetic properties of biopharmaceuticals. Plant expression platforms offer multiple advantages for the production of N-glycosylated proteins, but their use is impeded by the presence of plant-specific N-glycan epitopes, which raise concerns of possible immunogenicity to humans. In this study, N-glycoengineered Nicotiana benthamiana plants that produce more homogeneous N-glycans without plant-specific epitopes were generated using multiplex CRISPR/Cas9 genome editing. To achieve this N-glycosylation modification, ALG3 and GNTI, which function in N-glycosylation processes in the ER and Golgi, respectively, were characterised, and single- and double-knockout mutant plants were generated. Comprehensive N-glycan profiling revealed that while the ALG3-knockout plant line, alg3, maintained predominantly plant-specific N-glycans with altered mannose content, the GNTI-knockout line, gntI, produced exclusively high-mannose-type N-glycans. Notably, the alg3gntI double-knockout mutants yielded highly uniform trimannosidic N-glycans. To validate our N-glycoengineering approach, we expressed two model biopharmaceuticals, Varlilumab (anti-CD27 antibody) and β-glucocerebrosidase (GCase), in wild-type and mutant plants. While the antibodies expressed in alg3 and alg3gntI showed a certain level of glucosylated endoplasmic reticulum-type N-glycan, with increased non-N-glycosylated heavy chains, GCase exhibited a more consistent N-glycosylation profile, reflecting the engineered N-glycosylation pathway. Our findings provide valuable insights into N-glycan biosynthesis in N. benthamiana and demonstrate the potential of targeted N-glycoengineering for producing biopharmaceuticals with more homogeneous mannose-type N-glycan profiles.},
}
@article {pmid40905066,
year = {2025},
author = {Lin, XX and Gong, BQ and Wang, FZ and Wan, JB and Xiong, X and Li, JF},
title = {Versatile Applications of CRISPR-Based Programmable T-DNA Integration in Plants.},
journal = {Plant biotechnology journal},
volume = {23},
number = {12},
pages = {5950-5964},
doi = {10.1111/pbi.70353},
pmid = {40905066},
issn = {1467-7652},
support = {32125004//National Natural Science Foundation of China/ ; 2023B0303000022//Guangdong Major Project of Basic and Applied Basic Research grant/ ; },
mesh = {*Arabidopsis/genetics ; *DNA, Bacterial/genetics ; *CRISPR-Cas Systems/genetics ; Plants, Genetically Modified/genetics ; Gene Editing/methods ; Agrobacterium/genetics ; DNA Breaks, Double-Stranded ; },
abstract = {Agrobacterium-mediated T-DNA integration into plant genomes represents a cornerstone for transgenic expression in plant basic research and synthetic biology. However, random T-DNA integration can disrupt essential endogenous genes or compromise transgene expression, stressing the need for targeted integration strategies. Here we explored CRISPR-aided targeted T-DNA integration (CRISTTIN) in Arabidopsis, leveraging CRISPR-induced double-strand breaks (DSBs) to facilitate precise T-DNA insertion. Contrary to our initial hypothesis, conventional Cas9 outperformed a designed Cas9-adaptor fusion nuclease that may recruit Agrobacterium VirD2/T-DNA complexes to DSB sites via the adaptor-VirD2 interaction. Using Cas9-based CRISTTIN, we streamlined the parallel generation of FERONIA null alleles and in-locus complementation alleles expressing a wild-type or mutated gene. This enabled phenotypic comparisons under identical genomic contexts and significantly accelerated gene characterisation and critical residue identification. Additionally, CRISTTIN was employed to simultaneously knockout AGAMOUS and in-locus integrate a RUBY reporter, yielding plants with pink double-petaled flowers. CRISTTIN also enabled site-specific insertion of 35S enhancers for transcriptional upregulation of adjacent genes or reporter constructs for promoter activity monitoring. CRISTTIN's effectiveness was further validated in rice. These results demonstrated CRISTTIN as a versatile tool for gene functional studies and precise control of transgene expression in plants.},
}
@article {pmid40836480,
year = {2025},
author = {Qin, B and Hou, X and Fan, S and Li, C and Wang, X and Chen, Q and Yang, Y and Lin, P and Lu, LR and Fu, C and Yang, N and Gao, Q and Zheng, S and Liu, S},
title = {Simultaneous Knockout of Tk1-SST and Tk1-FFT via CRISPR/Cas9 Enhances the Natural Rubber Accumulation in Taraxacum kok-saghyz.},
journal = {Plant biotechnology journal},
volume = {23},
number = {12},
pages = {5654-5676},
doi = {10.1111/pbi.70329},
pmid = {40836480},
issn = {1467-7652},
support = {2023YFA0914801//National Key Research and Development Program of China/ ; 31970364//National Natural Science Foundation of China/ ; ZDYF2024XDNY213//Hainan Provincial Science and Technology Special Fund/ ; 324RC533//Hainan Provincial Natural Science Foundation of China/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; *Taraxacum/genetics/metabolism ; *Rubber/metabolism ; Gene Knockout Techniques ; Inulin/metabolism/biosynthesis ; Sucrose/metabolism ; *Plant Proteins/genetics/metabolism ; Gene Expression Regulation, Plant ; Plant Leaves/metabolism ; },
abstract = {Taraxacum kok-saghyz (TKS) synthesises natural rubber (NR) and inulin using sucrose as a carbon source. However, molecular mechanisms regulating inulin and NR accumulation remain largely unclear. Here, we report the generation of double-gene homozygous mutants, 1-sst1-fft, by simultaneously knocking out two key genes responsible for inulin biosynthesis (Tk1-SST and Tk1-FFT) using CRISPR/Cas9 technology. The 1-sst1-fft mutants exhibited significant increases in rosette leaf number, flower number, leaf area, whole-plant biomass and seed set. Moreover, inulin biosynthesis was abolished in 1-sst1-fft, leading to significant changes in sugar composition, particularly a marked increase in sucrose levels. Notably, NR accumulation more than doubled, with no significant change in molecular weight and most terpenoid accumulation also increased in 1-sst1-fft, both being positively correlated with sucrose levels. For the first time, this study reports the generation of an inulin synthesis-deficient mutant in plants, emphasising the essential roles of 1-SST and 1-FFT in regulating carbon partitioning and, consequently, modulating important traits and metabolite accumulation. Transcriptomic analysis revealed fundamental genes involved in sucrose metabolism, sugar signalling and transport and NR elongation were significantly upregulated in 1-sst1-fft; accompanied by enhanced enzymatic activities of sucrose-phosphate synthase and invertase. These findings demonstrate that blocking inulin biosynthesis in 1-sst1-fft redirects sucrose towards NR biosynthesis, highlighting the dual role of sucrose as both a carbon source and signalling molecule in modulating plant growth and development and metabolite synthesis. Our study provides a successful approach to enhancing NR accumulation by modifying carbon allocation in TKS, offering novel insights into high-yield breeding strategies.},
}
@article {pmid41309382,
year = {2025},
author = {Dao, TO and Park, HE and Lee, JH and Kim, KM and Trinh, MP and Kang, HL and Yoo, HS and Shin, MK},
title = {Advances and Challenges in Mycobacterial Genetic Engineering: Techniques for Knockout, Knockdown and Overexpression.},
journal = {Journal of microbiology and biotechnology},
volume = {35},
number = {},
pages = {e2507051},
doi = {10.4014/jmb.2507.07051},
pmid = {41309382},
issn = {1738-8872},
mesh = {*Genetic Engineering/methods ; *Gene Knockout Techniques/methods ; *Mycobacterium/genetics ; CRISPR-Cas Systems ; *Gene Knockdown Techniques/methods ; DNA Transposable Elements ; Homologous Recombination ; Gene Transfer Techniques ; },
abstract = {Genetic engineering of mycobacteria is challenging due to their hydrophobic cell wall structure and slow growth rates. Despite these obstacles, significant progress has been made to develop genetic engineering tools to study gene function and pathogenesis in these organisms. This review comprehensively explores the current methodologies employed in the genetic modification of mycobacteria, focusing on gene knockout, knockdown, and overexpression systems. Techniques covered include homologous recombination, recombineering, transposon mutagenesis, CRISPR-Cas systems, conditional expression strategies, and phage-mediated gene delivery. The mechanism, advantages, and limitations of those methods are critically analyzed, with particular emphasis on the adaptability of these tools to various mycobacterial species. By providing a detailed comparative analysis of available genetic tools, this review is a practical guide for researchers aiming to develop targeted and efficient genetic modifications in Mycobacterium species, accelerating discoveries in pathogenesis, drug resistance, and vaccine development.},
}
@article {pmid41167015,
year = {2025},
author = {Song, X and Peng, Q and Zhang, X and Zhao, Y and Zhang, G and Guo, R and Li, Y and Shen, Y and Yang, S and Hu, M and Zhang, X and Li, J and Fan, B and Li, B},
title = {Enhanced immunogenicity of swine coronavirus PEDV by engineering transcriptional regulatory sequences.},
journal = {Vaccine},
volume = {68},
number = {},
pages = {127914},
doi = {10.1016/j.vaccine.2025.127914},
pmid = {41167015},
issn = {1873-2518},
mesh = {Animals ; *Porcine epidemic diarrhea virus/immunology/genetics ; Swine ; Antibodies, Viral/blood/immunology ; *Coronavirus Infections/prevention &amp; control/veterinary/immunology ; *Viral Vaccines/immunology/genetics/administration &amp; dosage ; *Swine Diseases/prevention &amp; control/immunology/virology ; Antibodies, Neutralizing/blood/immunology ; Spike Glycoprotein, Coronavirus/immunology/genetics ; Female ; *Immunogenicity, Vaccine ; Vaccines, Inactivated/immunology/administration &amp; dosage ; Immunoglobulin G/blood ; CRISPR-Cas Systems ; Pregnancy ; Immunoglobulin A/blood ; Gene Editing ; },
abstract = {Porcine epidemic diarrhea virus (PEDV) is a porcine enteropathogenic coronavirus that can cause severe diarrhea and high mortality in neonatal piglets. Therefore, the development of safe and effective vaccines remains a top priority for controlling PEDV infection. Previous studies have found that the transcription regulatory sequence (TRS) of the viral M gene has the strongest regulatory ability for the expression of exogenous gene. In order to investigate whether the transcription level of the S gene is increased after swapping body TRS of the S gene (TRS-S) with the body-TRS of M gene (TRS-M), CRISPR/Cas9 gene editing technology were used to replace the TRS-S of PEDV with TRS-M, and found that replacing TRS-S by TRS-M, as well as the downstream bases of TRS-M, can effectively promote the expression of S protein. Subsequently, the recombinant strain with high expression of S protein was prepared into an inactivated vaccine, which induced higher levels of S-specific immunoglobulin G (IgG), IgA, and neutralizing antibodies in immunized pregnant sows. The newborn spiking pigs also obtained high levels of antibodies through breastfeeding, and after being challenged, it was found that clinical symptoms and microscopic lesions were significantly reduced. In conclusion, these data suggest that replacing TRS-S with the transcriptionally efficient TRS-M effectively promotes PEDV S protein expression, and this modified strain exhibits improved immunogenicity and better protection against viral challenge, which provides new method for the research and development of PEDV vaccines.},
}
@article {pmid41067593,
year = {2026},
author = {Wijerathna, HMSM and Jung, S and Lee, J},
title = {Enhancing Edwardsiella piscicida resistance through CRISPR/Cas9-mediated deletion of toll-like receptor 5a (tlr5a) in zebrafish.},
journal = {Fish &amp; shellfish immunology},
volume = {168},
number = {},
pages = {110915},
doi = {10.1016/j.fsi.2025.110915},
pmid = {41067593},
issn = {1095-9947},
mesh = {Animals ; *Toll-Like Receptor 5/genetics/immunology ; Edwardsiella/physiology ; *Fish Diseases/immunology/microbiology/genetics ; *Zebrafish/immunology/genetics ; *Enterobacteriaceae Infections/immunology/veterinary/genetics ; CRISPR-Cas Systems ; *Immunity, Innate/genetics ; *Disease Resistance/genetics ; *Zebrafish Proteins/genetics/immunology ; },
abstract = {Toll-like receptor 5 (TLR5), a pattern recognition receptor that detects bacterial flagellin, plays a critical role in innate immune responses. Teleost fish possess two paralogs, tlr5a and tlr5b, of which the functional role of tlr5a during flagellated bacterial infections remains unclear. In this study, we investigated the involvement of tlr5a in the immune response against Edwardsiella piscicida infection in zebrafish (Danio rerio). To this end, we generated tlr5a-deficient (tlr5a[-/-]) zebrafish using CRISPR/Cas9 technology. Survival analysis following E. piscicida challenge revealed significantly higher survival rates and reduced bacterial loads in tlr5a[-/-] larvae and adults compared to wild-type (WT) controls. tlr5a[-/-] fish exhibited significantly lower expression of pro-inflammatory cytokines (tnfa, il6, and il-1β), chemokine (il8), and pathway genes (nfκb, myd88, and mapk14a) at multiple time points post-infection (0, 6, 12, 24, 48, and 72 hpi). DCFH-DA staining and Sudan Black/Neutral Red staining revealed elevated reactive oxygen species (ROS) levels and immune cell recruitment, respectively, indicating reduced ROS production and diminished neutrophil and macrophage infiltration in tlr5a[-/-] larvae. Antioxidant gene expression analysis revealed reduced levels of cat and nrf2 in tlr5a[-/-] larvae compared to WT. The results of this study indicate that tlr5a knockout attenuates excessive inflammation and improves resistance against E. piscicida infection, likely by reducing bacterial adhesion and suppressing NF-κB-mediated pro-inflammatory pathways. This study highlights tlr5a as a potential immunomodulatory target to enhance disease resistance in teleost aquaculture.},
}
@article {pmid40834600,
year = {2025},
author = {Chang, Y and Huang, H and Zhao, R and Diao, L and Xiong, W and Wan, P},
title = {Development of the CRISPR/Cas9 system for genome editing in Riemerella anatipestifer.},
journal = {Poultry science},
volume = {104},
number = {11},
pages = {105696},
pmid = {40834600},
issn = {1525-3171},
mesh = {*Riemerella/genetics ; *CRISPR-Cas Systems ; *Gene Editing/methods/veterinary ; Animals ; Poultry Diseases/microbiology ; Flavobacteriaceae Infections/veterinary/microbiology ; },
abstract = {Riemerella anatipestifer (R. anatipestifer) is an important pathogen responsible for high mortality rates and severe economic losses in the poultry industry. Research on R. anatipestifer is constrained by limited genetic manipulation tools, highlighting the need for an effective genome editing toolkit. The Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)/Cas9 system from Streptococcus pyogenes has been widely used as a genome editing tool for various bacteria, but has not been used in R. anatipestifer. In this study, we developed a CRISPR/Cas9-based genome editing system, pCasRA-SacB, specifically for R. anatipestifer. This shuttle vector contains the replication origin from R. anatipestifer plasmid pRA0726 and p15A origin for replication in both R. anatipestifer and Escherichia coli (E. coli). It also includes chloramphenicol and cefoxitin resistance genes, which allow for selection in E. coli and R. anatipestifer. The vector features BsaI and SalI sites, enabling the cloning of single guide RNA (sgRNA) and homologous arms. In addition, it contains the high-expression promoter of B739_0921 gene and the promoter of rpsL gene, facilitating the expression of sgRNA and Cas9 protein. Furthermore, the vector features the sucrose-sensitive gene sacB and the oriT region, which enable plasmid curing and the transfer of plasmid via conjugation. Notably, the pCasRA-SacB system enables rapid, efficient, and scarless genome editing in R. anatipestifer, including gene deletion, insertion, and point mutation in the dprA gene, with editing efficiencies of 54.2 %, 100.0 %, and 50.0 %, respectively. In summary, the pCasRA-SacB system not only expands the genome editing toolbox of R. anatipestifer but also is helpful in fundamental research in R. anatipestifer.},
}
@article {pmid40659814,
year = {2025},
author = {Levesque, S and Bauer, DE},
title = {CRISPR-based therapeutic genome editing for inherited blood disorders.},
journal = {Nature reviews. Drug discovery},
volume = {24},
number = {12},
pages = {907-925},
pmid = {40659814},
issn = {1474-1784},
mesh = {Humans ; *Gene Editing/methods ; *Genetic Therapy/methods ; *CRISPR-Cas Systems/genetics ; Animals ; *Hematologic Diseases/therapy/genetics ; Anemia, Sickle Cell/therapy/genetics ; },
abstract = {Therapeutic genome editing promises to transform medicine. Pivotal discoveries have provided a diverse and versatile set of tools to correct pathogenic mutations or produce protective alleles using CRISPR-based technologies. These innovative therapies are especially adaptable for blood and immune disorders, where clinical methods allow haematopoietic stem cells (HSCs) to be mobilized, harvested, engineered ex vivo and transplanted back into a patient to permanently replace their blood system. This paradigm has been exemplified with the first US Food and Drug Administration (FDA)-approved CRISPR-Cas9 therapy for sickle cell disease and β-thalassaemia, exa-cel (Casgevy). Although promising, efficient delivery of gene edits involves complicated ex vivo manipulation and toxic myeloablative conditioning. The quiescent and elusive nature of HSCs also brings associated challenges. In this Review, we explore the state-of-the-art genome editing technologies of nucleases, base editors and prime editors, which hold promise to address unmet clinical needs for patients with inherited haematological disorders. We highlight the progress made for several disorders and discuss the challenges that remain for ex vivo and in vivo targeting of HSCs for next-generation gene therapies.},
}
@article {pmid41309229,
year = {2025},
author = {Song, BS and Baek, YH and Kim, EH and Kwon, HI and Kim, AH and Lee, SH and Son, YB and Kim, SH and Song, MS and Choi, YK and Park, SJ},
title = {Development of an RT-LAMP-CRISPR/Cas12a assay for rapid and specific detection of Bandavirus dabieense.},
journal = {Journal of microbiology (Seoul, Korea)},
volume = {63},
number = {11},
pages = {e2506013},
doi = {10.71150/jm.2506013},
pmid = {41309229},
issn = {1976-3794},
support = {2022R1C1C1004704//National Research Foundation of Korea/ ; RS-2023-00301974//Ministry of Education/ ; //Korea Institute of Planning and Evaluation for Technology in Food, Agriculture, and Forestry/ ; RS-2022-IP322088//Ministry of Agriculture, Food and Rural Affairs/ ; },
mesh = {*Molecular Diagnostic Techniques/methods ; *Nucleic Acid Amplification Techniques/methods ; Sensitivity and Specificity ; Humans ; *CRISPR-Cas Systems ; *Severe Fever with Thrombocytopenia Syndrome/diagnosis/virology ; RNA, Viral/genetics ; *RNA Viruses/genetics/isolation &amp; purification ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {Bandavirus dabieense, a single-stranded RNA virus, is the causative agent of severe fever with thrombocytopenia syndrome (SFTS), a disease associated with high fatality rates. Early and accurate diagnosis is essential for improving clinical outcomes, particularly given the limited therapeutic options and high mortality rates associated with SFTS. However, while highly sensitive, conventional diagnostic methods such as PCR and qRT-PCR require specialized laboratory facilities and trained personnel, making them impractical for rapid detection in resource-limited settings. To address these challenges, we developed a rapid and highly sensitive assay for Bandavirus dabieense detection by integrating reverse transcription loop-mediated isothermal amplification (RT-LAMP) with CRISPR/Cas12a technology. LAMP primers and guide RNA sequences were designed to target the L gene, ensuring broad detection across viral genotypes. The optimized assay demonstrated a detection limit of 5 RNA copies per reaction, showing more sensitivity than qRT-PCR, and exhibited 100% concordance with qRT-PCR results in clinical samples. Given its speed, accuracy, and field applicability, this LAMP-CRISPR/Cas12a-based assay represents a promising diagnostic tool for early SFTSV detection, particularly in resource-constrained environments where conventional molecular diagnostics are not readily available.},
}
@article {pmid41307588,
year = {2025},
author = {Kumar, V and Verma, P},
title = {Advances in microbial biotechnology for sustainable wastewater reclamation: recent trends and future prospects.},
journal = {World journal of microbiology &amp; biotechnology},
volume = {41},
number = {12},
pages = {478},
pmid = {41307588},
issn = {1573-0972},
mesh = {*Wastewater/microbiology/chemistry ; *Biotechnology/methods/trends ; *Water Purification/methods ; Biodegradation, Environmental ; Bacteria/metabolism/genetics ; Microalgae/metabolism ; Bioelectric Energy Sources ; Waste Disposal, Fluid/methods ; Microbial Consortia ; },
abstract = {The growing demand for freshwater, coupled with the increasing volume of industrial and municipal wastewater, has intensified the need for sustainable and eco-friendly reclamation strategies. Recent advancements in microbial biotechnology have emerged as promising tools for developing cost-effective, efficient, and environmentally sustainable wastewater treatment (WWT) strategies for reuse and safe disposal. This mini-review explores current innovations, such as microbial consortia, bioaugmentation, and the microalgae-bacteria nexus, which have shown promising results in nutrient removal, enhanced degradation of complex pollutants (including emerging contaminants), and biomass valorization. Moreover, bioelectrochemical systems, such as microbial fuel cells (MFC) and microbial electrolysis cells (MEC), have revolutionized WWT by facilitating pollutant degradation while simultaneously generating bioelectricity or biohydrogen. This article also critically examines the role of CRISPR-based tools and 'omics' approaches, which have enabled the development of novel microbial strains and degradative pathways, enhancing wastewater reclamation in challenging environments. Furthermore, advancements through the integration of multi-omics and artificial intelligence, digital twins, and Internet of Things (IoT) for microbial optimization and real-time process control are discussed. The review highlights the role of microbial systems in resource recovery, supporting a circular economy by transforming wastewater into valuable bioresources. Additionally, this review addresses the major challenges and proposes future research directions for effective wastewater treatment. The novelty of this manuscript is that no single review explores the cutting-edge microbial biotechnologies for wastewater reclamation, uniquely integrating CRISPR-Cas genome editing, multi-omics analyses, and artificial intelligence-driven optimization to advance pollutant degradation and real-time process control in one place. This study concludes that by implementing multi-omics and artificial intelligence (AI)-driven optimization process for wastewater treatment can be effective towards wastewater treatment while simultaneously minimizing the environmental pollution.},
}
@article {pmid41307501,
year = {2025},
author = {Sahu, S and Boukherroub, R and Ritzenthaler, C and Szunerits, S},
title = {Emerging technologies for in-field plant virus detection: innovations and future directions.},
journal = {The Journal of general virology},
volume = {106},
number = {11},
pages = {},
pmid = {41307501},
issn = {1465-2099},
mesh = {*Plant Viruses/isolation &amp; purification/genetics ; *Plant Diseases/virology ; Biosensing Techniques/methods ; High-Throughput Nucleotide Sequencing ; CRISPR-Cas Systems ; Nanotechnology/methods ; },
abstract = {Plant virus infections pose a substantial threat to crop quality and productivity, contributing to considerable economic losses in global agriculture annually. Traditionally, laboratories have widely adopted serological techniques, such as ELISA, and molecular methods, including quantitative PCR, for virus diagnostics. More recently, sophisticated next-generation sequencing approaches have been introduced to improve the efficiency and reliability of virus detection and identification. However, the development of sensitive, rapid and low-cost methods for the on-site detection, quantification and identification of plant viruses remains an ongoing challenge and is still in its early days. Point-of-care technologies have not fully realized their potential in agriculture due to numerous challenges, such as the elevated cost of development, lack of standardized validation and insufficient field testing. Therefore, future success depends on addressing these technical, economic and regulatory hurdles, as well as considering the specific user needs within the agricultural context. In this mini-review, recent advancements in biosensing for on-site plant virus monitoring, involving nanotechnology-based sensors, clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated (Cas) systems, electrochemical and modern field-effect transistor-based sensors offering high sensitivity, speed and portability, are discussed. These technologies, when integrated with smartphone applications and/or machine learning modules, could enable real-time, field-deployable diagnostics for early disease management and sustainable agriculture. The aim is to raise awareness among plant virologists about this panel of emerging diagnostic concepts that could help improve current methods, ultimately facilitating the management of plant viral diseases.},
}
@article {pmid41306592,
year = {2025},
author = {Shi, L and Chen, H and Zhang, Z and Wang, Y and Ren, W and Huang, J},
title = {Evolving HPV diagnostics: current practice and future frontiers.},
journal = {Frontiers in cellular and infection microbiology},
volume = {15},
number = {},
pages = {1681779},
pmid = {41306592},
issn = {2235-2988},
mesh = {Humans ; *Papillomavirus Infections/diagnosis/virology ; Female ; *Molecular Diagnostic Techniques/methods/trends ; *Papillomaviridae/genetics/isolation &amp; purification ; Uterine Cervical Neoplasms/virology/diagnosis ; Early Detection of Cancer/methods ; High-Throughput Nucleotide Sequencing ; Nucleic Acid Amplification Techniques/methods ; Sensitivity and Specificity ; Polymerase Chain Reaction/methods ; Mass Screening/methods ; },
abstract = {Human papillomavirus (HPV) infection serves as a primary causative agent of cervical cancer, highlighting the importance of early screening and detection in mitigating the incidence and mortality rates of HPV-related diseases. Over the past decades, HPV detection technologies have evolved considerably, transitioning from traditional methods to more advanced, patient-centered approaches. This review provides a comprehensive overview of both established and emerging HPV detection strategies, with a particular focus on their clinical applicability, technical advantages, and limitations. Conventional methods such as hybrid capture and PCR-based assays remain the backbone of clinical screening, offering robust sensitivity and specificity. However, their reliance on invasive sampling and centralized laboratory infrastructure limits accessibility and patient compliance, particularly in low-resource settings. To address these limitations, emerging technologies-including CRISPR/Cas systems, droplet digital PCR (ddPCR), next-generation sequencing (NGS), isothermal amplification techniques (IAT) and artificial intelligence (AI) combined with hpv screening offer enhanced accuracy, rapid turnaround, and the potential for point-of-care deployment. In parallel, innovations in sampling such as self-collected vaginal swabs and liquid biopsy using urine, blood, or extracellular vesicles are improving test acceptability and broadening screening coverage. By summarizing current progress and highlighting ongoing challenges, this review aims to guide the development of more precise, non-invasive, and scalable HPV detection strategies to reduce the global burden of HPV-related disease, support global prevention efforts, and guide public health policies.},
}
@article {pmid41305866,
year = {2025},
author = {Naderi, S and Williamson, J and Sun, H and Joshi, S and Spera, RJ and Zaib, S and Sharma, S and Sun, C and Brodovskiy, A and Zawar, I and Kapur, J},
title = {Hydroxycarboxylic Acid Receptor 2 Mediates β-hydroxybutyrate's Antiseizure Effect in Mice.},
journal = {Annals of neurology},
volume = {},
number = {},
pages = {},
doi = {10.1002/ana.78098},
pmid = {41305866},
issn = {1531-8249},
support = {R01NS120945//United States National Institute of Health (NINDS)/ ; R37N119012//United States National Institute of Health (NINDS)/ ; },
abstract = {OBJECTIVE: The ketogenic diet, a high-fat, low-carbohydrate regimen, is often used to treat drug-resistant seizures and is being studied for Alzheimer's disease and other neuropsychiatric disorders. However, its mechanism of action remains unclear. β-hydroxybutyrate, a primary circulating ketone body produced by the ketogenic diet, may mediate its effects on seizures by binding to a recently identified Gi-coupled receptor: hydrocarboxylic acid receptor 2 (HCAR2).<br><br>METHODS: RNAscope in situ hybridization assay and real-time quantitative polymerase chain reaction were used to assess HCAR2 expression in the mouse brain. We generated HCAR2[-]/[-] using the CRISPR-Cas technique on an S129 mouse background. Whole-cell current-clamp was performed to measure the passive and active membrane properties of hippocampal dentate granule cells. The voltage-clamp was performed to record synaptic currents. Two complementary in vivo mouse models-continuous hippocampal stimulation to induce status epilepticus (SE) and kindling-were used to induce seizures.<br><br>RESULTS: HCAR2 was localized in dentate granule cells and microglia. In mice with HCAR2, &#x3b2;-hydroxybutyrate reduced neuronal excitability by hyperpolarizing the resting membrane potential, raising the action potential threshold, and reducing the firing frequency of dentate granule cells. &#x3b2;-hydroxybutyrate suppressed excitatory synaptic transmission. These effects were nullified in HCAR2[-]/[-] mice. HCAR2[-]/[-] mice showed no cognitive impairment. Moreover, &#x3b2;-hydroxybutyrate did not affect seizures in HCAR2[-]/[-] mice. However, it diminished both the duration and severity of seizures in HCAR2[+]/[+] mice.<br><br>INTERPRETATION: These findings demonstrate that HCAR2 mediates &#x3b2;-hydroxybutyrate's antiseizure effects by regulating neuronal excitability and synaptic transmission. These studies propose a new mechanism for the antiseizure action of the ketogenic diet. ANN NEUROL 2025.},
}
@article {pmid41305525,
year = {2025},
author = {Verma, N and O'Mahony, A and Mohammad, R and Keiser, D and Mosman, CW and Holden, D and Starr, K and Bauer, J and Bauer, B and Suntisukwattana, R and Atthaapa, W and Tantituvanont, A and Nilubol, D and Gladue, DP},
title = {The First CRISPR-Based Therapeutic (SL_1.52) for African Swine Fever Is Effective in Swine.},
journal = {Viruses},
volume = {17},
number = {11},
pages = {},
pmid = {41305525},
issn = {1999-4915},
mesh = {Animals ; *African Swine Fever/therapy/virology ; Swine ; *African Swine Fever Virus/genetics ; *CRISPR-Cas Systems ; *Gene Editing/methods ; Genome, Viral ; },
abstract = {African swine fever virus (ASFV) is a high-consequence pathogen that causes African swine fever (ASF), for which mortality rates can reach 90-100%, with death typically occurring within 14 days. ASF is currently a highly contagious pandemic disease responsible for extensive losses in pig production in multiple affected countries suffering from extended outbreaks. While a limited number of vaccines to prevent ASF are in use in south-east Asia, vaccines are not widely available, are only effective against highly homologous strains of ASFV, and must be used prior to an outbreak on a farm. Currently, there is no treatment for ASF and culling affected farms is the only response to outbreaks on farms to try and prevent spreading. CRISPR/Cas systems evolved as an adaptive immune response in bacteria and archaea that function by cleaving and disrupting the genomes of invading bacteriophage pathogens. CRISPR technology has since been leveraged into an array of endonuclease-based systems used for nucleic acid detection, targeting, genomic cleavage, and gene editing, making them particularly well-suited for development as sequence-specific therapeutic modalities. The programmability of CRISPR-based therapeutics offers a compelling new way to rapidly and specifically target pathogenic viral genomes simply by using different targeting guide RNAs (gRNA) as an adaptable antiviral modality. Here, we demonstrate for the first time a specific CRISPR/Cas9 multiplexed gRNA system that targets the African swine fever viral genome, resulting in sequence-specific cleavage, leading to the reduction in the viral load in infected animals, and subsequent recovery from an otherwise lethal dose of ASFV. Moreover, animals that recovered had protective immunity to subsequent homologous ASFV infection.},
}
@article {pmid41062067,
year = {2025},
author = {Izumikawa, T and Moriya, A and Nakato, E and Yamamoto, K and Sano, R and Akiyama, T and Kinoshita-Toyoda, A and Toyoda, H and Nakato, H},
title = {In vivo analysis of Drosophila chondroitin sulfate biosynthetic genes.},
journal = {The Journal of biological chemistry},
volume = {301},
number = {11},
pages = {110783},
pmid = {41062067},
issn = {1083-351X},
mesh = {Animals ; *Chondroitin Sulfates/biosynthesis/genetics ; *Sulfotransferases/genetics/metabolism ; *N-Acetylgalactosaminyltransferases/genetics/metabolism ; *Drosophila Proteins/genetics/metabolism ; *Drosophila melanogaster/genetics/metabolism ; Mutation ; CRISPR-Cas Systems ; },
abstract = {Chondroitin sulfate (CS) is an evolutionarily conserved class of glycosaminoglycans and is found in most animal species. Previous studies of CS-deficient Drosophila models, Chondroitin sulfate synthase (Chsy), and Chondroitin polymerizing factor (Chpf) mutants demonstrated the importance of CS in the structural integrity of the basement membrane and organ shape maintenance. However, biosynthetic mechanisms of Drosophila CS remain to be elucidated. To investigate the CS biosynthesis in Drosophila, we generated mutants for two additional biosynthetic enzyme genes, CS N-acetylgalactosaminyltransferase (Csgalnact) and CS 4-O sulfotransferase (C4st), using CRISPR-Cas9 mutagenesis. Csgalnact-null mutants show moderate changes in CS biosynthesis, including reduced CS in the larval brain and altered CS chain length. We found that this gene is dispensable for normal viability and morphogenesis. On the other hand, C4st mutants show more severe defects, including a high level of lethality and a folded wing phenotype. The C4st mutation not only eliminates CS sulfation but increases production of unsulfated chondroitin, suggesting the existence of a compensatory feedback mechanism. Both Csgalnact and C4st mutants show impaired adult negative geotaxis behavior, consistent with the role of CS proteoglycan in the neuromuscular systems. Our study revealed unique and poorly understood features of invertebrate CS biosynthesis and provides novel in vivo toolsets to investigate CSPG functions in development.},
}
@article {pmid41005478,
year = {2025},
author = {Veit, MC and Stauder, R and Bai, Y and Gabhrani, R and Schmidt, M and Klähn, S and Lai, B},
title = {The necessity of multi-parameter normalization in cyanobacterial research: A case study of the PsbU in Synechocystis sp. PCC 6803 using CRISPRi.},
journal = {The Journal of biological chemistry},
volume = {301},
number = {11},
pages = {110763},
pmid = {41005478},
issn = {1083-351X},
mesh = {*Synechocystis/genetics/metabolism/growth &amp; development ; *Photosystem II Protein Complex/genetics/metabolism ; *Bacterial Proteins/genetics/metabolism ; Oxygen/metabolism ; Photosynthesis ; *CRISPR-Cas Systems ; Chlorophyll A/metabolism ; },
abstract = {Photosystem II (PSII) is a multiprotein complex and plays a central role in oxygenic photosynthesis. PsbU, a 12 kDa subunit of PSII, is associated with thermotolerance and structural stabilization of the oxygen-evolving complex in cyanobacteria. Corresponding knockout strains showed decreased oxygen evolution rates, although the growth was not impaired. In this study, we provide further insights into the consequences of PsbU perturbations and propose to revisit the impact of PsbU on cell physiology. We made use of CRISPRi to knock down the psbU gene in Synechocystis sp. PCC 6803, and assessed previously described effects referred to different biomass parameters including optical density, chlorophyll a content and cell number. After knocking down psbU, the growth rate was decreased by 15% based on counting the cell number, while this effect was not observed when monitoring optical density. Furthermore, the oxygen evolution rate per cell in the psbU knockdown strain did not show a significant difference compared to the control groups, which was probably due to its larger cell size and higher chlorophyll a content per cell. The decreased quantum efficiency of pigments was compensated by the increased pigment content on the single-cell level in the knockdown strain. Our results complement previous analyses and highlight the importance of evaluating cyanobacterial physiology based on different biomass quantitative units to avoid misinterpretation of the results.},
}
@article {pmid39938795,
year = {2025},
author = {Fu, ZH and Cheng, S and Li, JW and Zhang, N and Wu, Y and Zhao, GR},
title = {Synthetic tunable promoters for flexible control of multi-gene expression in mammalian cells.},
journal = {Journal of advanced research},
volume = {78},
number = {},
pages = {351-361},
doi = {10.1016/j.jare.2025.02.008},
pmid = {39938795},
issn = {2090-1224},
mesh = {*Promoter Regions, Genetic/genetics ; Humans ; *Synthetic Biology/methods ; Genes, Reporter ; CRISPR-Cas Systems/genetics ; Animals ; HEK293 Cells ; *Gene Expression Regulation/genetics ; },
abstract = {INTRODUCTION: Synthetic biology revolutionizes our ability to decode and recode genetic systems. The capability to reconstruct and flexibly manipulate multi-gene systems is critical for understanding cellular behaviors and has significant applications in therapeutics.<br><br>OBJECTIVES: This study aims to construct a diverse library of synthetic tunable promoters (STPs) to enable flexible control of multi-gene expression in mammalian cells.<br><br>METHODS: We designed and constructed synthetic tunable promoters (STPs) that incorporate both a universal activation site (UAS) and a specific activation site (SAS), enabling multi-level expression control via the CRISPR activation (CRISPRa) system. To evaluate promoter activity, we utilized Massively Parallel Reporter Assays (MPRA) to assess the basal strengths of the STPs and their activation responses. Next, we constructed a three-gene reporter system to assess the capacity of the synthetic promoters for achieving multilevel control of single-gene expression within multi-gene systems.<br><br>RESULTS: The promoter library contains 24,960 unique non-redundant promoters with distinct sequence characteristics. MPRA revealed a wide range of promoter activities, showing different basal strengths and distinct activation levels when activated by the CRISPRa system. When regulated by targeting the SAS, the STPs exhibited orthogonality, allowing multilevel control of single-gene expression within multi-gene systems without cross-interference. Furthermore, the combinatorial activation of STPs in a multi-gene system enlarged the scope of expression levels achievable, providing fine-tuned control over gene expression.<br><br>CONCLUSION: We provide a diverse collection of synthetic tunable promoters, offering a valuable toolkit for the construction and manipulation of multi-gene systems in mammalian cells, with applications in gene therapy and biotechnology.},
}
@article {pmid39914488,
year = {2025},
author = {Yu, H and Zhang, G and Liu, J and Liu, P and Peng, H and Teng, Z and Li, Y and Ren, X and Fu, C and Tang, J and Li, M and Wang, Y and Wang, L and Peng, L},
title = {A functional cascading of lignin modification via repression of caffeic acid O-methyltransferase for bioproduction and anti-oxidation in rice.},
journal = {Journal of advanced research},
volume = {78},
number = {},
pages = {1-9},
doi = {10.1016/j.jare.2025.01.048},
pmid = {39914488},
issn = {2090-1224},
mesh = {*Lignin/metabolism ; *Oryza/metabolism/genetics ; *Methyltransferases/metabolism/genetics ; Oxidation-Reduction ; Antioxidants/metabolism ; Cadmium/metabolism ; Biomass ; Coumaric Acids/metabolism ; Plant Proteins/metabolism/genetics ; Mutation ; CRISPR-Cas Systems ; },
abstract = {INTRODUCTION: Crop straws provide substantial biomass resources that are transformable for sustainable biofuels and valuable bioproducts. However, the natural lignocellulose recalcitrance results in an expensive biomass process and secondary waste liberation. As lignin is a major recalcitrant factor, genetic engineering of lignin biosynthesis is increasingly being implemented in bioenergy crops, but much remains unclear about the desired lignocellulose alteration and resulting function.<br><br>OBJECTIVES: This study attempted to explore the mechanisms of lignin modification responsible for efficient lignocellulose conversion in vitro and an effective plant anti-oxidation response in vivo.<br><br>METHODS: We initially selected specific rice mutants by performing modern CRISPR/cas9 editing with caffeic acid O-methyltransferase involved in the synthetic pathways of monolignols (G, S) and ferulic acid (FA), and then explored lignocellulose conversion and plant cadmium (Cd) accumulation using advanced chemical, biochemical and thermal-chemical analyses.<br><br>RESULTS: Notable lignin modification was achieved from the predominately synergistic down-regulation of S-monomer synthesis in three mutants. This consequently upgraded lignocellulose porosity by up to 1.8 folds to account for significantly enhanced biomass saccharification and bioethanol production by 20&#xa0;%-26&#xa0;% relative to the wild-type. The modified lignin also favors the dissection of diverse lignin nanoparticles with dimensions reduced by 1.5-1.9 folds, applicable for thermal-chemical conversion into the carbon quantum dots with increased yields by 15&#xa0;% and 31&#xa0;%. The proportions of G-monomers and FA were significantly increased in the mutants, and the lignin extractions were further assayed with higher activities for two standard antioxidants (DPPH and ABTS) in vitro compared to the wild-type, revealing a distinctively enhanced plant antioxidative capacity in the mutants. Water culture showed that young mutant seedlings accumulated more Cd than wild-type did (p&#xa0;<&#xa0;0.01, n&#xa0;=&#xa0;3), suggesting effective heavy metal phytoremediation in the mutants.<br><br>CONCLUSION: A hypothetical model of characteristic lignin modification for specific S-monomer reduction, accountable for improved lignocellulose recalcitrance, was proposed. It provides a powerful strategy for achieving high-yield biofuels and value-added bioproducts or enhancing plant antioxidative capacity for heavy metal phytoremediation.},
}
@article {pmid41305485,
year = {2025},
author = {Iftehimul, M and Hasan, NA and Bass, D and Bashar, A and Haque, MM and Santi, M},
title = {Combating White Spot Syndrome Virus (WSSV) in Global Shrimp Farming: Unraveling Its Biology, Pathology, and Control Strategies.},
journal = {Viruses},
volume = {17},
number = {11},
pages = {},
doi = {10.3390/v17111463},
pmid = {41305485},
issn = {1999-4915},
support = {2022/21/Other//Ocean Country Partnership Programme (OCPP), Blue Planet Fund/ ; },
mesh = {*White spot syndrome virus 1/pathogenicity/immunology/physiology/genetics ; Animals ; *Aquaculture/methods ; *Penaeidae/virology/immunology ; Immunity, Innate ; Viral Vaccines/immunology ; },
abstract = {White Spot Syndrome Virus (WSSV) is one of the most devastating viral pathogens affecting shrimp, causing severe economic losses to the global farmed shrimp trade. The globalization of live shrimp trade and waterborne transmission have facilitated the rapid spread of WSSV across major shrimp-producing countries since its initial emergence. The present review gives an updated account of WSSV biology, pathology, transmission dynamics, and recent developments in control measures. The virus, a double-stranded DNA virus of the Nimaviridae family, utilizes advanced immune evasion strategies, resulting in severe mortality. Shrimp lack adaptive immunity and hence rely predominantly on innate immunity, which is insufficient to mount an effective response against severe infections. Traditional disease control measures such as augmented biosecurity, selective breeding, and immunostimulants have, despite extensive research, achieved only limited success. New biotechnological tools such as RNA interference, CRISPR-Cas gene editing, and nanotechnology offer tremendous potential for disease mitigation. In parallel, the development of DNA and RNA vaccines targeting WSSV structural proteins, such as VP28, holds significant promise for stimulating the shrimp immune system. This review highlights the urgent need for a convergent approach to sustainable disease management in global shrimp aquaculture, with interdisciplinarity playing a pivotal role in shaping the future of WSSV control.},
}
@article {pmid41304933,
year = {2025},
author = {Yue, Y and Xu, Z and Soteyome, T and Premarathna, M and Yin, X and Liu, J},
title = {Phage Encapsulation and Delivery Technology: A Strategy for Treating Drug-Resistant Pathogenic Microorganisms.},
journal = {Pharmaceuticals (Basel, Switzerland)},
volume = {18},
number = {11},
pages = {},
doi = {10.3390/ph18111688},
pmid = {41304933},
issn = {1424-8247},
abstract = {Antimicrobial resistance (AMR) is one of the most critical challenges to global public health in the 21st century, posing a significant threat to healthcare systems and human health due to treatment failure and high mortality. The World Health Organization (WHO) estimates that, without effective interventions, AMR-associated infections could cause 10 million deaths annually and economic losses of up to 100 trillion US dollars by 2050. The rapid spread of drug-resistant strains, especially in hospital and community settings, has significantly reduced the efficacy of traditional antibiotics. With the continuous advancements in relevant research, bacteriophage (Phage) therapy is constantly innovating in the antimicrobial field. The application of frontier technologies, such as phage cocktails and engineered phages, has significantly enhanced the broad spectrum and high efficiency of phage therapy, which is gradually becoming a new generation of tools to replace antibiotics and effectively combat pathogenic bacteria. However, phage therapy is facing several challenges, including phage inactivation by gastric acid, enzymes, ultraviolet light, and mechanical stress, as well as the potential risk of bacterial phage resistance. Advanced encapsulation technologies such as electrospun fibers, liposomes, chitosan nanoparticles, and electrospray provide solutions to these problems by protecting phage activity and enabling controlled release and targeted delivery. This review addresses phage therapeutic studies of Salmonella, Pseudomonas aeruginosa, Staphylococcus aureus, Escherichia coli, and Listeria monocytogenes, summarizes the recent advances in phage research, and details the current development and applications of encapsulated phage technologies across various delivery modes.},
}
@article {pmid41304797,
year = {2025},
author = {Sharma, A and Sharma, V and Sharma, S and Sharma, S and Sharma, M and Sivanesan, I},
title = {Advanced Nanosystems and Emerging Therapies: Innovations in Tuberculosis Treatment and Drug Resistance.},
journal = {Pharmaceutics},
volume = {17},
number = {11},
pages = {},
doi = {10.3390/pharmaceutics17111459},
pmid = {41304797},
issn = {1999-4923},
abstract = {Tuberculosis (TB) remains a significant worldwide health challenge due to the limitations of conventional treatments and the rising incidence of drug-resistant Mycobacterium tuberculosis strains. This review consolidates the advancements in nanotechnology-based therapeutics, inhalable formulations, CRISPR-Cas tools, host-directed therapies (HDTs), and nanoparticle-based vaccine development aimed at enhancing TB management. Novel nanocarriers such as liposomes, solid-lipid nanoparticles (SLNs), dendrimers, and polymeric nanoparticles (NPs) offer enhanced bioavailability of drugs, sustained release, as well as targeted delivery to infected macrophages, thereby reducing systemic toxicity and dosing frequency. Inhalable nanomedicines provide localized delivery to the pulmonary site, enhancing the concentration of the drug at the primary site of infection. CRISPR-Cas technology is emerging as a transformative approach to disabling drug-resistant genes and enhancing diagnostic precision. HDTs, including agents like vitamin D and metformin, show potential in modulating host immune responses and enhancing pathogen clearance. Nanoparticle-based vaccines, including mRNA and antigen-conjugated platforms, aim to overcome the limitations of the BCG vaccine by enhancing antigen presentation and eliciting stronger, longer-lasting immunity. Collectively, these modalities mark a shift toward more personalized, effective, and less toxic TB therapies. However, challenges such as regulatory approval, safety, scalability, and accessibility remain. This review highlights the integrated potential of nanomedicine, gene editing, and immunomodulation to transform TB care and combat drug resistance, paving the way for more robust and durable treatment strategies.},
}
@article {pmid41304764,
year = {2025},
author = {Alidriss, OM and AlSudais, H and Alhumaidan, OS and Altwaijry, HD and Bakhsh, A and Almuhanna, Y and Alkudmani, ZS and Alqarni, IA and Alenazi, D and Aljasham, AT and Jamous, YF},
title = {Targeted Drug Delivery Strategies in Overcoming Antimicrobial Resistance: Advances and Future Directions.},
journal = {Pharmaceutics},
volume = {17},
number = {11},
pages = {},
doi = {10.3390/pharmaceutics17111426},
pmid = {41304764},
issn = {1999-4923},
abstract = {Antimicrobial resistance (AMR) is a present, pressing global public health crisis associated with rising morbidity and mortality rates due to previously curable infectious disease. Targeted drug delivery is an important approach to address AMR due to its ability to improve the therapeutic performance of antibiotics without leading to any adverse effects or organ toxicities. In this review we explore molecular mechanisms of AMR and drawbacks of conventional antibiotic therapies and discuss unique drug delivery approaches to compensate these. Nanoparticulate carrier systems, stimuli-responsive systems, antibody-drug conjugates, and CRISPR-Cas systems are some of the carrier method designs that are promising for tackling hard to treat infections related to pathogenic strains and biofilms due to their features. Many of these are among the most significant advances in the field. However, there are many challenges to be overcome, with biological limitations, scaling and regulatory challenges, etc., before they can be employed in commercial applications. Materials are being developed, and an approach standardized and applicable to future work is in development to improve the efficiency of targeted delivery systems. Controlled drug delivery, which could be the answer to an increasing AMR problem, will not only help in alerting awareness among individuals but will also help in prolonging the activity of antibiotics by providing synergistic interdisciplinary solutions. This review emphasizes the complementary role of targeted drug delivery in transitioning from laboratory investigations to clinical therapy. It addresses underrepresented aspects, including new materials, scalability, regulatory considerations, and ethical implications, while offering a roadmap for translating innovations into next-generation antimicrobials.},
}
@article {pmid41304743,
year = {2025},
author = {Zhang, H and Li, Y and Li, J and Li, X and Li, T},
title = {Base and Prime Editing for Inherited Retinal Diseases: Delivery Platforms, Safety, Efficacy, and Translational Perspectives.},
journal = {Pharmaceutics},
volume = {17},
number = {11},
pages = {},
doi = {10.3390/pharmaceutics17111405},
pmid = {41304743},
issn = {1999-4923},
support = {U22A20311//he National Natural Science Foundation of China/ ; 82571246//the National Natural Science Foundation of China/ ; 82388101//the National Natural Science Foundation of China/ ; 23J41900200//Science and Technology Commission of Shanghai Municipality/ ; SHWSRS(2025)_071//Shanghai "Rising Stars of Medical Talents" Youth Development Program/ ; },
abstract = {Inherited retinal diseases (IRDs) are a clinically and genetically heterogeneous spectrum of disorders that lead to progressive and irreversible vision loss. Gene therapy is the most promising emerging treatment for IRDs. While gene augmentation strategies have demonstrated clinical benefit and results within the first approved ocular gene therapy, their application is restricted by adeno-associated virus (AAV) packaging capacity and limited efficacy for dominant mutations. Recent breakthroughs in precision genome editing, particularly base editing (BE) and prime editing (PE), have provided alternatives capable of directly correcting pathogenic variants. BE enables targeted single-nucleotide conversions, whereas PE further allows for precise insertions and deletions, both circumventing the double-strand DNA cleavage or repair processes typically induced by conventional CRISPR-Cas editing systems, thereby offering advantages in post-mitotic retinal cells. Preclinical investigations across murine and non-human primate models have demonstrated the feasibility, molecular accuracy, and preliminary safety profiles of these platforms in targeting IRD-associated mutations. However, critical challenges remain before clinical application can be realized, including limited editing efficiency in photoreceptors, interspecies variability in therapeutic response, potential risks of off-target effects, and barriers in large-scale vector manufacturing. Moreover, the delivery of genome editors to the outer retina remains suboptimal, prompting intensive efforts in capsid engineering and the development of non-viral delivery systems. This review synthesizes the current progress in BE and PE optimization, highlights innovations in delivery platforms that encompass viral and emerging non-viral systems and summarizes the major barriers to clinical translation. We further discuss AI-driven strategies for the rational design of BE/PE systems, thereby outlining their future potential and perspectives in the treatment of IRDs.},
}
@article {pmid41303344,
year = {2025},
author = {Zhang, X and Che, J and Li, Z and Bao, B and Fan, C},
title = {The Mutation of piezo1 Weakens the Intermuscular Bones in Zebrafish and Crucian Carp.},
journal = {International journal of molecular sciences},
volume = {26},
number = {22},
pages = {},
doi = {10.3390/ijms262210851},
pmid = {41303344},
issn = {1422-0067},
support = {2023YFD2400300//National Key Research and Development Program of China/ ; 32170514//National Natural Science Foundation of China/ ; },
mesh = {Animals ; *Zebrafish/genetics ; *Carps/genetics ; *Ion Channels/genetics/metabolism ; *Mutation ; *Zebrafish Proteins/genetics/metabolism ; *Bone and Bones/metabolism ; CRISPR-Cas Systems ; *Fish Proteins/genetics/metabolism ; },
abstract = {Intermuscular bones (IBs), unique skeletal features found only in teleost fishes, pose significant challenges to food processing and consumption. While recent studies have identified several key genetic regulators of IB development, the role of mechanosensory mechanisms remains largely unexplored. This study investigated the role of Piezo1, a critical mechanosensitive ion channel, in IB formation using zebrafish and crucian carp models. Our findings demonstrated that piezo1 was expressed in the myoseptum of zebrafish, and CRISPR/Cas9-mediated knockout of this gene resulted in shorter and smaller IBs. Similar knockout experiments in crucian carp confirmed the conserved role of Piezo1 across cyprinid species. These results established Piezo1 as a key regulator of IB development, providing new insights into the molecular mechanisms underlying this process and suggesting potential strategies for breeding IB-free fish strains through modulation of mechanosensory pathways.},
}
@article {pmid41302913,
year = {2025},
author = {Ayaz, S and Kong, WW and Wang, J and Liu, SH and Xu, JP},
title = {Host Immunity Mechanisms Against Bacterial and Viral Infections in Bombyx mori.},
journal = {Insects},
volume = {16},
number = {11},
pages = {},
doi = {10.3390/insects16111167},
pmid = {41302913},
issn = {2075-4450},
abstract = {The domesticated silkworm, Bombyx mori, is a highly valued biodiversity and economic asset, acclaimed for its silk production, besides making important contributions to various scientific disciplines. However, the sericulture industry faces ongoing threats from bacterial and viral infections, which severely impact silkworm health and silk yield. This review provides a comprehensive overview of the innate immune response of B. mori against bacterial and viral pathogens, emphasizing the fundamental molecular and cellular defense mechanisms. We explore the humoral and cellular immune response using antimicrobial peptides (AMPs), pattern recognition receptors (PRRs) like peptidoglycan recognition protein (PGRP), and glucan recognition protein (GRP), which activate canonical signaling pathways. The review further highlights the molecular mechanisms underlying the silkworm's defense against viruses, incorporating RNA interference (RNAi), apoptosis, and distinct signaling pathways such as Toll and Imd, JAK/STAT, and STING. We also discussed the viral suppression strategies and modulation of host metabolism during infection. Furthermore, the review explores the recent use of CRISPR-Cas gene editing to enhance disease resistance, presenting a promising avenue for mitigating pathogen-induced losses in sericulture. By elucidating these mechanisms, the work provides a synthesis that is critical in terms of developing particular interventions and developing more resistant silkworm strains to ensure that the industry of sericulture becomes viable and productive.},
}
@article {pmid41301838,
year = {2025},
author = {Dziedzic, A and Kubina, R and Skonieczna, M and Madej, M and Fiegler-Rudol, J and Abid, M and Nadhim, D and Tanasiewicz, M},
title = {CRISPR Genome Editing in Personalized Therapy for Oral and Maxillofacial Diseases: A Scoping Review.},
journal = {Biomedicines},
volume = {13},
number = {11},
pages = {},
doi = {10.3390/biomedicines13112745},
pmid = {41301838},
issn = {2227-9059},
abstract = {Background: CRISPR/Cas genome editing is emerging as a powerful tool in oral and maxillofacial medicine, with potential applications in personalized therapies for conditions that currently lack durable treatments. Objectives: This scoping review aimed to map existing evidence on CRISPR-based applications in oral and maxillofacial fields, rather than to assess treatment effectiveness. Methods: A systematic search of PubMed, Scopus, Web of Science, and ClinicalTrials.gov (2012-2024) identified studies and registered trials involving CRISPR with oral health relevance. Eligible articles included peer-reviewed experimental reports and clinical trials. Results: From 1437 records, 121 studies met inclusion criteria: 106 preclinical reports and 15 clinical or translational studies. Investigated domains included oral cancer therapy, hereditary craniofacial syndromes, regenerative strategies, infectious disease models, and pathogen detection. Early clinical efforts focus mainly on CRISPR-edited T-cell immunotherapies in oncology. Major barriers include off-target effects, delivery challenges, regulatory complexity, and ethical concerns. Conclusions: CRISPR-based bioengineering shows strong promise for precision care in oral and maxillofacial medicine. However, current evidence remains largely preclinical and heterogeneous. No clinical recommendations can yet be made, and translation will depend on rigorous late-phase trials, ethical oversight, and health-economic evaluation.},
}
@article {pmid41301629,
year = {2025},
author = {Wang, Y and Li, L and Liang, Y and Xu, K and Ye, Y and He, M},
title = {Phage Therapy for Acinetobacter baumannii Infections: A Review on Advances in Classification, Applications, and Translational Roadblocks.},
journal = {Antibiotics (Basel, Switzerland)},
volume = {14},
number = {11},
pages = {},
doi = {10.3390/antibiotics14111134},
pmid = {41301629},
issn = {2079-6382},
support = {82302568//National Natural Science Foundation of China/ ; 2308085QH283//Anhui Provincial National Science Foundation/ ; 2022xkjT012//Basic and Clinical Collaboration Enhancement Program Foundation of Anhui Medical University/ ; },
abstract = {The global spread of carbapenem-resistant Acinetobacter baumannii (CRAB) poses a severe public health threat, driving growing interest in phage-based precision antibacterial strategies. This systematic review synthesizes recent advances in the field of A. baumannii phage. Modern taxonomy, based on whole-genome phylogeny, has reclassified the majority of A. baumannii phages into the class Caudoviricetes, revealing distinct evolutionary clades that correlate with host tropism and biological properties, superseding the traditional morphological families (Myoviridae, Siphoviridae, Podoviridae). To overcome limitations of natural phage therapy, such as narrow host range, cocktail therapies (ex vivo resistance mutation rates < 5%) and phage-antibiotic synergism (enabling antibiotic efficacy at 1/4 minimum inhibitory concentration) have significantly enhanced antibacterial efficacy. Preclinical models demonstrate that phage therapy efficiently clears pathogens in pneumonia models and promotes the healing of burn wounds and diabetic ulcers via immunomodulatory mechanisms. Technical optimizations include nebulized inhalation delivery achieving 42% alveolar deposition, and thermosensitive hydrogels enabling sustained release over 72 h. Genetic engineering approaches, such as host range expansion through tail fiber recombination and CRISPR/Cas-mediated elimination of lysogeny, show promise. However, the genetic stability of engineered phages requires further validation. Current challenges remain, including limited host spectrum, the absence of clinical translation standards, and lagging regulatory frameworks. Future efforts must integrate metagenomic mining and synthetic biology strategies to establish a precision medicine framework encompassing resistance monitoring and personalized phage formulation, offering innovative solutions against CRAB infections.},
}
@article {pmid41301547,
year = {2025},
author = {Mohammed, A and Ibrahim, NA and Basher, NS},
title = {Protein Engineering and Drug Discovery: Importance, Methodologies, Challenges, and Prospects.},
journal = {Biomolecules},
volume = {15},
number = {11},
pages = {},
doi = {10.3390/biom15111628},
pmid = {41301547},
issn = {2218-273X},
support = {IMSIU-DDRSP2501//Imam Mohammad ibn Saud Islamic University/ ; },
mesh = {*Drug Discovery/methods ; *Protein Engineering/methods ; Humans ; Animals ; Recombinant Proteins/therapeutic use/chemistry/genetics ; },
abstract = {Protein engineering is a rapidly evolving field that plays a critical role in transforming drug discovery and development. This innovative field harnesses the unique structural and functional properties of engineered proteins, such as monoclonal antibodies, nanobodies, therapeutic enzymes, and cytokines, to address complex diseases more effectively than traditional small-molecule drugs. These biologics not only enhance therapeutic specificity but also minimize adverse effects, marking a significant advancement in patient care. However, the journey of protein engineering is not without challenges. Issues related to protein folding, stability, and potential immunogenicity pose significant complications. Additionally, navigating the complex regulatory landscape can delay the transition from laboratory to clinical application. Addressing these hurdles requires the integration of cutting-edge technologies, including phage and yeast display technology, CRISPR, and advanced computational modeling, which enhance the predictability and efficiency of protein design. In this review, we explore the multifaceted impact of protein engineering on modern medicine, highlighting its potential to transform treatment paradigms, methodologies, challenges, and the successful development and approval of recombinant protein-based therapies. By navigating the complexities and leveraging technological advancements, the field is poised to unlock new therapeutic possibilities, ultimately improving patient outcomes and transforming healthcare.},
}
@article {pmid41301473,
year = {2025},
author = {Gibril, BAA and Chai, X and Xu, J},
title = {From Correlation to Causation: Defining Gene and RNA Function in Poultry Muscle Biology Using In Vivo Genetic Tools.},
journal = {Biomolecules},
volume = {15},
number = {11},
pages = {},
doi = {10.3390/biom15111554},
pmid = {41301473},
issn = {2218-273X},
support = {20242BCE50051//Science and Technology Research Project of the Education Department of Jiangxi Province/ ; },
mesh = {Animals ; *Muscle, Skeletal/metabolism ; Muscle Development/genetics ; *Poultry/genetics ; Muscular Diseases/genetics ; Transcriptome ; CRISPR-Cas Systems ; *RNA/genetics/metabolism ; },
abstract = {A central challenge in functional genomics is understanding the difference between correlative transcriptomic observations and definitive causal understanding of gene function in vivo. Poultry skeletal muscle, a system of significant agricultural and biological importance, demonstrates this challenge. While transcriptomic studies have cataloged extensive RNA expression dynamics during muscle development and in growth-related myopathies like wooden breast, establishing causative roles for these molecules is lacking. This review synthesizes how advanced genetic tools are now enabling a shift from correlation to causation in avian muscle biology. We detail how viral vectors (e.g., adenovirus, lentivirus, and RCAS) and CRISPR/Cas9 systems have provided direct in vivo validation of the functional roles of specific mRNAs, miRNAs, lncRNAs, and circRNAs in regulating myogenesis, hypertrophy, and atrophy. We contrast this success in fundamental biology with the study of myopathies, which remains largely descriptive. Here, a wealth of transcriptomic data has identified dysregulated pathways, including ECM remodeling, metabolism, and inflammation, but functional validation for most candidates is absent. We argue that the critical next step is to apply this established functional genomics toolkit to disease models. By defining causal mechanisms, this research will not only address a major agricultural issue but also provide a model for using genetic tools to dissect complex traits in a post-genomic era.},
}
@article {pmid41300819,
year = {2025},
author = {Kansal, R},
title = {Curing Sickle Cell Disease by Allogeneic Hematopoietic Stem Cell (HSC) Transplantation Toward In Vivo HSC Gene Therapy.},
journal = {Genes},
volume = {16},
number = {11},
pages = {},
doi = {10.3390/genes16111367},
pmid = {41300819},
issn = {2073-4425},
mesh = {*Anemia, Sickle Cell/therapy/genetics ; Humans ; *Hematopoietic Stem Cell Transplantation/methods ; *Genetic Therapy/methods ; Gene Editing/methods ; CRISPR-Cas Systems ; Transplantation, Homologous ; Animals ; Hematopoietic Stem Cells/metabolism ; },
abstract = {Sickle cell disease comprises a group of prevalent inherited disorders defined by an underlying sickle cell allele that forms sickle hemoglobin. The incidence of this disease is rising, with more than 500,000 children born with it globally. The disease carries significant morbidity and mortality. Its only curative treatment was an allogeneic hematopoietic stem cell (HSC) transplant (HSCT) until late 2023, when two one-time gene therapies were approved for treating patients aged 12 years or older with severe sickle cell disease. This work aims to inform readers about these two gene therapies: one lentiviral-based and the other nonviral. The latter is based on the Nobel Prize-winning discovery of clustered, regularly interspaced, short, palindromic repeats (CRISPR)/CRISPR-associated (Cas)9 proteins and single-guide RNA (sgRNA)-based genome editing. Both approved gene therapies require an autologous HSCT with ex vivo genetically edited autologous hematopoietic stem and progenitor cells. Therefore, access to these gene therapies is limited to specialized centers with expertise in HSCTs. This review is meant for students, researchers, and clinical practitioners. It explains the basis for both approved gene therapies, their mechanisms of action, differences, risks, and other lentiviral-based and CRISPR-Cas9-based ex vivo gene therapies for sickle cell disease in clinical development. Additionally, it discusses the current state of preclinical studies for in vivo HSC gene therapy for sickle cell disease, which utilize advanced genome editing technologies developed after CRISPR-Cas9-sgRNA-based genome editing. In vivo HSC gene therapy, after it is clinically developed, would eliminate the need for an HSCT in receiving gene therapy and vastly increase access for numerous patients worldwide, even in low-income countries with the most significant disease burden.},
}
@article {pmid41300715,
year = {2025},
author = {Sapakhova, Z and Kanat, R and Daurov, D and Daurova, A and Shamekova, M and Zhambakin, K},
title = {The Enhancement of Fungal Disease Resistance in Major Staple Crops Using CRISPR-Cas Technology.},
journal = {Genes},
volume = {16},
number = {11},
pages = {},
doi = {10.3390/genes16111263},
pmid = {41300715},
issn = {2073-4425},
support = {BR21882269//Ministry of Science and Higher Education of the Republic of Kazakhstan/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; *Disease Resistance/genetics ; *Plant Diseases/microbiology/genetics ; *Crops, Agricultural/genetics/microbiology ; Gene Editing/methods ; Fungi/pathogenicity/genetics ; Plants, Genetically Modified/genetics ; },
abstract = {Fungal pathogens represent a major constraint to global agricultural productivity, causing a wide range of plant diseases that severely affect staple crops such as cereals, legumes, and vegetables. These infections result in substantial yield losses, deterioration of grain and produce quality, and significant economic impacts across the entire agri-food sector. Among phytopathogens, fungi are considered the most destructive, causing a wide range of diseases such as powdery mildew, rusts, fusarium head blight, smut, leaf spot, rots, late blight, and other fungal pathogens. Traditional plant protection methods do not always provide long-term effectiveness and environmental safety, which requires the introduction of innovative approaches to creating sustainable varieties. CRISPR-Cas technology opens up new opportunities for targeted genome editing, allowing the modification or silencing of susceptibility genes and thus increasing plant resistance to fungal infections. This review presents current achievements and prospects for the application of CRISPR-Cas technology to increase the resistance of major agricultural crops to fungal diseases. The implementation of these approaches contributes to the creation of highly productive and resistant varieties, which is crucial for ensuring food security in the context of climate change.},
}
@article {pmid41300710,
year = {2025},
author = {Iksat, N and Madirov, A and Zhanassova, K and Masalimov, Z},
title = {Artificial Intelligence-Assisted CRISPR/Cas Systems for Targeting Plant Viruses.},
journal = {Genes},
volume = {16},
number = {11},
pages = {},
doi = {10.3390/genes16111258},
pmid = {41300710},
issn = {2073-4425},
support = {grant No. BR21882269//the Science Committee of the Ministry of Science and Higher Education of the Republic of Kazakhstan/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; *Artificial Intelligence ; *Plant Viruses/genetics ; *Gene Editing/methods ; *Plant Diseases/virology/genetics ; Crops, Agricultural/genetics/virology ; },
abstract = {Plant viral infections continue to pose a significant and ongoing threat to global food security, especially in the context of climatic instability and intensive agricultural practices. The CRISPR/Cas system has emerged as a powerful tool for developing virus-resistant crops by enabling precise modifications to viral genomes or plant susceptibility factors. Nonetheless, the efficacy and dependability of CRISPR-based antiviral approaches are limited by challenges in guide RNA design, off-target effects, insufficiently annotated datasets, and the intricate biological dynamics of plant-virus interactions. This paper summarizes the latest advancements in the incorporation of artificial intelligence (AI) methodologies, including machine learning and deep learning algorithms, into the CRISPR design and optimization framework. It examines how convolutional and recurrent neural networks, transformer architectures, and generative models like AlphaFold2, RoseTTAFold, and ESMFold can be used to predict protein structures, score sgRNAs, and model host-virus interactions. AI-enhanced methods have been proven to improve target specificity, Cas protein performance, and in silico validation. This paper aims to establish a foundation for next-generation genome editing strategies against plant viruses and promote the adoption of AI-powered CRISPR technologies in sustainable agriculture.},
}
@article {pmid41299842,
year = {2025},
author = {Shi, C and Yu, Z and Tan, H and Li, W and Wang, Y and Wang, Y and Zhang, Q and Man, Y},
title = {One-Pot CRISPR-Based Isothermal Amplification for Nucleic Acid Detection: A Comparative Review of Different Strategies.},
journal = {ACS sensors},
volume = {},
number = {},
pages = {},
doi = {10.1021/acssensors.5c00806},
pmid = {41299842},
issn = {2379-3694},
abstract = {Nucleic acid detection plays an important role in pathogen monitoring and disease diagnosis. CRISPR one-pot assays combined with isothermal amplification are emerging as promising point-of-care technologies that simplify workflows while increasing sensitivity and specificity. However, the incompatibility inherent in the one-pot reaction of isothermal amplification and CRISPR detection limits their practical application. This review comprehensively analyzes diverse advanced one-pot CRISPR-based isothermal amplification strategies developed to overcome this fundamental challenge. These strategies primarily encompass physical separation strategies (utilizing lid-bottom, internal ledge, nested tube, and membrane approaches), phase separation strategies (employing glycerol, sucrose, and gel matrices), reaction system optimization strategies (fine-tuning reaction parameters and incorporating specialized additives), non-PAM and suboptimal PAM strategies, improved Cas enzyme strategies (enhanced Cas12 and Cas13 variants), light-controlled approaches (PC-oligonucleotides, NPOM-dt modification, and acylation modification), and microfluidic chip integration strategies (centrifugal microfluidic chips, droplet microfluidic chips, and microarray chips). These methodological approaches have achieved important advances in simplifying operational processes, enhancing sensitivity, shortening detection cycles, and minimizing cross-contamination risks. The review further synthesizes critical insights regarding current opportunities, technical challenges, and future directions for one-pot CRISPR-based isothermal amplification technologies in nucleic acid detection, providing valuable guidance for researchers and practitioners in this evolving field.},
}
@article {pmid41298934,
year = {2025},
author = {Lee, SY and Birkholz, N and Lee, JH and Fineran, PC and Park, HH},
title = {Regulation of anti-CRISPR operons by structurally distinct families of Aca proteins.},
journal = {Communications biology},
volume = {8},
number = {1},
pages = {1698},
pmid = {41298934},
issn = {2399-3642},
support = {RS-2025-02316334//National Research Foundation of Korea (NRF)/ ; PE25150//Korea Polar Research Institute (KOPRI)/ ; },
mesh = {*Operon ; *CRISPR-Cas Systems/genetics ; *Gene Expression Regulation, Bacterial ; *Viral Proteins/genetics/metabolism/chemistry ; *CRISPR-Associated Proteins/genetics/metabolism/chemistry ; Protein Binding ; Bacteriophages/genetics ; Crystallography, X-Ray ; Models, Molecular ; },
abstract = {CRISPR-Cas systems provide bacteria with adaptive immunity against bacteriophages and mobile genetic elements, driving an evolutionary arms race in which phages deploy anti-CRISPR (Acr) proteins. Acr proteins are often co-encoded in operons with anti-CRISPR-associated (Aca) proteins, which coordinate the regulation of acr gene expression. Here, we reveal the molecular basis of DNA binding that mediates transcriptional repression by two distinct Aca family members: Aca7 and Aca11. Crystal structures of Aca7 and Aca11 highlight conserved helix-turn-helix (HTH) motifs within α-helix bundles, providing a universal DNA-binding platform. Aca7 forms a symmetrical dimer to recognize a 19-bp inverted repeat (IR) within the acrIF11-aca7 operon. Strikingly, Aca11 binds 22-bp IRs in two distinct promoters, suggesting that Aca proteins can control multiple target operons. Mutagenesis and electrophoretic mobility shift assays (EMSAs) confirm that dimerization and sequence-specific IR recognition are essential for DNA binding. Despite mechanistic similarities, these and other Aca proteins exhibit notable differences. Structural comparisons across Aca families reveal that while monomer structures are generally similar with conserved HTH motifs, the structures of their dimeric functional units vary significantly. These structural differences might be essential for Aca proteins to bind to various promoters and regulate the expression of different Acr proteins.},
}
@article {pmid41298570,
year = {2025},
author = {Kiseleva, AA and Timonova, EM and Berezhnaya, AA and Kolozhvari, AE and Kochetov, AV and Salina, EA},
title = {Fine tuning wheat heading time through genome editing of transcription factor binding sites in Ppd-1 gene promoter.},
journal = {Scientific reports},
volume = {15},
number = {1},
pages = {42034},
pmid = {41298570},
issn = {2045-2322},
support = {FWNR&#x2011;2024&#x2011;0009//The Ministry of Science and Higher Education of the Russian Federation/ ; 075&#x2011;15&#x2011;2025&#x2011;51//The Kurchatov Genomic Center of ICG SB RAS/ ; },
mesh = {*Triticum/genetics/growth &amp; development ; *Promoter Regions, Genetic ; *Gene Editing/methods ; *Transcription Factors/metabolism/genetics ; Binding Sites ; Gene Expression Regulation, Plant ; *Plant Proteins/genetics/metabolism ; CRISPR-Cas Systems ; Mutation ; Photoperiod ; },
abstract = {Increasing the productivity and adaptability of agricultural plants often depends on optimizing heading time. To develop common wheat lines with accelerated heading and investigate its regulation, we targeted the PPD-1 genes, which control photoperiod sensitivity. Large deletions in the promoter regions of these genes are known to disrupt their expression, resulting in early heading. Using CRISPR/Cas9 genome editing, we generated wheat plants with mutations in the promoter regions of the Ppd-D1 and Ppd-B1 genes. These mutations included nucleotide substitutions, deletions, and insertions ranging from several to hundreds of base pairs, occurring within probable transcription factor binding sites, that may influence gene expression. Under short-day conditions, we assessed PPD-1 gene expression in T0 plants and T2 lines with different mutations. Our analysis revealed that deletions spanning the CHE transcription repressor binding sites altered gene expression patterns, supporting the hypothesis regarding the role of these cis-elements in regulating PPD-1 expression. Furthermore, plants with different mutations displayed distinct diurnal expression patterns, suggesting the involvement of additional transcription factors in the regulation of this gene. Evaluation of heading time in T1 and T2 families with different mutations demonstrated that plants with mutations affecting the "core region", including the CHE binding sites, initiated heading significantly earlier than those without mutations.},
}
@article {pmid41296365,
year = {2025},
author = {Chen, W and Fan, L and Dong, M and Gao, L and Du, X and Wang, L and Su, S and Liu, LE and Wu, Y and Ding, L},
title = {Emerging CRISPR/Cas-Based Strategies for Extracellular Vesicle Detection: A Comprehensive Review.},
journal = {ACS sensors},
volume = {},
number = {},
pages = {},
doi = {10.1021/acssensors.5c02441},
pmid = {41296365},
issn = {2379-3694},
abstract = {Extracellular vesicles (EVs), which carry a variety of molecules such as proteins and nucleic acids, have great potential for broad application in liquid biopsy. However, achieving highly sensitive detection of biomarkers within EVs remains a significant challenge. The emergence of CRISPR/Cas systems─adaptive immune mechanisms found in bacteria and archaea that defend against foreign genetic elements─offers new opportunities to address this issue through powerful nucleic acid recognition and cleavage capabilities. Compared to other EV detection techniques, CRISPR/Cas-based biosensors exhibit superior sensitivity, specificity, and operational efficiency, making them a compelling platform for clinical translation. Thus, to promote the application of EVs in disease diagnosis, disease monitoring, and therapeutic evaluation, this review focuses on the state-of-the-art CRISPR/Cas systems (specifically CRISPR/Cas9, CRISPR/Cas12, CRISPR/Cas13, and CRISPR/Cas14) as well as the latest applications of CRISPR/Cas-based EV detection techniques.},
}
@article {pmid41294842,
year = {2025},
author = {Nagaoka, K and Kobayashi, Y and Kakimi, K},
title = {NeoPAIR-T: Functional Mapping of Neoantigen-TCR Pairs Using a CRISPR-Engineered Jurkat Reporter System.},
journal = {Cells},
volume = {14},
number = {22},
pages = {},
pmid = {41294842},
issn = {2073-4409},
support = {24ama221321h0002//Japan Agency for Medical Research and Development (AMED)/ ; 23H02762//JSPS KAKENHI/ ; },
mesh = {Humans ; Jurkat Cells ; *Receptors, Antigen, T-Cell/metabolism/genetics ; *Antigens, Neoplasm/immunology/genetics/metabolism ; Genes, Reporter ; *CRISPR-Cas Systems/genetics ; Coculture Techniques ; Antigen-Presenting Cells/immunology/metabolism ; },
abstract = {Targeting mutation-derived neoantigens is a promising strategy for personalized immunotherapies. However, identifying true neoantigens and cognate T cell receptors (TCRs) remains challenging because computational prediction of neoantigen peptides is uncertain and most tumor-infiltrating lymphocytes are bystanders rather than tumor-reactive, necessitating functional validation. Here, we developed NeoPAIR-T (Neoantigen-TCR Pairing Assay using reporter T cells), a functional assay based on co-culture of TCR-T reporter cells and autologous antigen-presenting cells (APCs) to screen neoantigen-TCR pairs. Reporter T cells are Jurkat-derived cells engineered to express a luciferase/eGFP dual reporter, providing quantitative readouts of TCR activation, while APCs are immortalized autologous cells transfected with tandem minigenes (TMGs) encoding predicted neoantigens, bypassing peptide synthesis. NeoPAIR-T also includes TCRα-knockout with targeted knock-in of candidate TCRs at the TCRβ locus to prevent mispairing and enables parallel testing of multiple reporter T cell clones co-cultured with the same APCs for efficient identification of functional pairs. Using lung cancer samples, whole-exome and RNA sequencing predicted 63 candidate peptides assembled into three TMGs. Single-cell RNA/TCR sequencing identified eight TCR clonotypes, introduced into reporter T cells and tested in parallel. Co-culture with TMG-expressing APCs revealed two functional neoantigen-TCR pairs validated by peptide assays (EC50: 10[-9.2]-10[-6.7] M). Collectively, NeoPAIR-T streamlines neoantigen-TCR identification for vaccine and TCR-T applications.},
}
@article {pmid41294822,
year = {2025},
author = {Bucheeri, S and Alcibahy, Y and Bucheeri, Y and Bucheeri, S and Alhermi, A and Butler, AE},
title = {CRISPR as a Tool to Uncover Gene Function in Polycystic Ovary Syndrome: A Literature Review of Experimental Models Targeting Ovarian and Metabolic Genes.},
journal = {Cells},
volume = {14},
number = {22},
pages = {},
pmid = {41294822},
issn = {2073-4409},
mesh = {*Polycystic Ovary Syndrome/genetics/metabolism ; Animals ; Female ; Humans ; *CRISPR-Cas Systems/genetics ; Gene Editing/methods ; Disease Models, Animal ; *Ovary/metabolism/pathology ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; },
abstract = {Polycystic ovary syndrome (PCOS) is a complex disorder characterized by reproductive abnormalities such as hyperandrogenism, ovulatory dysfunction, and polycystic ovarian morphology, and is frequently accompanied by metabolic disturbances such as insulin resistance, obesity and dyslipidemia. Genome-wide association studies (GWASs) have identified several susceptibility loci, yet little is known about their functional implications. Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (CRISPR/Cas9) has emerged as a powerful gene editing tool in bridging this gap by allowing researchers to directly target candidate genes in ovarian and metabolic pathways. For instance, experimental models have highlighted the role of CYP17A1 and DENND1A.V2 in androgen excess, anti-Müllerian hormone (AMH) in follicular arrest, and insulin receptor substrate 1 (IRS1) and PPARγ in insulin signaling and adipogenesis. To highlight the multifactorial nature of PCOS, animal models, including zebrafish and rodents, have been used to reveal interactions between reproductive and metabolic phenotypes. Nevertheless, most studies remain restricted to single-gene models, and dual-gene models or combined gene editing and hormonal induction models remain underexplored. Future research integrating precision editing, multi-omic platforms, and patient-derived organoids may provide more accurate disease models and novel therapeutic strategies.},
}
@article {pmid41294729,
year = {2025},
author = {Guo, W and Jiang, M and Xie, Y and Xu, H and Sun, Z},
title = {Recognition Element-Based Strategies for Rapid Detection of Foodborne Pathogens: Recent Progress and Perspectives.},
journal = {Biosensors},
volume = {15},
number = {11},
pages = {},
pmid = {41294729},
issn = {2079-6374},
support = {BK20241924//Natural Science Foundation of Jiangsu Province/ ; SH2024010//Key Research and Development Program of Zhenjiang City/ ; SH2024112//Key Research and Development Program of Zhenjiang City/ ; NY2023002//Key Research and Development Program of Zhenjiang City/ ; },
mesh = {*Biosensing Techniques/methods ; *Food Microbiology ; Humans ; *Foodborne Diseases/microbiology/diagnosis ; Nucleic Acid Amplification Techniques ; CRISPR-Cas Systems ; Molecular Imprinting ; },
abstract = {The detection of foodborne pathogens is of great significance for safeguarding food safety and public health. In recent years, rapid detection technologies based on diverse recognition elements have advanced considerably, driven by progress in molecular biology, materials science, and information technology. This review takes recognition elements as the central theme and systematically outlines the mechanisms and research progress of antibodies, nucleic acid aptamers, nucleic acid amplification techniques, CRISPR/Cas systems, molecular imprinting technology, peptides, and small-molecule receptors in foodborne pathogen detection, while comparing their performance in terms of specificity, sensitivity, stability, and applicability. In addition, this review further elaborates on the developmental trends of detection platforms, including multi-target and multimodal integration, microfluidics combined with portable point-of-care testing (POCT) systems, and intelligent terminals empowered by artificial intelligence algorithms. These trends provide new perspectives for improving detection systems in terms of throughput, portability, and intelligence. Overall, this review aims to serve as a comprehensive reference for the development of rapid, accurate, and intelligent detection systems for foodborne pathogens.},
}
@article {pmid41293956,
year = {2025},
author = {Zang, J and Niklaus, S and Neuhauss, SCF},
title = {An EAAT2b/SLC1A2b-mediated chloride leak current enables rapid cone photoreceptor signalling.},
journal = {Open biology},
volume = {15},
number = {11},
pages = {250347},
pmid = {41293956},
issn = {2046-2441},
support = {/SNSF_/Swiss National Science Foundation/Switzerland ; },
mesh = {Animals ; *Retinal Cone Photoreceptor Cells/metabolism ; Zebrafish/genetics/metabolism ; *Excitatory Amino Acid Transporter 2/metabolism/genetics ; *Signal Transduction ; Electroretinography ; *Chlorides/metabolism ; *Zebrafish Proteins/metabolism/genetics ; CRISPR-Cas Systems ; Gene Knockout Techniques ; Gene Editing ; },
abstract = {Excitatory amino acid transporters not only mediate high-affinity glutamate uptake but also conduct an uncoupled chloride current. In zebrafish, a whole-genome duplication gave rise to two eaat2 paralogues with distinct roles. Excitatory amino acid transporter 2a (SLC1A2b, GLT-1) functions primarily in Müller glia as a glutamate transporter, whereas excitatory amino acid transporter 2b is expressed in cone photoreceptors and exhibits a prominent glutamate-independent chloride current. We hypothesized that this leak current stabilizes the cone resting membrane potential, thereby supporting rapid visual signalling. In order to test this hypothesis, we generated eaat2b knockout zebrafish using CRISPR-Cas9-mediated genome editing. While eaat2b mutants showed no gross morphological abnormalities, they exhibited reduced electroretinogram b-wave amplitudes. Consistent with our hypothesis, eaat2b-deficient larvae displayed a significant reduction in flicker fusion electroretinogram power at each stimulus frequency, indicating impaired temporal processing likely due to delayed repolarization of cone photoreceptors. Our findings reveal a critical role for an excitatory amino acid transporter 2b-mediated chloride anion leak current in regulating the kinetics of photoreceptor responses. This functional innovation, enabled by a whole-genome duplication in the teleost lineage, highlights how gene duplications can lead to the acquisition of physiologically relevant new functions.},
}
@article {pmid41292787,
year = {2025},
author = {Majumder, P and Cahir, CW and Roberts, CG and Patel, DJ and Marraffini, LA},
title = {Cap1 forms a cyclic tetra-adenylate-induced membrane pore during the type III-A CRISPR-Cas immune response.},
journal = {bioRxiv : the preprint server for biology},
volume = {},
number = {},
pages = {},
doi = {10.1101/2025.11.13.688252},
pmid = {41292787},
issn = {2692-8205},
abstract = {During type III CRISPR-Cas immunity in prokaryotes, RNA-guided recognition of viral (phage) transcripts stimulates the Cas10 complex to convert ATP into cyclic oligoadenylates. These act as signaling molecules that bind to CARF proteins and activate their effector domains. Here, we report the structure and function of the Cap1 effector, composed of a pair of transmembrane helices (TM1/2), a CARF-like (CARFL) domain and a domain of unknown function (DUF4579). Cryo-EM studies on apo- and ligand-bound states of Cap1 in glyco-diosgenin detergent revealed the formation of tetrameric complexes in both states, with one cyclic tetra-adenylate molecule bound in a pocket composed by the four CARFL domains. Binding of cA 4 triggers conformational changes that widen an otherwise narrow pore formed by the four TM1/2 domains. In vivo , Cap1 activation results in membrane depolarization, a growth arrest of the bacterial host and the abrogation of the viral lytic cycle. Our findings reveal the mechanistic basis of membrane depolarizarion mediated by cyclic nucleotide signaling during the type III CRISPR-Cas response.},
}
@article {pmid41291345,
year = {2025},
author = {Wang, Z and Zhang, Y and Xu, N and Liu, L and Zhang, M and Huang, S and Su, C and Liu, T and Duan, K},
title = {Improving soybean fatty acid profiles by CRISPR/Cas12a-mediated gene editing of GmFAD2 and GmFAD3.},
journal = {Plant cell reports},
volume = {44},
number = {12},
pages = {282},
pmid = {41291345},
issn = {1432-203X},
support = {32172499//National Natural Science Foundation of China/ ; JSSCTD202342//Shuangchuang Program of Jiangsu Province/ ; },
mesh = {*Glycine max/genetics/metabolism ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; *Fatty Acids/metabolism ; Soybean Oil/metabolism ; Plants, Genetically Modified ; Linoleic Acid/metabolism ; *Plant Proteins/genetics/metabolism ; Oleic Acid/metabolism ; Fatty Acid Desaturases/genetics/metabolism ; },
abstract = {Using CRISPR/Cas12a, we engineered novel soybean germplasms by knocking out GmFAD2 (GmFAD2-1A, GmFAD2-1B) and GmFAD3 (GmFAD3A, GmFAD3B) genes, yielding elevated oleic or linoleic acid content. Soybean oil contains high levels of polyunsaturated fatty acids (PUFAs), which are known to reduce cholesterol levels and help prevent hypertension, thereby contributing significantly to human health. However, the chemical instability of PUFAs makes them susceptible to oxidation, a process that generates harmful trans-fatty acids. To address this issue, precise modulation of fatty acid composition in soybeans becomes critically important for health applications. In this study, we employed CRISPR/Cas12a gene editing technology to selectively knock out the GmFAD2 (GmFAD2-1A, GmFAD2-1B) and GmFAD3 (GmFAD3A, GmFAD3B) genes in soybean. This approach successfully created novel soybean germplasms with distinct fatty acid profiles: one with elevated oleic acid content and another with increased linoleic acid levels. These engineered variants provide valuable options for utilizing soybean oil with optimized fatty acid compositions tailored for specific health and nutritional purposes.},
}
@article {pmid41291135,
year = {2025},
author = {Hann, E and Majumdar, D and Layton, D and Fareh, M and Cahill, DM and Ziemann, M and Ujvari, B and Schat, KA and Challagulla, A},
title = {Systematic evaluation of CrRNA design parameters for optimized Cas13d-mediated RNA targeting in chicken cells.},
journal = {Functional &amp; integrative genomics},
volume = {25},
number = {1},
pages = {256},
pmid = {41291135},
issn = {1438-7948},
mesh = {Animals ; Chickens/genetics ; *CRISPR-Cas Systems ; Cell Line ; RNA, Messenger/genetics ; },
abstract = {The CRISPR-Cas13 system has emerged as a powerful platform for programmable RNA targeting, offering efficient and sequence-specific silencing of coding and non-coding transcripts. The RNA-targeting capabilities of CRISPR-Cas13 have been harnessed to silence transcripts harbouring pathogenic mutations and combat infectious diseases. However, the molecular basis of on-target and collateral activity are not completely understood, limiting the utility of Cas13 systems. In this study, we delineate the principles for the development of effective crRNAs by targeting DsRed fluorescence reporter and synthetic influenza mRNA in chicken fibroblast DF1 cells. To systematically determine the optimal design for RfxCas13d crRNA, we investigated the minimum length of the crRNA, importance of protospacer flanking sequence, degree of mismatch tolerance, and off target effects. Our data reveal variable knockdown levels between crRNAs, in which several crRNAs achieved over 95% target knockdown. We show that crRNAs exhibit a high degree of tolerance to single-nucleotide mismatches, regardless of their position in the spacer sequence. However, 4-nt mismatches between the spacer and the target significantly reduces targeting efficacy, whereas eight nucleotide mismatches completely abolish the activity of RfxCas13d. Finally, we compared targeting efficiency and collateral activity of two widely used RfxCas13d and HfCas13d variants. Our data extend current understanding of Cas13d-mediated RNA targeting and offer a framework for rational crRNA design to enhance effectiveness in diverse applications, including antiviral strategies.},
}
@article {pmid41290964,
year = {2025},
author = {El Menofy, NG and Payoumi, AN and Eissa, MA and El-Sharif, A},
title = {Association of the existence of CRISPR-Cas system and antimicrobial resistance in multi-drug resistant Klebsiella pneumoniae in Egypt.},
journal = {Scientific reports},
volume = {15},
number = {1},
pages = {41814},
pmid = {41290964},
issn = {2045-2322},
mesh = {*Klebsiella pneumoniae/genetics/drug effects/isolation &amp; purification ; Egypt ; *Drug Resistance, Multiple, Bacterial/genetics ; *CRISPR-Cas Systems/genetics ; *Anti-Bacterial Agents/pharmacology ; Humans ; Microbial Sensitivity Tests ; *Klebsiella Infections/microbiology/drug therapy/epidemiology ; beta-Lactamases/genetics ; Colistin/pharmacology ; Bacterial Proteins/genetics ; },
abstract = {The CRISPR-Cas systems are supposed to be associated with antibiotic susceptibility. Klebsiella pneumoniae (K. pneumoniae) is a major multidrug-resistant (MDR) pathogen that may cause severe infections. This study aimed to detect the antimicrobial resistance (AMR) of K. pneumoniae isolates in addition to determine the association between the existence of CRISPR-Cas systems and the presence of AMR in Egypt. The antibiotic susceptibility patterns of 100 K. pneumoniae isolates were determined using the Kirby Bauer disc diffusion and broth microdilution methods. The frequency of carbapenem resistance encoding genes (blaKPC, blaOXA, blaIMP, blaNDM, and blaVIM), ESBLs encoding genes (blaTEM), aminoglycoside resistance encoding genes (aac(3)-Ia, aac(3)-IIa, colistin resistance encoding genes (mcr-1, mcr-2) and tetracycline resistance encoding genes (tetB) were determined using polymerase chain reaction (PCR). The presence or absence of CRISPR-Cas systems was determined by detection of Cas genes (Cas1 or Cas3) in conjunction with one of CRISPR arrays 1, 2 or 3. Kirby Bauer disc diffusion revealed that 95% of isolates were MDR. The resistance rates of K. pneumoniae isolates to amikacin, meropenem, and colistin were 76%, 67%, and 41% respectively by broth microdilution assay. Among selected 41 K. pneumoniae, the frequency of ESBLs; blaTEM was 92.7%, while the frequency of blaNDM blaOXA blaVIM blaIMP and blaKPC was 95.1%, 95.1%, 39%, 19.5% and 14.6% respectively. The frequency of mcr-1 and mcr-2 was 70.7% and 65.9%. Additionally, the frequency of aac(3)-Ia was 12.2%, and aac(3)-IIa was 87.8%, while the frequency of tetB was 100%. Our isolates exhibited varied profiles for CRISPR-Cas systems, where 65.9% were positive for CRISPR-Cas system. No general significant positive correlation between AMR and the presence ofCRISPR-Cas system was detected; however, a significant difference is present for imipenem, colistin and chloramphenicol phenotypic resistance andfor aac(3)-IIa and mcr-1 genes (P value > 0.1). A significant positive correlation was detected between AMR for imipenem, colistin and chloramphenicol and for aac(3)-IIa and mcr-1 genes and thepresence of CRISPR-Cas system.},
}
@article {pmid41230612,
year = {2025},
author = {Hao, J and Gong, X and Duan, X and Qin, W and Ren, H and ShenTu, X and Ye, Z and Yu, X},
title = {Ultrasensitive electrochemical detection of parvovirus B19 DNA by combining CRISPR-Cas12a and multivalent framework nucleic acids.},
journal = {Analytical methods : advancing methods and applications},
volume = {17},
number = {46},
pages = {9357-9364},
doi = {10.1039/d5ay01285f},
pmid = {41230612},
issn = {1759-9679},
mesh = {*Electrochemical Techniques/methods ; *CRISPR-Cas Systems/genetics ; *Parvovirus B19, Human/genetics/isolation &amp; purification ; *DNA, Viral/analysis/genetics ; *Biosensing Techniques/methods ; Humans ; Limit of Detection ; CRISPR-Associated Proteins ; *Endodeoxyribonucleases/genetics ; Bacterial Proteins ; },
abstract = {The rapid and ultrasensitive detection of parvovirus B19 (B19V) DNA is critical for preventing severe complications in high-risk populations, such as fetal hydrops in pregnant women and aplastic crisis in immunocompromised patients. The absence of clinically approved vaccines or antivirals against B19V thus mandates the urgent development of accessible in vitro diagnostics to enable time-critical interventions and contain community transmission. Herein, we developed an electrochemical biosensor as a proof-of-concept for B19V by integrating CRISPR-Cas12a with multivalent framework nucleic acids (FNAs), namely, 12 nm tetrahedral DNA nanostructures (TDNs). Target B19V DNA activates Cas12a to indiscriminately cleave the biotin-modified ssDNA protruding from the four vertices of the TDNs, while the TDNs precisely orient ssDNA probes on electrodes, minimizing nonspecific adsorption. This method takes advantage of the target-specific cleavage ability of CRISPR-Cas12a (Cas12a-crRNA complex, 10-12 nm) and the unique structural and functional features of 12 nm TDNs. The comparable dimensions of the Cas12a-crRNA complex and the TDN suggest a potential synergistic effect, which contributes to the observed signal amplification and high detection sensitivity. The developed platform is user-friendly, has a low detection limit (2.19 fM), and shows high selectivity. This work establishes a foundational biosensing platform, demonstrating potential for ultrasensitive nucleic acid detection. By combining the accuracy of CRISPR-Cas12a and the benefits of FNAs, this method provides a more efficient, amplification-free, and reliable approach that holds promise for future development in point-of-care diagnostics and other applications.},
}
@article {pmid41038164,
year = {2025},
author = {Cheng, Y and Dang, S and Zhang, Y and Chen, Y and Yu, R and Liu, M and Jin, S and Han, A and Katz, S and Wang, S},
title = {Sequencing-free whole-genome spatial transcriptomics at single-molecule resolution.},
journal = {Cell},
volume = {188},
number = {24},
pages = {6953-6970.e12},
doi = {10.1016/j.cell.2025.09.006},
pmid = {41038164},
issn = {1097-4172},
mesh = {Animals ; Humans ; Mice ; In Situ Hybridization, Fluorescence/methods ; *Transcriptome/genetics ; *Gene Expression Profiling/methods ; Single-Cell Analysis/methods ; RNA, Guide, CRISPR-Cas Systems/genetics/metabolism ; Single Molecule Imaging/methods ; Genome ; CRISPR-Cas Systems ; },
abstract = {Recent breakthroughs in spatial transcriptomics technologies have enhanced our understanding of diverse cellular identities, spatial organizations, and functions. Yet existing spatial transcriptomics tools are still limited in either transcriptomic coverage or spatial resolution, hindering unbiased, hypothesis-free transcriptomic analyses at high spatial resolution. Here, we develop reverse-padlock amplicon-encoding fluorescence in situ hybridization (RAEFISH), an image-based spatial transcriptomics method with whole-genome coverage and single-molecule resolution in intact tissues. We demonstrate the spatial profiling of transcripts from 23,000 human or 22,000 mouse genes in single cells and tissue sections. Our analyses reveal transcript-specific subcellular localization, cell-type-specific and cell-type-invariant zonation-dependent transcriptomes, and gene programs underlying preferential cell-cell interactions. Finally, we further develop our technology for the direct spatial readout of guide RNAs (gRNAs) in an image-based, high-content CRISPR screen. Overall, these developments offer a broadly applicable technology that enables high-coverage, high-resolution spatial profiling of both long and short, native and engineered RNAs in many biomedical contexts.},
}
@article {pmid41034579,
year = {2025},
author = {Sullivan, AE and Nabhani, A and Izrailevsky, DS and Schinkel, K and Hoffman, CRK and Robbins, LK and Nagy, TA and Duncan, ML and Ledvina, HE and Erbse, AH and Kibby, EM and Tak, U and Dinh, DM and Ednacot, EMQ and Nguyen, CM and Burroughs, AM and Aravind, L and Whiteley, AT and Morehouse, BR},
title = {The Panoptes system uses decoy cyclic nucleotides to defend against phage.},
journal = {Nature},
volume = {647},
number = {8091},
pages = {988-996},
pmid = {41034579},
issn = {1476-4687},
mesh = {*Nucleotides, Cyclic/metabolism ; Models, Molecular ; Dinucleoside Phosphates/metabolism ; Crystallography, X-Ray ; *Bacteriophages/physiology/immunology ; *Second Messenger Systems ; *Operon/genetics ; CRISPR-Cas Systems/genetics ; Protein Domains ; Viral Proteins/metabolism/chemistry ; },
abstract = {Bacteria combat phage infection using antiphage systems and many systems generate nucleotide-derived second messengers upon infection that activate effector proteins to mediate immunity[1]. Phages respond with counter-defences that deplete these second messengers, leading to an escalating arms race with the host. Here we outline an antiphage system we call Panoptes that indirectly detects phage infection when phage proteins antagonize the nucleotide-derived second-messenger pool. Panoptes is a two-gene operon, optSE, wherein OptS is predicted to synthesize a nucleotide-derived second messenger and OptE is predicted to bind that signal and drive effector-mediated defence. Crystal structures show that OptS is a minimal CRISPR polymerase (mCpol) domain, a version of the polymerase domain found in type III CRISPR systems (Cas10). OptS orthologues from two distinct Panoptes systems generated cyclic dinucleotide products, including 2',3'-cyclic diadenosine monophosphate (2',3'-c-di-AMP), which we showed were able to bind the soluble domain of the OptE transmembrane effector. Panoptes potently restricted phage replication, but phages that had loss-of-function mutations in anti-cyclic oligonucleotide-based antiphage signalling system (CBASS) protein 2 (Acb2) escaped defence. These findings were unexpected because Acb2 is a nucleotide 'sponge' that antagonizes second-messenger signalling. Our data support the idea that cyclic nucleotide sequestration by Acb2 releases OptE toxicity, thereby initiating inner membrane disruption, leading to phage defence. These data demonstrate a sophisticated immune strategy that bacteria use to guard their second-messenger pool and turn immune evasion against the virus.},
}
@article {pmid41034576,
year = {2025},
author = {Doherty, EE and Adler, BA and Yoon, PH and Hsieh, K and Loi, K and Armbruster, EG and Lahiri, A and Bolling, CS and Wilcox, XE and Akkati, A and Iavarone, AT and Pogliano, J and Doudna, JA},
title = {A miniature CRISPR-Cas10 enzyme confers immunity by inhibitory signalling.},
journal = {Nature},
volume = {647},
number = {8091},
pages = {997-1004},
pmid = {41034576},
issn = {1476-4687},
mesh = {Humans ; *CRISPR-Cas Systems/genetics/immunology ; *Signal Transduction/immunology ; Oligonucleotides/biosynthesis/immunology ; Dinucleoside Phosphates/metabolism ; Membrane Proteins/metabolism/chemistry ; Immune Evasion ; Cell Death ; Adenylyl Cyclases/metabolism/chemistry ; HEK293 Cells ; Models, Molecular ; },
abstract = {Microbial and viral co-evolution has created immunity mechanisms involving oligonucleotide signalling that share mechanistic features with human antiviral systems[1]. In these pathways, including cyclic oligonucleotide-based antiphage signalling systems (CBASSs) and type III CRISPR systems in bacteria and cyclic GMP-AMP synthase-stimulator of interferon genes (cGAS-STING) in humans, oligonucleotide synthesis occurs upon detection of virus or foreign genetic material in the cell, triggering the antiviral response[2-4]. Here, in an unexpected inversion of this process, we show that the CRISPR-related enzyme mCpol synthesizes cyclic oligonucleotides constitutively as part of an active mechanism that represses a toxic effector. Cell-based experiments demonstrated that the absence or loss of mCpol-produced cyclic oligonucleotides triggers cell death, preventing the spread of viruses that attempt immune evasion by depleting host cyclic nucleotides. Structural and mechanistic investigation revealed mCpol to be a di-adenylate cyclase whose product, c-di-AMP, prevents toxic oligomerization of the effector protein 2TMβ. Analysis of cells by fluorescence microscopy showed that lack of mCpol allows 2TMβ-mediated cell death due to inner membrane collapse. These findings unveil a powerful defence strategy against virus-mediated immune suppression, expanding our understanding of the role of oligonucleotides in immunity.},
}
@article {pmid41005744,
year = {2025},
author = {Li, E and Wen, L and Yin, C and Wang, N and Yang, S and Feng, W and Chen, M},
title = {Copper ionophore-autophagy interference nanoregulators for tumor self-defense reprograming to amplify cuproptotic stress and antitumor immunity.},
journal = {Journal of controlled release : official journal of the Controlled Release Society},
volume = {388},
number = {Pt 1},
pages = {114262},
doi = {10.1016/j.jconrel.2025.114262},
pmid = {41005744},
issn = {1873-4995},
mesh = {Humans ; *Copper/administration &amp; dosage/chemistry ; Autophagy/drug effects ; Animals ; Autophagy-Related Protein 5/genetics ; *Neoplasms/immunology/drug therapy ; CRISPR-Cas Systems ; Cell Line, Tumor ; *Naphthoquinones/administration &amp; dosage/chemistry ; *Antineoplastic Agents/administration &amp; dosage ; Hyaluronan Receptors/metabolism ; Polyethyleneimine/chemistry/administration &amp; dosage ; Female ; Mice ; },
abstract = {Cuproptosis as a copper-dependent cell death modality driven by pathological aggregation of lipoylated proteins and proteotoxic stress resulting from destabilization of iron-sulfur (FeS) cluster proteins, represents a promising therapeutic strategy for cancer. However, the therapeutic efficacy of cuproptosis can be compromised by the intrinsic compensatory mechanisms within cancers, particularly low intracellular copper ion concentration and protective autophagy, which facilitates cellular adaptation and survival under stress. To overcome this limitation, a self-amplifying cuproptosis nanoregulator (SHK-Cu/[TK]PF/pATG5@HA, abbreviated as SC/TpA@HA) for CD44-targeted delivery is developed that integrate shikonin‑copper (SHK-Cu) coordination complexes with CRISPR/Cas9 plasmids targeting ATG5, condensed by fluorinated polyethyleneimine ([TK]PF)-condensed to enhance cancer therapy. Briefly, Cu[+] is released from the dissociated SHK-Cu complex upon intracellular GSH activation induces dihydrolipoamide S-acetyltransferase (DLAT) oligomerization and reduces FeS cluster proteins, triggering tricarboxylic acid (TCA) cycle collapse and irreversible mitochondrial damage. Concurrently, CRISPR/Cas9-mediated ATG5 knockout prevents autophagosome formation, creating an autophagic flux trap that accumulates copper-damaged mitochondria. Notably, such mitochondrial dysfunction as induced by copper overload combined with impaired cellular damage clearance from autophagy blockade elevates cuproptosis. In addition, the immunogenic cell death through cuproptosis in cancer cells, as validated by the exposure of calreticulin and the extracellular release of HMGB1, triggers a potent anti-tumor immune response. This response is enhanced through autophagy inhibition, as ATG5 deletion blocks the downstream signaling of copper-activated Unc-51-like autophagy activating kinase 1/2 (ULK1/2), ultimately amplifying cytotoxic T lymphocyte infiltration. Therefore, this dual intervention through copper overload and autophagy blockade potentiates both cuproptosis and anti-tumor immune effect, representing an innovative strategy of cuproptosis treatment.},
}
@article {pmid40848222,
year = {2025},
author = {Liu, L and Song, L and Qi, C and Cao, X and Huang, S and Zhang, G and Chen, G and Men, X and Zhang, H},
title = {Enhanced production of sabinene by engineered Saccharomyces cerevisiae from corn hydrolysates.},
journal = {Brazilian journal of microbiology : [publication of the Brazilian Society for Microbiology]},
volume = {56},
number = {4},
pages = {2327-2337},
pmid = {40848222},
issn = {1678-4405},
support = {2022YFC2106200//National Key R&amp;D Program of China/ ; ZR2023MC163//Natural Science Foundation of Shandong Province/ ; QNESL 0P202308//Qingdao New Energy Shandong Laboratory/ ; TSQN201909159//Young Taishan Scholars/ ; Y2021063//Youth Innovation Promotion Association CAS/ ; },
mesh = {*Saccharomyces cerevisiae/metabolism/genetics ; *Zea mays/metabolism/chemistry ; *Metabolic Engineering ; Fermentation ; CRISPR-Cas Systems ; Saccharomyces cerevisiae Proteins/genetics/metabolism ; *Monoterpenes/metabolism ; },
abstract = {Sabinene is a type of monoterpene that is widely used in flavors, fragrances and pharmaceuticals. Though sabinene biosynthesis has been investigated in a variety of microorganisms, application of sabinene is still limited due to its high production cost and lesser yielding strains. The baker's yeast Saccharomyces cerevisiae, which is generally recognized as safe (GRAS), is a suitable cell factory for the food and beverage industries. In this study, we aimed to enhance the production of sabinene from corn hydrolysates by employing genetic engineering techniques on S. cerevisiae. Here, we engineered S. cerevisiae for the production of sabinene by overexpressing sabinene synthase (SabS) and geranyl diphosphate synthase (GPPS) via CRISPR-Cas9, which is a simple and efficient tool for targeted and marker-free genome engineering. Subsequently, the culture medium and process conditions were optimized to enhance sabinene production and achieve ~ 23.6 mg/L under flask fermentation conditions. Based on the optimized culture conditions, we further investigated the production of sabinene from corn hydrolysates, which is a major source of dietary nutrients worldwide and an inexpensive source of sugars, and a high-level production of 60.0 mg/L was achieved in shake-flask fermentation. Our results implied that corn hydrolysates was a suitable medium for sabinene production and that CRISPR-Cas9 could boost the marker-free engineered yeast strain, which was more suitable for the food and beverage industry. Altogether, our work represents the progress in the bioproduction of food-grade sabinene from an inexpensive raw material.},
}
@article {pmid41288772,
year = {2025},
author = {Sun, W and Ren, X and Huang, J and Wang, Y and Liu, S},
title = {B-PER tandem assembly CRISPR/Cas12a cascade amplification strategy based fluorescence/colorimetric dual-mode for detection of MC-LR.},
journal = {Mikrochimica acta},
volume = {192},
number = {12},
pages = {853},
pmid = {41288772},
issn = {1436-5073},
support = {32072330//National Natural Science Foundation of China/ ; ZR2022MB066//Natural Science Foundation of Shandong Province/ ; },
mesh = {*Microcystins/analysis ; Quantum Dots/chemistry ; *Biosensing Techniques/methods ; *CRISPR-Cas Systems ; *Colorimetry/methods ; Limit of Detection ; *Nucleic Acid Amplification Techniques/methods ; Marine Toxins ; Fluorescence ; Spectrometry, Fluorescence/methods ; Cadmium Compounds/chemistry ; },
abstract = {Microcystins are the most common, most powerful and most toxic types of cyanobacteria, which seriously threaten the public health and ecological environment. Biosensor has been widely used in Microcystin-Leucine-Arginine (MC-LR) detection. In this study, we report an efficient single-hairpin with double primers (the trigger chain T hybridizes with the left end a, and the amplified product ab hybridizes with the right end a) for bidirectional PER (B-PER) amplification strategy for uniform visual and fluorescence detection of trace amounts of MC-LR in the environment using quantum dots (QDs) as signal reporters. The biosensor is triggered by a single stranded DNA of MC-LR specificity to initiate the amplification by B-PER. From the long product of the B-PER, the trans-cleavage activity of CRISPR/Cas12a is activated, and the Ag[+] is released from the C-Ag[+]-C. The released Ag[+] undergoes cation exchange reaction (CER) with CdTe QDs (QDs), quenching QDs fluorescence and generating visual and fluorescent dual signals. The biosensor can simultaneously complete the display of dual signals of naked eyes and fluorescence, and can successfully used for the detection of the actual environment sample. In addition, the biosensor has low detection limits, high sensitivity, good accuracy and high selectivity. The detection range under the best conditions was 0.05-500 nM, and the detection limit was 0.705 pM. In summary, this strategy provides a general detection platform for detecting trace pollutants in the environment by using biosensors.},
}
@article {pmid41288291,
year = {2025},
author = {Liu, H and Liu, Y and Feng, R and Qian, M and Li, Y and Zhai, S and Song, J and Qiu, X},
title = {Homogeneous Femtomolar Detection of P-tau181 via Proximity Extension and CRISPR/Cas Technique.},
journal = {Analytical chemistry},
volume = {},
number = {},
pages = {},
doi = {10.1021/acs.analchem.5c04856},
pmid = {41288291},
issn = {1520-6882},
abstract = {Accurate quantification of site-specific tau phosphorylation in plasma holds great promise for the noninvasive early diagnosis of Alzheimer's disease (AD). Here, we integrated the proximity extension assay (PEA) with nucleic acid amplification techniques-polymerase chain reaction (PCR) and recombinase polymerase amplification (RPA)-and coupled them with CRISPR/Cas12a-mediated fluorescence detection to enable quantitative and homogeneous measurement of threonine-181-phosphorylated tau (p-tau181), a key biomarker of AD. Binding of two PEA probes to a single p-tau181 molecule induces proximity-mediated probe hybridization and extension, thereby converting the protein signal into an amplifiable nucleic acid signal. The resulting double-stranded DNA is subsequently amplified by PCR or RPA and detected through Cas12a trans-cleavage activity. The limits of detection (LODs) for the PEA-PCR-CRISPR/Cas and PEA-RPA-CRISPR/Cas assays were 149.0 fM (6.8 pg·mL[-1]) and 45.4 fM (2.1 pg·mL[-1]), respectively. In fetal bovine serum, LODs of 231.4 fM (10.6 pg·mL[-1]) and 139.2 fM (6.3 pg·mL[-1]) were achieved, demonstrating excellent antimatrix performance. The accuracy of the PEA-RPA-CRISPR/Cas assay in human serum was further validated using a commercial enzyme-linked immunosorbent assay (ELISA) kit. This homogeneous, wash-free approach combines operational simplicity with ultrahigh sensitivity, showing great potential for routine clinical detection and early stage monitoring of AD biomarkers.},
}
@article {pmid41288175,
year = {2025},
author = {Prokhorova, PV and Vlasova, NN and Yuzhakov, AG and Gulyukin, AM},
title = {Modern approaches to the construction and use of recombinant viruses.},
journal = {Voprosy virusologii},
volume = {70},
number = {5},
pages = {417-430},
doi = {10.36233/0507-4088-323},
pmid = {41288175},
issn = {2411-2097},
mesh = {Humans ; *Genetic Vectors/genetics ; *Oncolytic Viruses/genetics ; *CRISPR-Cas Systems/genetics ; *Genetic Therapy/methods ; *Vaccines, Synthetic/genetics/immunology ; Animals ; Oncolytic Virotherapy/methods ; Homologous Recombination ; Plasmids/genetics ; },
abstract = {The review describes certain viral vectors and considers various methods for constructing recombinant viruses with special attention paid to the homologous recombination and CRISPR/Cas9 system, and also describes the capabilities of using various cloning vectors (different plasmids, BAC etc.). The review also presents a comparative analysis of the effectiveness and safety of using various viral vectors, both for creating recombinant vaccines and for obtaining oncolytic viruses, as well as medicines for gene therapy.},
}
@article {pmid41285890,
year = {2025},
author = {Kim, S and Won, H and Bae, J and Kim, J and Choi, J and Richar, H and Kim, YG and Choi, HJ},
title = {Structural and functional insights into internal domain replacement in SpCas9 for protein engineering.},
journal = {Scientific reports},
volume = {15},
number = {1},
pages = {41528},
pmid = {41285890},
issn = {2045-2322},
support = {SRFC-MA1801-09//Samsung Science and Technology Foundation/ ; },
mesh = {*Protein Engineering/methods ; *CRISPR-Associated Protein 9/genetics/chemistry/metabolism ; CRISPR-Cas Systems ; Gene Editing/methods ; *Streptococcus pyogenes/enzymology/genetics ; Escherichia coli/genetics/enzymology ; Protein Domains ; },
abstract = {The CRISPR-Cas9 system has emerged as a powerful tool for precise genome editing, with ongoing research focused on enhancing its reliability and expanding its versatility. One effective strategy involves the integration of foreign functional domains into Cas9 to confer new capabilities. However, successful integration requires identification of insertion sites that preserve the protein's structural integrity and function. In this study, we identified a C-terminal region of Streptococcus pyogenes Cas9 (SpCas9), spanning residues 1242-1263, as a viable site for domain replacement. Structural and biochemical analyses of a SpCas9 variant lacking this region confirmed its dispensability for SpCas9 activity. As a proof of concept, we substituted this segment with the evolved E. coli tRNA adenosine deaminase (TadA), a key component of adenine base editors. Functional evaluation of this engineered SpCas9-TadA variant demonstrated deamination efficiency comparable to that of the ABE8e, with the potential to modulate the editing window through linker design. These results highlight the potential of targeted engineering of this region to develop more precise and versatile genome editing tools.},
}
@article {pmid41285767,
year = {2025},
author = {Rahimov, F and Ghosh, S and Petiwala, S and Schmidt, M and Nyamugenda, E and Shi, M and Tam, J and Verduzco, D and Singh, S and Avram, V and Modi, A and Espinoza, CA and Lu, C and Wang, J and Keller, A and Macoritto, M and Mahi, NA and Anton, T and Chung, N and Flister, MJ and Katlinski, KV and Biswas, A and den Hollander, AI and Waring, JF and Stender, JD},
title = {A genome-wide CRISPR screen identifies the TNRC18 gene locus as a regulator of inflammatory signaling.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {10346},
pmid = {41285767},
issn = {2041-1723},
mesh = {Humans ; *Signal Transduction/genetics ; *Interleukin-1beta/metabolism/genetics ; *Inflammation/genetics/metabolism ; Lipopolysaccharides/pharmacology ; CRISPR-Cas Systems ; U937 Cells ; Finland ; Gene Knockout Techniques ; Genome-Wide Association Study ; Clustered Regularly Interspaced Short Palindromic Repeats ; Gene Expression Regulation ; },
abstract = {Interleukin-1β (IL-1β) is dysregulated in chronic inflammatory diseases, yet the genetic factors influencing IL-1β production remain largely unknown. Myeloid-derived cells are the primary producers of IL-1β, which prompted a genome-wide CRISPR knockout screen in the human myeloid-derived U937 cells treated with lipopolysaccharide (LPS) to mimic inflammatory conditions and sorted for high and low intracellular IL-1β levels. A total of 295 genes are identified as regulators of IL-1β production, with 57 overlapping loci associated with inflammatory diseases, including the TNRC18 gene locus associated with multiple diseases in the Finnish population. U937 cells engineered with the Finnish-enriched rs748670681 risk allele demonstrate decreased expression of TNRC18 and an adjacent gene WIPI2, reduction in LPS-dependent gene activation and cytokine production, but elevation of interferon-responsive gene programs. Transcriptomic profiles for individual knockouts of TNRC18 and WIPI2 attribute the loss of LPS-dependent signaling primarily to TNRC18, which occurs through the modulation of H3K27 acetylation around inflammatory regulatory regions via TNRC18 and its protein interaction network. In contrast, the loss of WIPI2 is characterized by an exacerbation of interferon signaling. These findings delineate the global regulatory mechanisms of IL-1β production and provide molecular insights to the role of the rs748670681 variant in inflammatory diseases.},
}
@article {pmid41285753,
year = {2025},
author = {Mameli, E and Samantsidis, GR and Viswanatha, R and Kwon, H and Hall, DR and Butnaru, M and Hu, Y and Mohr, SE and Perrimon, N and Smith, RC},
title = {A genome-wide CRISPR screen in Anopheles mosquito cells identifies fitness and immune cell function-related genes.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {10323},
pmid = {41285753},
issn = {2041-1723},
support = {AI166857//U.S. Department of Health &amp; Human Services | NIH | National Institute of Allergy and Infectious Diseases (NIAID)/ ; 2336877//National Science Foundation (NSF)/ ; GM132087//U.S. Department of Health &amp; Human Services | NIH | National Institute of General Medical Sciences (NIGMS)/ ; GM132087//U.S. Department of Health &amp; Human Services | NIH | National Institute of General Medical Sciences (NIGMS)/ ; },
mesh = {Animals ; *Anopheles/genetics/immunology/drug effects ; *Mosquito Vectors/genetics/immunology/drug effects ; Liposomes ; Clodronic Acid/pharmacology ; CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Malaria/transmission/prevention &amp; control ; Genome, Insect ; *Genetic Fitness ; Female ; },
abstract = {Anopheles mosquitoes are the sole vector of malaria, the most burdensome vector-borne disease worldwide. At present, strategies for reducing mosquito populations or limiting their ability to transmit disease show the most promise for disease control. Therefore, improving our understanding of mosquito biology and immune function may aid new approaches to limit malaria transmission. Here, we perform genome-wide CRISPR screens in Anopheles mosquito cells to identify genes required for fitness and that confer resistance to clodronate liposomes, which are used to ablate immune cells. The cellular fitness screen identifies 1280 fitness-related genes (393 at highest confidence) that are highly enriched for roles in fundamental cell processes. The clodronate screen identifies resistance factors that impair clodronate liposome function. For the latter, we confirm roles in liposome uptake and processing through in vivo validation in Anopheles gambiae that provide new mechanistic detail of phagolysosome formation and clodronate liposome processing. Altogether, we present a genome-wide CRISPR knockout platform in a major malaria vector and identify genes important for fitness and immune-related processes.},
}
@article {pmid41285544,
year = {2026},
author = {Gong, C and Wang, Z and Gao, X and He, S},
title = {A review of CRISPR-Cas as a "molecular catcher" for tracking circulating tumor cells and extracellular vesicles.},
journal = {Analytica chimica acta},
volume = {1381},
number = {},
pages = {344675},
doi = {10.1016/j.aca.2025.344675},
pmid = {41285544},
issn = {1873-4324},
mesh = {*Neoplastic Cells, Circulating/pathology/metabolism ; Humans ; *Extracellular Vesicles/metabolism/chemistry ; *CRISPR-Cas Systems ; Biomarkers, Tumor/genetics ; },
abstract = {BACKGROUND: Liquid biopsy is reshaping the landscape of cancer diagnostics by turning a simple blood sample into a rich source of real-time molecular insights. Among its most promising targets are circulating tumor cells (CTCs) and extracellular vesicles (EVs), which carry valuable clues about tumor progression, metastasis, and treatment response. However, traditional workflows for analyzing CTCs and EVs typically rely on immunoaffinity-based enrichment followed by molecular assays such as quantitative Polymerase Chain Reaction (qPCR) or Enzyme-Linked Immunosorbent Assay (ELISA). These approaches are often limited by low sensitivity, high costs, and complex procedures, hindering their widespread clinical use.<br><br>RESULTS: Recent advances in Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas technologies offer a multifaceted approach to biomarker analysis. Cas9 is primarily used for functional gene interrogation to identify and validate targets, while Cas12a and Cas13a serve as direct diagnostic tools, enabling ultrasensitive signal amplification for DNA, RNA, and protein markers. By integrating these distinct Cas effectors with aptamer-based recognition, nanomaterial-assisted enrichment, and hybrid amplification techniques like Hybridization Chain Reaction (HCR), Rolling Circle Amplification (RCA), researchers have developed highly sensitive and programmable platforms for analyzing CTCs and EVs.<br><br>SIGNIFICANCE: Despite ongoing challenges such as off-target effects, Protospacer Adjacent Motif (PAM) sequence limitations, and clinical variability, the field is rapidly evolving. The convergence of CRISPR diagnostics with artificial intelligence, device miniaturization, and multiplexed sensing is accelerating clinical translation. Collectively, these innovations are paving the way for a new era of precision oncology that is fast, flexible, and achievable with only a drop of blood.},
}
@article {pmid41284662,
year = {2025},
author = {Abad Santos, JC and Garimella, SS and Khanchandani, AN and Shah, PS},
title = {Rapid Optimization of a Light-Inducible System to Control Mammalian Gene Expression.},
journal = {Journal of visualized experiments : JoVE},
volume = {},
number = {225},
pages = {},
doi = {10.3791/68779},
pmid = {41284662},
issn = {1940-087X},
mesh = {Humans ; HEK293 Cells ; *Optogenetics/methods ; Light ; *CRISPR-Cas Systems ; Flow Cytometry/methods ; },
abstract = {Inducible gene expression tools can open novel applications in human health and biotechnology, but current options are often expensive, difficult to reverse, and have undesirable off-target effects. Optogenetic systems use light-responsive proteins to control the activity of regulators such that expression is controlled with the "flip of a switch". This study optimizes a simplified light activated CRISPR effector (2pLACE) system, which provides tunable, reversible, and precise control of mammalian gene expression. The OptoPlate-96 enables high-throughput screening via flow cytometry for single-cell analysis and rapid optimization of 2pLACE. This study demonstrates how to use the 2pLACE system with the OptoPlate-96 in HEK293T cells to identify the optimal component ratios for maximizing dynamic range and to find the blue light intensity response curve. Similar workflows can be developed for other mammalian cells and for other optogenetic systems and wavelengths of light. These advancements enhance the precision, scalability, and adaptability of optogenetic tools for biomanufacturing applications.},
}
@article {pmid41281758,
year = {2025},
author = {Jia, Z and Wu, J and Zhang, J and Zheng, P and Zhang, H and Lin, Y and Pan, T and Wu, M and Song, Y},
title = {Precision Reprogramming in CAR-T Cell Therapy: Innovations, Challenges, and Future Directions of Advanced Gene Editing.},
journal = {International journal of biological sciences},
volume = {21},
number = {15},
pages = {6884-6906},
pmid = {41281758},
issn = {1449-2288},
mesh = {Humans ; *Gene Editing/methods ; *Immunotherapy, Adoptive/methods ; *Receptors, Chimeric Antigen/genetics ; CRISPR-Cas Systems/genetics ; Neoplasms/therapy ; T-Lymphocytes ; Animals ; },
abstract = {Chimeric antigen receptor (CAR)-T cell therapy represents a breakthrough in cancer immunotherapy, demonstrating impressive clinical outcomes, particularly for hematologic malignancies. However, its broader therapeutic application, especially against solid tumors, remains limited. Key challenges include T cell exhaustion, limited persistence, cytokine-mediated toxicities, and logistical hurdles associated with manufacturing autologous products. Emerging gene editing technologies, such as CRISPR/Cas systems, base editing, and prime editing, offer novel approaches to optimize CAR-T cells, aiming to enhance efficacy while managing toxicity and improving accessibility. This review comprehensively examines the current landscape of these gene editing tools in CAR-T cell therapy, highlighting the latest advancements, persisting challenges, and future directions. Leveraging gene editing holds the potential to transform CAR-T therapy into a more potent, safer, and broadly applicable modality for cancer and beyond.},
}
@article {pmid41281724,
year = {2025},
author = {Barraclough, A and Bär, I and van Duijl, T and Fijnvandraat, K and Eikenboom, JCJ and Leebeek, FWG and Bierings, R and Voorberg, J and Trasanidou, D},
title = {Correction: Rewriting the script: gene therapy and genome editing for von Willebrand Disease.},
journal = {Frontiers in genome editing},
volume = {7},
number = {},
pages = {1719330},
doi = {10.3389/fgeed.2025.1719330},
pmid = {41281724},
issn = {2673-3439},
abstract = {[This corrects the article DOI: 10.3389/fgeed.2025.1620438.].},
}
@article {pmid41232934,
year = {2025},
author = {Li, Z and Li, F and Hua, L and Chai, F and Xie, L and Wang, D and Zhang, S and Zheng, C and Wang, Z and Jiang, X},
title = {Unlocking Zeptomolar Single-Molecule Detection by Synergizing Digital Microfluidics and Digital CRISPR.},
journal = {Journal of the American Chemical Society},
volume = {147},
number = {47},
pages = {43870-43883},
doi = {10.1021/jacs.5c15767},
pmid = {41232934},
issn = {1520-5126},
mesh = {Humans ; Immunoassay/methods ; Limit of Detection ; *CRISPR-Cas Systems ; Natriuretic Peptide, Brain/blood ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Tumor Necrosis Factor-alpha/blood ; *Single Molecule Imaging ; Interleukin-6/blood ; *Microfluidics ; Biomarkers/blood ; Peptide Fragments/blood ; },
abstract = {Accurate diagnosis relies on the highly sensitive and quantitative detection of multiple immune-related biomarkers. However, current detection methods still face significant limitations in sensitivity, specificity, and background signal control. Here, we introduce DDA (Dual-Digital immunoAssay), a fully automated, universal immunoassay platform that synergizes digital microfluidics with digital Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-based amplification. This "dual-digital" strategy pushes the detection limit into the zeptomolar (zM) regime, enabling unprecedented sensitivity for single-molecule analysis. The DDA platform is built upon a digital microfluidic microwell array chip, integrating magnetic bead-based immunocapture with RNA-guided CRISPR/Cas13a signal amplification. This system enables a fully automated, "sample-in, answer-out" workflow. By systematically optimizing the entire process, DDA significantly reduces background noise and enhances detection sensitivity, achieving a limit of detection (LOD) down to 100 zM for key protein biomarkers. This represents a >100-fold improvement over leading commercial ultrasensitive assays. With single-molecule resolution and full automation, DDA provides a robust solution for the precise quantification of low-abundance immune biomarkers. As a proof-of-concept, we demonstrate its ability to accurately quantify key heart-failure-associated biomarkers, including NT-proBNP (LOD: 1 aM), IL-6 (LOD: 1.5 aM), and TNF-α (LOD: 2.5 aM), directly in complex serum samples. This platform holds great promise for automated multibiomarker screening and risk assessment, showcasing its powerful potential for the early diagnosis of major diseases such as cardiovascular diseases, cancers, neurodegenerative disorders, and infectious diseases.},
}
@article {pmid41232706,
year = {2026},
author = {Puri, B and Majumder, S and Gaikwad, AB},
title = {Targeting LncRNA MEG3 to modulate ER stress and autophagy: A CRISPR/Cas9-based strategy in AKI-to-CKD transition.},
journal = {Experimental cell research},
volume = {454},
number = {1},
pages = {114826},
doi = {10.1016/j.yexcr.2025.114826},
pmid = {41232706},
issn = {1090-2422},
mesh = {*Endoplasmic Reticulum Stress/genetics ; *Autophagy/genetics ; *RNA, Long Noncoding/genetics/metabolism ; *CRISPR-Cas Systems/genetics ; Animals ; *Acute Kidney Injury/genetics/pathology/metabolism ; Rats ; *Renal Insufficiency, Chronic/genetics/pathology/metabolism ; Fibrosis ; Cell Line ; Apoptosis/genetics ; },
abstract = {Acute kidney injury (AKI) to chronic kidney disease (CKD) transition is a progressive, long-term kidney dysfunction driven by complex pathophysiological processes, including persistent endoplasmic reticulum (ER) stress and impaired autophagy, contributing to fibrosis. Long non-coding RNA (lncRNA) maternally expressed gene 3 (MEG3) has been implicated in endoplasmic reticulum (ER) stress and autophagy in several diseases, but its role in kidney injury and fibrosis during AKI-to-CKD transition remains unclear. Our previous transcriptomic analysis revealed that MEG3 is dysregulated during this transition, prompting us to explore its functional role. In this study, we investigated the function of MEG3 in ER stress-autophagy crosstalk during the AKI-to-CKD transition. Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR/Cas9)-mediated MEG3 knockout in NRK-52E cells was confirmed by T7 endonuclease assay, quantitative real-time polymerase chain reaction (qRT-PCR), and fluorescence in-situ hybridization (FISH) assay. Functionally, MEG3 knockout markedly attenuated ER stress and apoptosis, as shown by reduced expression of BiP, CHOP, ATF6, ATF4, p-PERK, p-eIF2α, along with restoration of the Bax/Bcl-2 balance. Autophagy activity was restored, with increased Beclin-1 and LC3B expression and decreased p62 accumulation. Furthermore, fibrotic progression was reduced, as indicated by lower levels of fibronectin and collagen I. Notably, tauroursodeoxycholic acid (TUDCA, 400 μM) acted synergistically with MEG3 knockout, further suppressing ER stress and fibrosis markers compared to either treatment alone. These findings demonstrate that MEG3 promotes maladaptive ER stress and impaired autophagy in tubular epithelial cells, driving AKI-to-CKD transition. Targeting MEG3 through CRISPR-based strategies or in combination with TUDCA may represent a promising therapeutic strategy to mitigate fibrosis and slow disease progression.},
}
@article {pmid41211945,
year = {2025},
author = {Laffin, LJ and Nicholls, SJ and Scott, RS and Clifton, PM and Baker, J and Sarraju, A and Singh, S and Wang, Q and Wolski, K and Xu, H and Nielsen, J and Patel, N and Duran, JM and Nissen, SE},
title = {Phase 1 Trial of CRISPR-Cas9 Gene Editing Targeting ANGPTL3.},
journal = {The New England journal of medicine},
volume = {393},
number = {21},
pages = {2119-2130},
doi = {10.1056/NEJMoa2511778},
pmid = {41211945},
issn = {1533-4406},
mesh = {Humans ; Angiopoietin-Like Protein 3 ; Male ; Middle Aged ; Female ; *Angiopoietin-like Proteins/genetics ; Adult ; *Gene Editing ; *CRISPR-Cas Systems ; Aged ; Loss of Function Mutation ; *Dyslipidemias/genetics/drug therapy ; Hypercholesterolemia/drug therapy/genetics ; Hypertriglyceridemia/drug therapy/genetics ; },
abstract = {BACKGROUND: Angiopoietin-like protein 3 (ANGPTL3) inhibits lipoprotein and endothelial lipases. ANGPTL3 loss-of-function genetic variants are associated with decreased levels of low-density lipoprotein cholesterol and triglycerides and a decreased lifetime risk of atherosclerotic cardiovascular disease.<br><br>METHODS: We conducted an ascending-dose phase 1 trial to assess the safety and efficacy of CTX310, a lipid-nanoparticle-encapsulated clustered regularly interspaced short palindromic repeats-Cas9 endonuclease (CRISPR-Cas9) messenger RNA (mRNA) and guide RNA targeting hepatic ANGPTL3 to induce a loss-of-function mutation. Adults who had uncontrolled hypercholesterolemia, hypertriglyceridemia, or mixed dyslipidemia and were receiving maximally tolerated lipid-lowering therapy received a single intravenous dose of CTX310 (0.1, 0.3, 0.6, 0.7, or 0.8 mg per kilogram of body weight). The primary end point was adverse events, including dose-limiting toxic effects.<br><br>RESULTS: A total of 15 participants received CTX310 and had at least 60 days of follow-up. No dose-limiting toxic effects related to CTX310 occurred. Serious adverse events occurred in two participants (13%): one participant had a spinal disk herniation, and the other died suddenly 179 days after treatment with the 0.1-mg-per-kilogram dose. Infusion-related reactions were reported in three participants (20%), and one participant (7%) who had elevated levels of aminotransferases at baseline had a transient elevation in aminotransferases to between three times and five times as high as those at baseline, peaking on day 4 and returning to baseline by day 14. The mean percent change in ANGPTL3 level was 9.6% (range, -21.8 to 71.2) with the dose of 0.1 mg per kilogram, 9.4% (range, -25.0 to 63.9) with 0.3 mg per kilogram, -32.7% (range, -51.4 to -19.4) with 0.6 mg per kilogram, -79.7% (range, -86.8 to -72.5) with 0.7 mg per kilogram, and -73.2% (range, -89.0 to -66.9) with 0.8 mg per kilogram.<br><br>CONCLUSIONS: Editing of ANGPTL3 was associated with few adverse events and resulted in reductions from baseline in ANGPTL3 levels. (Funded by CRISPR Therapeutics; Australia New Zealand Clinical Trials Registry number, ACTRN12623000809639.).},
}
@article {pmid41064856,
year = {2025},
author = {Jiang, Z and Su, L and Chen, C and He, R and Jiang, L and Shu, Y and Dai, D and Wu, M and Guo, A and Liu, J and Liu, S and Liu, Z},
title = {Atf3 Deficiency Promotes Mesodermal Commitment and Enhances Endothelial Differentiation in Embryonic Stem Cells.},
journal = {Arteriosclerosis, thrombosis, and vascular biology},
volume = {45},
number = {12},
pages = {2226-2242},
pmid = {41064856},
issn = {1524-4636},
mesh = {*Activating Transcription Factor 3/genetics/deficiency/metabolism ; Animals ; *Cell Differentiation ; Mice, Knockout ; Mice ; *Endothelial Cells/metabolism ; *Mouse Embryonic Stem Cells/metabolism ; *Mesoderm/metabolism/cytology ; Cells, Cultured ; CRISPR-Cas Systems ; Cell Lineage ; Signal Transduction ; },
abstract = {BACKGROUND: Ischemic diseases have become a major threat to global health, with endothelial cell (EC) damage closely associated with their pathogenesis and progression. Cell therapies targeting endothelial repair have thus become a treatment approach of great interest, yet the procurement of clinically approved ECs for these applications has not been fully established. Modulating the expression of Atf3 (activating transcription factor 3) represents a potential strategy for deriving ECs from stem cells; however, its precise function in the development and differentiation of ECs from stem cells remains elusive. In the present study, we sought to elucidate the potential role of Atf3 in the differentiation of embryonic stem cells into ECs.<br><br>METHODS: CRISPR-Cas9 (clustered regularly interspaced short palindromic repeats/clustered regularly interspaced short palindromic repeat-associated 9) system was used to knockout Atf3 (Atf3KO [Atf3 knockout]) in mouse embryonic stem cells. EC differentiation was initially induced using the hanging drop method to promote embryoid bodies formation, followed by embryoid bodies attachment onto culture slides. The expression changes of EC markers during differentiation were assessed by RNA sequencing, Western blotting, immunofluorescence staining, flow cytometry, and reverse transcription quantitative polymerase chain reaction. Functional comparisons of differentiated ECs were performed by assessing LDL (low-density lipoprotein) uptake and NO production. Potential molecular mechanisms were further explored via bioinformatic analysis of RNA sequencing data.<br><br>RESULTS: Atf3KO led to a significant upregulation in the expression levels of progenitor and mesoderm cell markers on days 3 and 6 of differentiation. By day 9, the expression of mature EC markers also exhibited a notable increase. Moreover, Atf3KO enhanced the functional properties of differentiated Atf3KO ECs. In addition, our findings revealed that the activation of the Rap1 (Ras-related protein 1) signaling pathway, triggered by Atf3KO, contributed to ECs development and maturation.<br><br>CONCLUSIONS: Atf3KO directs embryonic stem cells toward the mesodermal lineage and activates the Rap1 signaling pathway, thereby promoting ECs development. These findings highlight a key role of Atf3 in regulating early stage of vascular endothelial development.},
}
@article {pmid40842147,
year = {2025},
author = {Ye, S and Kim, JS and Kim, M and Kim, KY and Won, YH and Park, T and An, S and Jeong, H and Chung, HJ and Lee, IS and Kang, MH and Kang, CY and Kim, MY and Chung, JH and Gim, JA and Hwang, W and Kim, Y and Kim, SC and Lee, S and Hur, JK and Hur, JW},
title = {MUTE-Seq: An Ultrasensitive Method for Detecting Low-Frequency Mutations in cfDNA With Engineered Advanced-Fidelity FnCas9.},
journal = {Advanced materials (Deerfield Beach, Fla.)},
volume = {37},
number = {47},
pages = {e05208},
doi = {10.1002/adma.202505208},
pmid = {40842147},
issn = {1521-4095},
support = {2021R1A6A1A03040260//National Institute for International Education/ ; RS-2023-00260529//National Institute for International Education/ ; RS-2024-00435385//Korea National Institute of Health/ ; RS-2021-KD000007//Korea Medical Device Development Fund/ ; RS-2021-NR056589//National Research Foundation of Korea/ ; RS-2023-00260529//National Research Foundation of Korea/ ; RS-2023-00262309//National Research Foundation of Korea/ ; RS-2025-02218918//National Research Foundation of Korea/ ; RS-2022-KH129266//Korea Ministry of Health &amp; Welfare/ ; K2125811//Korea University/ ; },
mesh = {Humans ; *Mutation ; CRISPR-Cas Systems ; Carcinoma, Non-Small-Cell Lung/genetics ; *Cell-Free Nucleic Acids/genetics ; Lung Neoplasms/genetics ; Leukemia, Myeloid, Acute/genetics ; Pancreatic Neoplasms/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; },
abstract = {In this study, we present the development of the Mutation tagging by CRISPR-based Ultra-precise Targeted Elimination in Sequencing (MUTE-Seq) method. We engineered a highly precise advanced-fidelity FnCas9 variant, named FnCas9-AF2, to effectively discriminate single-base mismatches at all positions of the single guide RNA (sgRNA) target sequences. FnCas9-AF2 exhibited significantly lower off-target effects compared to existing high-fidelity CRISPR-Cas9 variants. MUTE-Seq leverages FnCas9-AF2 for the enrichment of mutant DNA through the exclusive cleavage of perfectly matched wild-type DNA, allowing for sensitive detection of low-frequency cancer-associated mutant alleles. MUTE-Seq enabled sensitive monitoring of minimal residual disease (MRD) from the bone marrow of patients with Acute Myeloid Leukemia (AML). Furthermore, MUTE-Seq was applied in a multiplexed manner on cell-free DNA (cfDNA) from patients diagnosed with non-small cell lung cancer (NSCLC) and pancreatic cancer. This approach demonstrated a significant improvement in the sensitivity of simultaneous mutant detection and highlighted its clinical utility for early-stage cancer patients with extremely low levels of circulating tumor DNA (ctDNA). We anticipate that the FnCas9-AF2-based MUTE-Seq could offer a valuable clinical tool to facilitate improved molecular diagnosis, prognosis evaluation, and treatment planning for cancers in various stages.},
}
@article {pmid41280798,
year = {2025},
author = {Fronza, F and Verardo, R and Schneider, C},
title = {RepFluo, a Fast Fluorescent In Vitro Assay of Cas9 Activity Exploiting Melting Curve Analysis.},
journal = {ACS omega},
volume = {10},
number = {45},
pages = {53816-53826},
pmid = {41280798},
issn = {2470-1343},
abstract = {Demand for less labor-intensive in vitro assays of the activity of CRISPR/Cas proteins is rising to extend the potential applications of CRISPR in the field of diagnostics. RNA guided DNA endonucleases of the Cas family generate double-strand breaks in the target DNA, which results in two shorter DNA fragments. We hypothesized that this cleavage event could be studied using melting curve analysis, and using SpyCas9, we demonstrate that it is possible to evaluate the activity of Cas proteins by measuring the melting curves of their products. We present here a novel assay for the in vitro activity of Cas9 that exploits melting curve analysis (MCA) to be fast, inexpensive, and widely accessible. The assay can, in fact, be performed with readily available components(?)in its simplest form a real-time thermal cycler and an intercalating dye (SYBR Green I)(?)and produces reliable results with a run-time of 15 min. It does not require external intervention to stop the reaction, which is done by thermal denaturation of the protein directly in the thermal cycler machine. The described advantages, combined with the provided data analysis package, make the assay robust and amenable to high-throughput applications. To increase the accessibility of our assay, we provided an R package that simplifies the analytical process.},
}
@article {pmid41280279,
year = {2026},
author = {Li, X and Ye, C and Liu, T and Li, S and Zhang, M and Zhao, Y and Jin, Y and Cheng, J and Yang, G and Li, P},
title = {Engineering genetic elements for microbial protein expression systems: Advances, challenges, applications, and prospects.},
journal = {Synthetic and systems biotechnology},
volume = {11},
number = {},
pages = {370-384},
pmid = {41280279},
issn = {2405-805X},
abstract = {The rising global demand for sustainable protein sources poses critical challenges across food, pharmaceutical, and industrial biotechnology sectors. Microbial expression systems provide scalable and versatile platforms for producing recombinant proteins, including enzymes, therapeutic molecules, and functional food ingredients. These platforms enable efficient biosynthesis of high-value proteins from renewable substrates often via precision fermentation, surpassing conventional methods in yield, cost-efficiency, and environmental sustainability. This review summarizes the genetic regulatory elements that govern gene expression in microbial hosts, with comparative coverage of prokaryotic and eukaryotic systems at transcriptional and translational levels. Key regulatory components, such as promoters, ribosome binding sites (RBS), untranslated regions (UTRs), signal peptides, and terminators, are discussed in the context of host-specific engineering strategies. We highlight advanced engineering approaches, including artificial intelligence (AI) assisted sequence design, CRISPR-Cas-based genome editing, and modular combinatorial optimization of genetic elements. Particular attention is given to the integration of high-throughput screening and predictive modeling tools that accelerate the rational design and optimization of microbial production systems. The review also discusses practical applications in food, pharmaceutical, and industrial enzyme production, emphasizing how genetic element engineering bridges fundamental research and biomanufacturing. Finally, key challenges and future prospects are analyzed to guide the development of next-generation microbial cell factories for sustainable protein production and industrial innovation.},
}
@article {pmid41279041,
year = {2025},
author = {Vera, AO and Avilés-Vázquez, FJ and Ha, T and Choudhary, A and Raines, RT},
title = {Nuclear Localization Signals Enable the Cellular Delivery of an Anti-CRISPR Protein to Control Genome Editing.},
journal = {bioRxiv : the preprint server for biology},
volume = {},
number = {},
pages = {},
doi = {10.1101/2025.10.28.685205},
pmid = {41279041},
issn = {2692-8205},
abstract = {Precise regulation of Cas9 activity is essential to minimize off-target effects, mosaicism, chromosomal alterations, immunogenicity, and genotoxicity in genome editing. Although type II anti-CRISPR proteins (Acrs) can inhibit and regulate Cas9, their size and anionic charge generally prevent them from crossing the cell membrane. Existing Acr delivery methods employing vectors or electroporation are either slow and persistent or require external equipment, limiting their therapeutic utility. To address these challenges, we developed a cell-permeable Acr (6×NLS-Acr), which uses nuclear localization signals (NLSs) to cross the cell membrane. We conjugated 6×NLS-Acr to a fluorescent dye to elucidate its cellular entry mechanism and directly visualized its binding to a fluorescent Cas9·gRNA complex to study its inhibitory mechanism. 6×NLS-Acr (IC 50 = 0.47 µM) directly transduces human cells, including immortalized cell lines, embryonic stem cells, and 3D cell cultures, within 5 min, inhibiting up to 99% of Cas9 activity and increasing genome-editing specificity by nearly 100%. We further compared 6×NLS-Acr with our anthrax-derived Acr delivery platform. Our results demonstrate that 6×NLS-Acr is the most efficacious cell-permeable CRISPR-Cas inhibitor, significantly enhancing the precision and therapeutic potential of CRISPR-based genome editing.},
}
@article {pmid41277735,
year = {2025},
author = {Zhang, L and He, X and Hu, J and Bai, H and Yao, Y and Hu, WW},
title = {Recent advances in nanozyme-CRISPR/Cas biosensors.},
journal = {Chemical communications (Cambridge, England)},
volume = {},
number = {},
pages = {},
doi = {10.1039/d5cc05585g},
pmid = {41277735},
issn = {1364-548X},
abstract = {The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein (Cas) system, renowned for precise DNA recognition and potent trans-cleavage activity, has become a promising tool for biosensing. Nanozymes, a new class of artificial enzymes that combine the physicochemical properties of nanomaterials with the catalytic functions of natural enzymes, offer high surface area and versatile functionalization, enabling efficient target binding and catalysis to markedly boost biosensing performance. With advances across disciplines, the integration of nanozymes and CRISPR/Cas has become prominent in biosensing. Nanozyme-enhanced CRISPR/Cas biosensors can substantially improve detection sensitivity and expand sensing modes for bioanalysis, with potential for direct target detection without pre-amplification. In this review, we comprehensively examine recent strategies in nanozyme-enhanced CRISPR/Cas biosensing, with particular emphasis on advances in bioanalytical applications. We further critically assess the challenges and prospects of using nanozymes to improve CRISPR/Cas performance for biosensing and offer insights for the design of next-generation biosensors for precise, rapid, on-site detection of nucleic acids, proteins, and small molecules in clinical samples.},
}
@article {pmid41277692,
year = {2025},
author = {Jang, H and Kang, JE and Kim, H and Kim, JR and Park, J and Go, SR and Lee, YH and Kang, H and Park, Y and Kim, S and Jung, Y and Kim, SJ and Lim, EK and Jung, J and Woo, EJ and Park, KH and Kang, T},
title = {CRISPR/Cas12a2 enables ultra-sensitive amplification-free RNA detection.},
journal = {Nucleic acids research},
volume = {53},
number = {21},
pages = {},
pmid = {41277692},
issn = {1362-4962},
support = {//National Research Foundation of Korea/ ; 2021M3H4A1A02051048//Ministry of Science and ICT/ ; 2023R1A2C2005185//Ministry of Science and ICT/ ; RS-2024-00438316//Ministry of Science and ICT/ ; RS-2024-00348576//Ministry of Science and ICT/ ; RS-2024-00459749//Ministry of Science and ICT/ ; RS-2025-00554718//Ministry of Science and ICT/ ; 2021M3A9G802559922//Ministry of Science and ICT/ ; RS-2022-NR071772//Ministry of Science and ICT/ ; RS-2021-NR059435//Ministry of Science and ICT/ ; CRC22024-500//Ministry of Science and ICT/ ; 2020R1A5A1018052//Ministry of Science and ICT/ ; GTL25061-000//Ministry of Science and ICT/ ; //Korea Evaluation Institute of Industrial Technology/ ; RS-2022-00154853//Ministry of Trade, Industry and Energy/ ; RS-2024-00432382//Ministry of Trade, Industry and Energy/ ; RS-2024-00403563//Ministry of Trade, Industry and Energy/ ; //Korea Environmental Industry and Technology Institute/ ; 2021003370003//Ministry of Environment/ ; //Korea Health Industry Development Institute/ ; RS-2025-02213315//Ministry of Health and Welfare/ ; RS-2024-00401639//Korea Institute of Planning and Evaluation for Technology in Food, Agriculture/ ; KGM1322511//Korea Research Institute of Bioscience and Biotechnology Research Initiative Program/ ; KGM1032511//Korea Research Institute of Bioscience and Biotechnology Research Initiative Program/ ; KGM1062511//Korea Research Institute of Bioscience and Biotechnology Research Initiative Program/ ; KGM5382531//Korea Research Institute of Bioscience and Biotechnology Research Initiative Program/ ; //Research Institute of Bioscience and Biotechnology/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; *RNA, Viral/genetics/analysis ; *SARS-CoV-2/genetics/isolation &amp; purification ; *CRISPR-Associated Proteins/genetics/metabolism ; Humans ; *COVID-19/diagnosis/virology ; Limit of Detection ; Endodeoxyribonucleases ; RNA, Guide, CRISPR-Cas Systems/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats ; Bacterial Proteins ; },
abstract = {Advances in clustered regularly interspaced short palindromic repeat (CRISPR) technologies have led to the use of diverse CRISPR-associated (Cas) proteins in diagnostic applications. Herein, we present a CRISPR/Cas12a2-based amplification-free RNA detection method that exhibits sub-attomolar sensitivity and substrate versatility. Cas12a2, a recently characterized RNA-guided nuclease, uniquely integrates bimolecular recognition through CRISPR RNA (crRNA)-target complementarity and protospacer flanking sequence identification, enabling highly specific trans-cleavage of single-stranded DNA, double-stranded DNA, and RNA. We have optimized key biochemical parameters, including pH, ionic strength, and temperature, to enhance the catalytic efficiency of Cas12a2. Based on the optimal activity conditions of Cas12a2, we have achieved ultra-sensitive viral RNA detection with a limit of detection of 46.7 aM through the strategic design and cooperative activation of crRNAs targeting conserved regions of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) genome. The diagnostic accuracy of the Cas12a2-based assay has been demonstrated for 26 SARS-CoV-2 variants, and it has further resulted in the definitive diagnosis of 317 clinical samples. This work establishes Cas12a2 as a promising molecular diagnostic tool that provides an amplification-free, rapid, and versatile solution for RNA detection. The adaptability and simplicity of the platform render it particularly well suited for point-of-care applications, paving the way for next-generation CRISPR diagnostics.},
}
@article {pmid41222440,
year = {2025},
author = {Ming, W and Zhu, Y and Li, L and Wang, T and Pan, A and Xu, Q and Ji, H and Qin, Y and Wu, L},
title = {Allele-Specific CRISPR-Cas9-Based Ratiometric Fluorescence Platform for Portable EGFR L858R Mutation Detection.},
journal = {Analytical chemistry},
volume = {97},
number = {46},
pages = {25832-25839},
doi = {10.1021/acs.analchem.5c05548},
pmid = {41222440},
issn = {1520-6882},
mesh = {Humans ; ErbB Receptors/genetics ; *CRISPR-Cas Systems/genetics ; Alleles ; Mutation ; *Lung Neoplasms/genetics/diagnosis ; *Carcinoma, Non-Small-Cell Lung/genetics/diagnosis ; Fluorescence ; Point-of-Care Testing ; },
abstract = {Effective detection of low-abundance EGFR L858R mutation from circulating tumor DNA (ctDNA) is critical for early stage NSCLC diagnosis. Here, a portable CRISPR-Cas9-based ratiometric fluorescence sensor was proposed. Typically, allele-specific activation of Cas9 and the trans-cleavage of Cy5/BHQ2-labeled blocker DNAs were achieved by engineering sgRNA to position the L858R mutation within the PAM region of Cas9, resulting in increased Cy5 fluorescence. Simultaneously, the attenuated fluorescence of HBC-530 was observed because the released input RNA bound to the Pepper aptamer, which resulted from the decreased melting temperature (Tm) of the blocker DNA-RNA hybrid. Thus, a dual-channel ratiometric readout can be readily attained. Ultimately, visual point-of-care testing (POCT) of L858R at 0.01% allele frequency with single-nucleotide specificity was realized using a compact 3D-printed device. The validation result of 22 collected plasma samples demonstrated 100% concordance with the clinical diagnoses. This platform provides a cost-effective and accessible solution for NSCLC screening, making it particularly suitable for resource-limited healthcare settings.},
}
@article {pmid40854986,
year = {2025},
author = {Lu, Z and Chen, C and Zhang, H and Li, B and Liu, Y and Guo, J and Xu, R and Shi, K and Ma, Q and Zhang, M and Cai, Y and Huang, J and Geng, H and Fan, L and Ning, C and Li, Y and Chen, S and Tian, W and Hu, K and Li, H and Yang, X and Huang, C and Wei, Y and Zhu, X and Li, X and Xiong, Z and Cai, M and Wang, X and Zhang, S and Chen, H and Dai, M and Chen, K and Jin, M and Jin, M and Zhu, Y and Tian, J and Miao, X},
title = {Characterization of cis-regulatory elements and functional variants in colorectal cancer using epigenomics and CRISPRi screenings.},
journal = {Nature cancer},
volume = {6},
number = {11},
pages = {1777-1799},
pmid = {40854986},
issn = {2662-1347},
support = {NSFC-82130098//National Science Foundation of China | Key Programme/ ; },
mesh = {Humans ; *Colorectal Neoplasms/genetics/pathology ; *Epigenomics/methods ; Epigenesis, Genetic ; Gene Expression Regulation, Neoplastic ; *Regulatory Sequences, Nucleic Acid/genetics ; Transcription Factor 7-Like 2 Protein/genetics ; Repressor Proteins/genetics ; CRISPR-Cas Systems ; Polymorphism, Single Nucleotide ; Cell Proliferation/genetics ; Cell Line, Tumor ; Genetic Predisposition to Disease ; },
abstract = {Genetic variants associated with colorectal cancer (CRC) are primarily noncoding and reside in cis-regulatory elements (CREs), yet their underlying mechanisms remain elusive. Here we established a dynamic epigenetic atlas using multiomics data from 533 colorectal tissues spanning normal to advanced adenoma to cancer, identifying 7,492 differential CREs linked to 5,490 target genes. High-throughput CRISPR interference screening revealed 265 functional CREs involved in CRC cell proliferation. A polygenic risk score (PRS) based on functional CRE variants effectively predicted CRC and precancerous lesions among 476,770 individuals. Notably, the functional variant rs10871066 was significantly associated with increased risk of precancerous lesions and CRC (odds ratio = 1.27, P = 1.03 × 10[-13]). Mechanistically, rs10871066 triggers silencer-to-enhancer switching mediated by FOXP1 and TCF7L2, distally upregulating KLF5 to activate oncogenic pathways and PIBF1 to suppress natural killer cell cytotoxicity. Our study provides a comprehensive resource of dynamic epigenomic atlas, a functionally informed PRS for risk prediction and insights into epigenetic mechanisms underlying CRC development.},
}
@article {pmid40669499,
year = {2025},
author = {Tadokoro, T and Olson, EN and Liu, N},
title = {Gene Editing Applications as Future Cardiovascular Therapies.},
journal = {Annual review of genetics},
volume = {59},
number = {1},
pages = {119-145},
doi = {10.1146/annurev-genet-011725-094039},
pmid = {40669499},
issn = {1545-2948},
mesh = {*Gene Editing/methods/trends ; Humans ; CRISPR-Cas Systems/genetics ; *Cardiovascular Diseases/therapy/genetics ; *Genetic Therapy/methods/trends ; Animals ; },
abstract = {Cardiovascular disease is the leading cause of global morbidity and mortality, despite advances in pharmacological and surgical interventions. The emergence of CRISPR-Cas9 genome editing technology offers promising approaches for correcting genetic causes of hereditary cardiovascular disorders and modulating pathogenic signaling pathways implicated in various heart diseases. However, several challenges with respect to in vivo delivery of gene editing components, as well as important safety considerations, remain to be addressed in the path toward possible clinical application. We review current gene editing strategies, their potential therapeutic applications in the context of a variety of cardiovascular disorders, and their respective merits, limitations, and regulatory considerations. The rapid advances in this field combined with the many opportunities for deploying gene editing therapies for cardiovascular disorders augur well for the future of this transformative technology.},
}
@article {pmid41277686,
year = {2025},
author = {Stohr, AM and Hansen, H and Richards, B and Park, H and Goncalves, AG and Agrawal, A and Blenner, M and Chen, W},
title = {Metabolite-responsive scaffold RNAs for dynamic CRISPR transcriptional regulation.},
journal = {Nucleic acids research},
volume = {53},
number = {21},
pages = {},
doi = {10.1093/nar/gkaf1290},
pmid = {41277686},
issn = {1362-4962},
support = {MCB2317398//National Science Foundation/ ; GM133803/NH/NIH HHS/United States ; //U.S. Department of Defense/ ; P200A210065//GAANN Fellowship Program/ ; },
mesh = {*CRISPR-Cas Systems ; Theophylline/metabolism/pharmacology ; *RNA, Guide, CRISPR-Cas Systems/genetics/metabolism ; Tryptophan/metabolism/pharmacology ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Transcriptional Activation ; Transcription, Genetic ; *Gene Expression Regulation ; },
abstract = {CRISPR activation is a powerful tool to upregulate a vast array of genes in many different contexts. However, there are few dynamic CRISPR transcriptional programs, which limit its usage in the creation of living biosensors, self-regulating microbial factories, or conditional therapeutics. Here, we address this limitation by embedding a molecular switch directly into a guide RNA to create a combined sensor-actuator called a metabolite-responsive scaffold RNA (MR-scRNA). We demonstrate the regulatory potential for MR-scRNAs by conditionally activating genes in three different kingdoms of life. We create MR-scRNAs responsive to two distinct metabolites, theophylline and tryptophan, by swapping the molecular switch used. MR-scRNAs respond quickly in a dose-dependent manner specifically to their target metabolite and enhance biochemical production when used as a dynamic regulator of pathway enzyme expression. The broad functionality and ease of design of the MR-scRNAs offer a promising tool for dynamic cellular regulation.},
}
@article {pmid41277071,
year = {2025},
author = {Hotta, M and Inoue, YU and Asami, J and Hoshino, M and Inoue, T},
title = {Generation of a Triple Tag Knock-In Mouse to Visualize Precise Protein Localization Patterns for Type II Classic Cadherins During Brain Development.},
journal = {Genes to cells : devoted to molecular &amp; cellular mechanisms},
volume = {30},
number = {6},
pages = {e70070},
doi = {10.1111/gtc.70070},
pmid = {41277071},
issn = {1365-2443},
support = {NCNP 3-9//Intramural Research Grant for Neurological and Psychiatric Disorders of National Center of Neurology and Psychiatry/ ; NCNP 6-9//Intramural Research Grant for Neurological and Psychiatric Disorders of National Center of Neurology and Psychiatry/ ; },
mesh = {Animals ; *Cadherins/metabolism/genetics ; Mice ; *Brain/metabolism/embryology ; Gene Knock-In Techniques ; Gene Expression Regulation, Developmental ; Mice, Transgenic ; CRISPR-Cas Systems ; },
abstract = {Classic cadherin cell-cell adhesion molecules with self-organizing activities play roles in segregating distinct populations of cells at developing brain regions and/or boundaries. However, the protein dynamics of each cadherin subclass in the mouse embryonic brain is poorly described due to the low antigenicity. Here, we generate Cdh6-HA and Cdh8-PA tag knock-in (KI) mice by CRISPR/Cas9-mediated genome editing and establish Cdh6[HA/HA]; Cdh8[PA/PA]; Cdh11[EGFP/EGFP] triple tag KI homo mice with normal viability and fertility. Immunostaining with specific antibodies for these tags reveals differential protein expression profiles almost comparable with mRNA in situ hybridization (ISH) results during embryonic brain development. We can additionally detect considerable levels of immunostaining signals outside the mRNA ISH-positive areas, specifically along the nerve tracts, suggesting physiological accumulation of these type II cadherin proteins along axons. By using super-resolution imaging, we further evaluate cadherin subcellular localization dynamics around the zona limitans intrathalamica to confirm that the prosomere 2/3 compartment boundary at E12.5 is maintained by the distinctive integration of Cdh6 or Cdh11 at apical attachment sites of the ventricular cells. These results highlight the value of the genetic tag KI strategy for proteins with low antigenicity and the functional relevance of type II classic cadherins in brain development.},
}
@article {pmid41276731,
year = {2025},
author = {Eren Eroglu, AE and Toklu, K and Yasa, &#x130;},
title = {Functional genomics of a food-related thermotolerant Acetobacter oryzifermentans strain AAB5: genetic determinants of stress response, CAZyme repertoire, and CRISPR-Cas system.},
journal = {Functional &amp; integrative genomics},
volume = {25},
number = {1},
pages = {253},
pmid = {41276731},
issn = {1438-7948},
}
@article {pmid41276041,
year = {2025},
author = {Khan, MF and Javed, M and Kaur, J and Badwal, AK and Singh, S},
title = {CRISPR-Cas mediated targeting of resistance genes for combating ESKAPE pathogen infections: A Review.},
journal = {International journal of biological macromolecules},
volume = {},
number = {},
pages = {149180},
doi = {10.1016/j.ijbiomac.2025.149180},
pmid = {41276041},
issn = {1879-0003},
abstract = {Advancements in the treatment of antimicrobial infections have highlighted the importance of the CRISPR-Cas system in targeting resistance genes in bacterial pathogens resistant to conventional drugs. Various CRISPR-Cas techniques, such as CRISPR-Cas9, Cas3, dCas9 and the mini-CRISPR system, have been utilized for this purpose in ESKAPE pathogens. Novel strategies like Associates Toxin Antitoxin and CRISPR-Cas to kill multidrug resistant pathogens-CRISPR-regulated toxin antitoxin module (ATTACK-CreTA) and CRISPR interference refine CRISPR-Cas efficacy. This review explores the mechanism of action of resistance genes (e.g., tetM, ermB, VanA, aph-3, aac3, oxa23, blaNDM etc.) prevalent within these pathogens and highlights the notable achievements of CRISPR-Cas technology in targeting these genes, thereby offering a pathway to sensitize resistant bacteria. This article also discusses various delivery approaches for CRISPR components in pathogens, mainly focusing on engineered bacteriophages, including phagemids, temperate phages and virulent phages. Additionally nanoparticles, bacterial conjugation and natural phages hold promise for administering the CRISPR system inside bacteria. Specific targeting of resistance genes in resistant pathogens via CRISPR-Cas based methods would pave a way for combating ESKAPE pathogen infections by reversing the resistance phenotype.},
}
@article {pmid41273791,
year = {2025},
author = {Espinoza-Erazo, VP and Vela-Chauvin, MG and Collantes-Vela, JC and Zapata-Mena, S and Machado, A},
title = {Biofilms of Salmonella: Implications for Food Safety and Public Health.},
journal = {Foodborne pathogens and disease},
volume = {},
number = {},
pages = {},
doi = {10.1177/15353141251389597},
pmid = {41273791},
issn = {1556-7125},
abstract = {Salmonella enterica is a leading cause of foodborne illness worldwide, responsible for an estimated 93.8 million cases and approximately 155,000 deaths annually, according to the World Health Organization. This foodborne pathogen imposes a significant burden on public health and the global economy. A key factor contributing to the persistence and widespread impact of S. enterica is its potential to form biofilms, which may enhance its survival in clinical, industrial, and agricultural environments, making it a major and ongoing public health concern. Biofilms are structured microbial communities encapsulated in a self-produced extracellular matrix that protects against environmental stressors, disinfectants, and antimicrobial agents. This complex phenotype enables Salmonella to colonize food-contact surfaces, medical devices, and host tissues, hampering efforts to eliminate contamination and control transmission. The poultry industry, a key component of the global food supply, is particularly vulnerable to emerging Salmonella strains with increased virulence, stress tolerance, and disinfectant resistance, making biofilm control a top priority. This review aims to provide an updated and comprehensive overview of the mechanisms involved in Salmonella biofilm formation, its implications for food safety, and recent advances in detection and control strategies. Emerging technologies such as CRISPR-Cas systems are receiving particular attention due to their potential as precise molecular tools for investigating genes implicated in biofilm formation. By integrating current findings, this review underscores the urgent need for novel and effective strategies for biofilm control. It highlights the importance of a One Health approach that links human, animal, and environmental health to address the risks posed by Salmonella biofilms in the food production and public health sectors.},
}
@article {pmid41273435,
year = {2025},
author = {Yu, SM and Li, TT and Fu, BQ and Zhang, NZ},
title = {Molecular diagnosis of Trichinella spp.: current status and future prospects.},
journal = {Parasitology research},
volume = {124},
number = {11},
pages = {136},
pmid = {41273435},
issn = {1432-1955},
support = {2023YFD1801000//National Key Research and Development Program of China/ ; QYXTZX-RKZ2024-03-3//Science and Technology Project of Tibet Autonomous Region/ ; },
mesh = {Animals ; *Trichinellosis/diagnosis/parasitology/veterinary ; *Trichinella/genetics/isolation &amp; purification ; *Molecular Diagnostic Techniques/methods/trends ; CRISPR-Cas Systems ; Humans ; },
abstract = {Trichinellosis, a significant parasitic zoonotic disease, poses a risk to public health as well as economic implications for the safety of animal feed. Consumption of raw or undercooked meat containing Trichinella larvae can lead to trichinellosis infection. Many molecular diagnostic methods have been developed to identify Trichinella spp. at muscle or intestine stages. However, no molecular diagnostic technique is currently advised for routine testing on Trichinella infection in food animals, particularly at the early stage of infection. Here, the authors review the development of molecular diagnostic techniques of Trichinella spp., such as PCR, RT-PCR, LAMP, RPA and other methods to detect Trichinella DNA. Recently, the Clustered Regularly Interspaced Short Palindromic Repeats and CRISPR-associated proteins (CRISPR-Cas) technology holds great promise for diagnostic testing by providing rapid, sensitive and specific methods for detection. Diagnosis of Trichinella spp. based on CRISPR-Cas system may be a promising method meeting the needs of individual testing.},
}
@article {pmid41273433,
year = {2025},
author = {Schreiber, D and Yang, R and Guan, X and Schalper, KT and Hou, C and Li, Z and Hegde, P and Liu, C},
title = {3D-Printed CRISPR-based detection system powered by a reusable handwarmer.},
journal = {Biomedical microdevices},
volume = {27},
number = {4},
pages = {53},
pmid = {41273433},
issn = {1572-8781},
support = {U01CA269147/NH/NIH HHS/United States ; UConn Research Excellence Program (REP) award//University of Connecticut/ ; },
mesh = {*Printing, Three-Dimensional ; Humans ; Human papillomavirus 16/genetics ; *CRISPR-Cas Systems/genetics ; *Lab-On-A-Chip Devices ; DNA, Viral/analysis/genetics ; Equipment Reuse ; },
abstract = {Nucleic acid-based molecular diagnostics are essential for the prevention, early detection, and treatment of cancer and infectious diseases. In this study, we developed a 3D-printed, electricity-free detection system for CRISPR-based nucleic acid detection. To eliminate the need for costly electrical heaters, we developed a reusable heating platform powered by a sodium acetate-based handwarmer. To maintain optimal temperatures for the CRISPR reaction, we designed and fabricated a 3D-printed heatsink filled with docosane wax to regulate the temperature. The fully 3D-printed microfluidic chip integrates finger-activated fluid transport via a 3D-printed flexible blister, a CRISPR reaction chamber, and a lateral flow strip for visual readout. We demonstrated the system's analytical performance by detecting HPV-16 DNA with a sensitivity as low as 1 femtomolar. Additionally, we validated its clinical pilot feasibility using clinical cervical samples, achieving results consistent with standard PCR assays. Overall, this low-cost, reusable, and electricity-free detection system offers a practical solution for point-of-care molecular testing, particularly in resource-limited settings.},
}
@article {pmid41273185,
year = {2025},
author = {Verma, A and Kaur, L and Kandoth, PK},
title = {Agrobacterium rhizogenes-Mediated Hairy Root Transformation for Genome Editing in Recalcitrant Legume Lathyrus sativus.},
journal = {Current protocols},
volume = {5},
number = {11},
pages = {e70256},
doi = {10.1002/cpz1.70256},
pmid = {41273185},
issn = {2691-1299},
mesh = {*Lathyrus/genetics ; *Agrobacterium/genetics ; *Plant Roots/genetics ; *Gene Editing/methods ; *Transformation, Genetic ; CRISPR-Cas Systems ; Plants, Genetically Modified/genetics ; },
abstract = {Lathyrus sativus, commonly known as the grass pea, is a nutritious legume that is resilient to climate change, allowing it to grow in drought, waterlogged, and saline soils. However, developing effective functional genomic tools for this crop has been challenging, primarily due to the absence of reliable and stable transformation protocols. Agrobacterium rhizogenes-mediated hairy root transformation provides a practical and rapid method for validating gene functions using the CRISPR/Cas system. This method has not been applied to grass pea despite its potential. In this article, we present the first protocol for A. rhizogenes-mediated hairy root transformation and CRISPR/Cas genome editing aimed at the functional characterization of candidate genes in L. sativus. © 2025 Wiley Periodicals LLC. Basic Protocol 1: Designing CRISPR/Cas9 construct for targeted gene editing in L. sativus Support Protocol 1: Escherichia coli competent cell preparation and transformation Support Protocol 2: A. rhizogenes competent cell preparation and transformation Basic Protocol 2: A. rhizogenes-mediated hairy root transformation in L. sativus Basic Protocol 3: Screening of transgenic hairy root lines Support protocol 3: DNA isolation from L. sativus hairy roots.},
}
@article {pmid41272318,
year = {2025},
author = {Duan, B and Jin, X and An, X and Xiao, Y and Yang, Q and Zhao, H and Huang, Y and Wang, J and Wang, Q and Du, F and Lu, L and Sun, L and Chen, Z and Zhao, B},
title = {Molecular basis of SAM-AMP synthesis and degradation in the type III-B CRISPR-Cas system.},
journal = {Nature chemical biology},
volume = {},
number = {},
pages = {},
pmid = {41272318},
issn = {1552-4469},
support = {2021hwyq36//National Natural Science Foundation of China (National Science Foundation of China)/ ; },
abstract = {Upon sensing nonself target RNA, the CorA-associated type III-B CRISPR-Cas system catalyzes S-adenosyl methionine (SAM) and ATP to synthesize SAM-AMP, which activates the effector CorA and triggers immune responses. SAM-AMP can be degraded by NrN and SAM lyase, potentially deactivating the system. Here we find that the type III-B effector complex from Bacteroides fragilis uses a specific mechanism to recognize nonself target RNA and synthesize SAM-AMP. The 3' anti-tag of nonself target RNA induces conformational changes in the Cmr2 subunit, triggering SAM-AMP synthesis independently of the stalk loop of Cmr3 subunit. SAM-AMP binding induces NrN to transit from an open to a closed conformation, enabling hydrolysis of the 3'-5' phosphodiester bond. SAM lyase forms a triangular trimer that specifically degrades SAM-AMP into 5'-methylthioadenosine-AMP and homoserine lactone. These findings unveil unique mechanisms for SAM-AMP synthesis and degradation and provide deeper insights into the molecular basis of type III CRISPR-Cas signaling.},
}
@article {pmid41271814,
year = {2025},
author = {Ueno, R and Ito, S and Oyama, T},
title = {A CRISPR/Cas9-induced restoration of bioluminescence reporter system for single-cell gene expression analysis in plants.},
journal = {Scientific reports},
volume = {15},
number = {1},
pages = {41271},
pmid = {41271814},
issn = {2045-2322},
support = {JPMJSP2110//Japan Science and Technology Agency/ ; JPMJAL1108//Japan Science and Technology Agency/ ; JP20K06342//Japan Society for the Promotion of Science/ ; 17KT0022//Japan Society for the Promotion of Science/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; *Arabidopsis/genetics/metabolism ; *Single-Cell Analysis/methods ; *Genes, Reporter ; Plants, Genetically Modified/genetics ; *Luminescent Measurements/methods ; *Gene Expression Regulation, Plant ; Luciferases/genetics/metabolism ; Single-Cell Gene Expression Analysis ; },
abstract = {Bioluminescence monitoring techniques have greatly contributed to revealing a variety of biological regulatory systems in living organisms, including circadian clocks. In plant science, these techniques are applied to long-term quantitative analyses of gene expression behavior. Transient transfection with a luciferase reporter using the particle bombardment method has been used for bioluminescence observations at the single-cell level. This allows for capturing heterogeneity and temporal fluctuations in cellular gene expression, although bioluminescence could fluctuate according to variation in physiological factors associated with the luciferase reaction. We developed a novel CRISPR/Cas9-induced restoration of bioluminescence reporter system, CiRBS, to monitor cellular bioluminescence from a reporter gene in the genome of transgenic Arabidopsis. In this method, the enzymatic activity of an inactive luciferase mutant, LUC40Ins26bp, which has a 26-bp insertion at the 40th codon, was restored by introducing an indel at the insertion site using CRISPR/Cas9. We succeeded in long-term monitoring of the cellular bioluminescence of Arabidopsis plants expressing LUC40Ins26bp, which was restored by transient transfection with CRISPR/Cas9-inducible constructs using particle bombardment. Recombination events via indels were mostly complete within 24 h of CRISPR/Cas9 induction, and 7.2% of CRISPR/Cas9-transfected cells restored bioluminescence. It was estimated that 94% of the bioluminescence-restored cells carried only one chromosome having the optimal recombination construction. Thus, CiRBS allows for reliable single-cell gene expression analysis of cell-to-cell heterogeneity and temporal fluctuations from a single locus.},
}
@article {pmid41271724,
year = {2025},
author = {Elsharkasy, OM and Hegeman, CV and Driedonks, TAP and Liang, X and Lansweers, I and Cotugno, OL and de Groot, IY and de Wit, ZEMNJ and Garcia-Guerra, A and Moorman, NJA and Boonstra, SH and Bosman, EDC and Lefferts, JW and de Voogt, WS and François, JJ and van Wesel, ACW and El Andaloussi, S and Schiffelers, RM and Kooijmans, SAA and Mastrobattista, E and Vader, P and de Jong, OG},
title = {A modular strategy for extracellular vesicle-mediated CRISPR-Cas9 delivery through aptamer-based loading and UV-activated cargo release.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {10309},
pmid = {41271724},
issn = {2041-1723},
support = {VI.Veni.192.174//Nederlandse Organisatie voor Wetenschappelijk Onderzoek (Netherlands Organisation for Scientific Research)/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; *Extracellular Vesicles/metabolism ; *Aptamers, Nucleotide/metabolism/genetics ; Humans ; *Gene Editing/methods ; Ultraviolet Rays ; RNA, Guide, CRISPR-Cas Systems/genetics/metabolism ; CRISPR-Associated Protein 9/metabolism/genetics ; HEK293 Cells ; },
abstract = {CRISPR-Cas9 gene editing technology offers the potential to permanently repair genes containing pathological mutations. However, efficient intracellular delivery of the Cas9 ribonucleoprotein complex remains a major hurdle in its therapeutic application. Extracellular vesicles (EVs) are biological nanosized membrane vesicles that play an important role in intercellular communication, and have an innate capability of intercellular transfer of biological cargos, including proteins and RNA. Here, we present a versatile, modular strategy for EV-mediated loading and delivery of Cas9. We leverage the high affinity binding of MS2 coat proteins fused to EV-enriched proteins to MS2 aptamers incorporated into guide RNAs, in combination with a UV-activated photocleavable linker domain, PhoCl. Moreover, we demonstrate that Cas9 can readily be exchanged for other variants, including transcriptional activator dCas9-VPR and adenine base editor ABE8e. Taken together, we describe a robust, modular strategy for successful Cas9 delivery, which can be applied for CRISPR-Cas9-based genetic engineering and transcriptional regulation.},
}
@article {pmid41271690,
year = {2025},
author = {Huang, Z and Dong, Y and Yang, Y and Han, X and Wang, F and Lyon, CJ and Ding, S and Peng, Y and Zhang, G and Hu, C and Huang, H and Yang, L and Zhao, G and Fan, XY and Lu, S and Hu, T and Wang, J},
title = {Thermally programmed one-pot CRISPR assay for on-site pandemic surveillance.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {10286},
pmid = {41271690},
issn = {2041-1723},
support = {31922046//National Natural Science Foundation of China (National Science Foundation of China)/ ; },
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Pandemics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Point-of-Care Systems ; Sensitivity and Specificity ; Temperature ; },
abstract = {The ongoing monkeypox virus outbreak highlights the need for rapid and accurate diagnostics to enhance epidemic control. CRISPR-based assays hold promise, but clinical translation is hindered by high complexity and low throughput. Here, we describe a thermally regulated asynchronous CRISPR-enhanced (TRACE) assay that rapidly and sensitively detects multiple DNA targets in a streamlined, one-pot format. TRACE exhibits a 2.5 copies/test limit of detection - 40 times lower than a canonical one-pot CRISPR. When applied to clinical samples, it achieves 99.5% accuracy across diverse sample types, and can detect MPXV within 11 minutes. Point-of-care TRACE assays meet ASSURED criteria and deliver comparable performance to qPCR, with a fivefold reduced report time, in outpatient settings. Moreover, TRACE enables simultaneous detection of pathogen and host genes at comparable sensitivity to address a critical limitation of current CRISPR assays, which lack internal controls. TRACE thus enables rapid, on-site surveillance to facilitate bench-to-bedside translation of CRISPR diagnostics.},
}
@article {pmid41271362,
year = {2025},
author = {Rahman, MA and Akter, S and Ashrafudoulla, M and Jung, SJ and Rapak, MT and Ha, SD},
title = {CRISPR-Cas systems as emerging tools for precision biofilm control for food safety: Mechanisms and applications.},
journal = {Food research international (Ottawa, Ont.)},
volume = {222},
number = {Pt 2},
pages = {117803},
doi = {10.1016/j.foodres.2025.117803},
pmid = {41271362},
issn = {1873-7145},
mesh = {*Biofilms/growth &amp; development ; *CRISPR-Cas Systems ; *Food Safety/methods ; Gene Editing/methods ; *Food Microbiology/methods ; },
abstract = {Biofilms on food-contact surfaces pose persistent challenges to sanitation, safety, and product quality within food processing. Traditional cleaning methods and broad-spectrum antimicrobials often fail to disrupt the resilient matrix and multispecies communities characteristic of these biofilms. Clustered regularly interspaced short palindromic repeat (CRISPR)-CRISPR-associated (Cas) systems offer a transformative approach to enhancing food safety, enabling precise modulation of microbial gene networks with applications in diagnostics, programmable sanitation, and targeted microbial control. This review synthesizes recent advances in CRISPR-Cas technology, encompassing Cas9/Cas12-based gene editing, Cas13-mediated RNA targeting, and dead Cas9 (dCas9)-based transcriptional regulation (CRISPR interference/activation, CRISPRi/a), and evaluates their relevance to biofilm prevention and eradication in food environments. We critically assess delivery platforms, including plasmids, nanocarriers, phagemids, and conjugative systems, for their efficiency in complex biofilm settings. The review highlights innovations such as multiplexed repression of redundant pathways, activation of latent antibiofilm functions. These genetic strategies are increasingly being integrated with omics-based analytics (e.g., transcriptomics, proteomics, metabolomics) to reveal systems-level cellular responses and regulatory shifts triggered by biofilm-targeted interventions. We also address the practical limitations, such as delivery barriers, off-target effects, regulatory hurdles, and ethical considerations specific to food applications. Ultimately, we propose a framework for translating CRISPR-Cas technology into scalable, safety-compliant tools for precision control of biofilms in food processing environments. This review aims to guide future research and inform stakeholders on leveraging CRISPR-Cas technology for safe, sustainable, and targeted management of food-associated biofilms.},
}
@article {pmid41202983,
year = {2026},
author = {Hocq, R and Chartier, G and Lopes Ferreira, N and Wasels, F},
title = {CRISPR/anti-CRISPR genome editing in Clostridium beijerinckii.},
journal = {Journal of biotechnology},
volume = {409},
number = {},
pages = {165-169},
doi = {10.1016/j.jbiotec.2025.11.002},
pmid = {41202983},
issn = {1873-4863},
mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; *Clostridium beijerinckii/genetics/drug effects ; Bacterial Proteins/genetics/metabolism ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Listeria monocytogenes/genetics ; Genome, Bacterial ; CRISPR-Associated Protein 9/genetics ; },
abstract = {The development of CRISPR technologies has revolutionized genome editing. However, in bacteria, CRISPR-based methods can be difficult to implement due to the cytotoxicity of CRISPR-associated proteins, which often impair or entirely prevent transformation. In this work, we combine inducible expression of classical CRISPR-Cas9 components with the anti-CRISPR protein AcrIIA4 from Listeria monocytogenes to tightly regulate Cas9 activity. Using this approach, we demonstrate efficient and iterative genome editing in the genetically recalcitrant Clostridium beijerinckii DSM 6423. While deletion of upp alone was not sufficient to render the strain sensitive to 5-fluorouracil, the additional deletion of a second gene involved in the uracil salvage pathway conferred resistance to the drug and validated our gene editing strategy. Collectively, our results show that CRISPR/anti-CRISPR systems can overcome a key limitation of CRISPR-based genome editing and may offer a broadly applicable strategy for engineering otherwise intractable bacterial species.},
}
@article {pmid41202696,
year = {2025},
author = {Ito, S and Nakamura, K and Murata, K and Nakajima, R and Kanou, M and Koketsu, M and Yamana, K and Yamanouchi, K and Ueda, H},
title = {Generation of Cre/LoxP-mediated extracellular TurboID knock-in rats with CRISPR/Cas9 system.},
journal = {Biochemical and biophysical research communications},
volume = {791},
number = {},
pages = {152898},
doi = {10.1016/j.bbrc.2025.152898},
pmid = {41202696},
issn = {1090-2104},
mesh = {Animals ; *CRISPR-Cas Systems/genetics ; *Gene Knock-In Techniques/methods ; Rats ; *Integrases/metabolism/genetics ; Biotinylation ; Cell Membrane/metabolism ; },
abstract = {The plasma membrane plays a central role in regulating signal transduction from the extracellular environment to the intracellular space and represents a major site of drug targeting. Proximity-dependent biotinylation with biotin ligases such as BioID and its derivatives, including TurboID and AirID, enables identification of novel protein‒protein interactions by fusion to a target protein. In vivo application of biotin ligases for cell membrane analysis has been attempted; however, methods for expressing biotin ligases on the target cell membrane remain largely limited to viral vector delivery. In this study, we inserted a Cre-dependent TurboID expression cassette into the rat genome to express TurboID on the cell surface via the CRISPR/Cas9 system. We generated TurboID knock-in (KI) rats that express TurboID on the cell surface, enabling biotinylation of extracellular proteins. The TurboID KI rats thus provide a valuable model for in vivo analysis of cell-surface molecules and may facilitate identification of novel drug targets or antigens.},
}
@article {pmid41130365,
year = {2026},
author = {Zou, M and Tao, Y and Shi, B and Xu, R and Zhu, D and Li, Y and Han, R and Wang, R},
title = {CRISPR/Cas9-based gene deletion and targeted metabolomics reveal ectoine flux reprogramming in Halomonas campaniensis.},
journal = {Journal of biotechnology},
volume = {409},
number = {},
pages = {67-76},
doi = {10.1016/j.jbiotec.2025.10.006},
pmid = {41130365},
issn = {1873-4863},
mesh = {*Halomonas/genetics/metabolism ; *Amino Acids, Diamino/metabolism/biosynthesis/genetics ; Metabolomics/methods ; Gene Deletion ; *CRISPR-Cas Systems/genetics ; Betaine/metabolism ; Bacterial Proteins/genetics/metabolism ; },
abstract = {Ectoine and betaine are widely used compatible solutes. In Halomonas campaniensis XH26, the hom gene is involved in betaine biosynthesis, and the doeA gene participates in ectoine degradation. Deletion of hom and doeA may lead to poorly understood changes in metabolic flux within the ectoine biosynthesis pathway. The metabolically deficient XH26/Δhom and XH26/Δhom/ΔdoeA strains were constructed using a CRISPR/Cas9 approach. Comparative analyses of colony morphology, growth characteristics, and intracellular ectoine yield were conducted to evaluate the regulatory roles of the hom and doeA genes. RT-qPCR and targeted metabolomics were used to assess changes in gene expression related to ectoine biosynthesis and shifts in central carbon metabolic flux. The metabolically deficient strains XH26/Δhom and XH26/Δhom/ΔdoeA were constructed. Compared to the strain XH26, both mutant strains exhibited smaller colony diameters and shorter, broader cells. Intracellular ectoine yield increased by 13.3 % and 33.3 %, respectively, while betaine yield significantly decreased by 73.08 % and 76.92 %. RT-qPCR analysis revealed the significant upregulation of asd, lysC, ectA, ectB, and ectC, suggesting an enhanced metabolic flux toward ectoine biosynthesis. Targeted metabolomics indicated that the differentially abundant metabolites were mainly involved in four key energy metabolism pathways. These results indicate that knocking out the key genes hom and doeA in the ectoine biosynthesis pathway led to the restructuring of carbon metabolic flux in H. campaniensis. More carbon entered the ectoine biosynthesis pathway, resulting in the enhanced production of ectoine and a concomitant reduction in its degradation. These findings offer theoretical support for engineering high-yield ectoine-producing strains.},
}
@article {pmid41048015,
year = {2025},
author = {Li, Y and Zhao, W and Wu, Y and Li, R and Zhang, J and Xie, H and Zhang, K and Li, J},
title = {In vivo CRISPR biosensing.},
journal = {Chemical Society reviews},
volume = {54},
number = {23},
pages = {10977-11016},
doi = {10.1039/d5cs00921a},
pmid = {41048015},
issn = {1460-4744},
mesh = {*Biosensing Techniques/methods ; *CRISPR-Cas Systems/genetics ; Humans ; Animals ; *Clustered Regularly Interspaced Short Palindromic Repeats ; DNA/genetics/analysis ; Gene Editing ; },
abstract = {In vivo biosensing is essential for real-time monitoring of biological processes and disease progression within living organisms. Leveraging the programmable specificity and multifunctionality of CRISPR effectors, in vivo CRISPR-based biosensing has emerged as a powerful tool for highly sensitive and target-specific detection in complex physiological environments. This review presents the fundamental principles, design strategies, and bioanalytical applications of these advanced sensors, focusing on three key approaches: CRISPR-mediated highly efficient in vivo sequence recognition, CRISPR-driven trans-cleavage activity for signal amplification, and the use of base editors and prime editors for sensing-coupled genetic modulation. Critical design parameters-including delivery strategies, intracellular dynamics, and signal amplification mechanisms-are discussed in detail. We further highlight a broad range of applications, including in vivo DNA/RNA imaging, quantification of proteins and small molecules, gene-controlled drug release, dynamic signal recording, environmental response sensing, and lineage tracing in embryogenesis and tumor progression. The current challenges and outlining future directions are also discussed, underscoring the transformative potential of in vivo CRISPR biosensing in both fundamental biology and clinical translation.},
}
@article {pmid40555270,
year = {2025},
author = {Liang, L and Yang, Y and Jacqueline Elise, F and Yu, J and Yin, X and Lu, G and Chen, B and Xing, J},
title = {Potential Applications of the CRISPR-Cas9 System for Research and Treatment of Osteoarthritis.},
journal = {Zeitschrift fur Orthopadie und Unfallchirurgie},
volume = {163},
number = {6},
pages = {510-517},
doi = {10.1055/a-2616-0819},
pmid = {40555270},
issn = {1864-6743},
support = {2308085QH292//Anhui Natural Science Foundation/ ; 82305280//This research did receive grant from National Natural Science Foundation of China/ ; },
mesh = {*Osteoarthritis/therapy/genetics ; *CRISPR-Cas Systems/genetics ; Humans ; Animals ; *Genetic Therapy/methods ; *Gene Editing/methods ; Disease Models, Animal ; },
abstract = {Osteoarthritis is a common degenerative disease of joint cartilage that affects millions of people in the world, especially the elderly. Progression of osteoarthritis is associated with a plethora of genetic and non-genetic factors. The CRISPR/Cas9 system is emerging as a powerful tool for genome engineering and has remarkable potential for guiding further research into osteoarthritis and may be a viable means for treating the disease. This review discusses existing and potential applications of the CRISPR/Cas9 system in osteoarthritis studies and treatments. Firstly, we briefly summarize the current status and mechanism of this technology. Next, we focus on the latest advances in the application of CRISPR/Cas9 system in elucidating the contributions of various factors to the pathogenesis of osteoarthritis as demonstrated through in vitro studies and animal models. Finally, we provide our perspective on the direction and challenges of studying and treating osteoarthritis with CRISPR/Cas9.},
}
@article {pmid41270733,
year = {2025},
author = {Wei, Z and Lan, Y and Meng, L and Wang, H and Li, L and Li, Y and Zhang, N and Lu, R and Cui, Z and Song, Y and Wang, Y and Li, Y and Yue, Z and Fan, G and Li, Q and Gu, Y and Liu, S and Qian, PY and Meng, L and Shao, C},
title = {Hologenomic insights into the molecular adaptation of deep-sea coral Bathypathes pseudoalternata.},
journal = {Cell host &amp; microbe},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.chom.2025.10.020},
pmid = {41270733},
issn = {1934-6069},
abstract = {Deep-sea coral ecosystems support biodiversity and nutrient cycling through interactions with symbionts. However, their molecular mechanisms remain unexplored. Here, hologenomic analyses of Bathypathes pseudoalternata are applied to uncover molecular adaptations underpinning host-symbiont interactions. Genomic evidence reveals that B. pseudoalternata exhibits adaptations in nutrient transport, immune response, and lysosomal digestion, reflecting its genomic adjustments for a stable symbiosis. Candidatus Nitrosopumilus bathypathes (78.43% ± 3.65%) is inferred to oxidize host-derived ammonia to synthesize amino acids and vitamins to provision the host. The presence of CRISPR-Cas and restriction-modification (R-M) systems suggests that Ca. Bathyplasma bathypathes and Ca. Thalassoplasma bathypathes (10.68% ± 2.99%) may protect the host from viral infections. Ca. Bathybacter bathypathes (8.39% ± 1.53%) is hypothesized to synthesize heme, lipoic acid, and glutathione, which serve dual functions as antioxidants and nutrients. These findings collectively provide insights into how the hologenome contributes to the survival of B. pseudoalternata in the extreme environment.},
}
@article {pmid41270608,
year = {2025},
author = {Zhou, Y and Zhai, J and Chen, H and Qu, Y and Fang, Z and Chen, B and Bao, Z and Chen, D},
title = {Dz-SiG CRISPR: A DNAzyme-Switched G-quadruplex-lock CRISPR system for isothermal and rapid detection of lead ions.},
journal = {Talanta},
volume = {299},
number = {},
pages = {129139},
doi = {10.1016/j.talanta.2025.129139},
pmid = {41270608},
issn = {1873-3573},
abstract = {Lead (Pb[2+]) poses serious risks to health and ecosystems, necessitating rapid, ultrasensitive detection. CRISPR/Cas12a systems offers exceptional specificity and intrinsic signal amplification. Nevertheless, their adaptation to Pb[2+] is hindered by the lack of programmable interfaces to convert small-molecule binding into Cas12a activation. Herein, we report a DNAzyme-Switched G-quadruplex-locked CRISPR (Dz-SiG CRISPR) strategy, enabling ultrasensitive, femtomolar-level detection of Pb[2+]. A RNA G-quadruplex (RG4) structure is conjugated to the 5' end of the crRNA, serving as a conformational lock that suppresses Cas12a's trans-cleavage activity. Upon Pb[2+] binding, the GR-5 DNAzyme catalyzes a hydrolytic cleavage that acts as a molecular switch, releasing the RG4 domain and unleashing active crRNA to trigger Cas12a-mediated cleavage of a fluorogenic reporter, yielding a sharp "off-to-on" fluorescent signal. The Dz-SiG CRISPR system achieves an ultralow limit of detection of 18.91 fM for Pb[2+] and demonstrates outstanding performance in real water and soil samples, with recovery rates ranging from 94.44 % to 99.03 %. The assay can be completed within 30 min, making it highly suitable for rapid on-site lead ion detection. Importantly, the modular Dz-SiG CRISPR framework can be readily reprogrammed for other small molecules by simply substituting the DNAzyme module, offering a generalizable strategy for rapid, ultrasensitive environmental monitoring.},
}
@article {pmid41202468,
year = {2025},
author = {Zingarelli, F and Nanni, J and Cristiano, G and Zannoni, L and Curti, A},
title = {CRISPR-Cas9 in acute myeloid leukaemia: Current state-of-art and future perspectives.},
journal = {Current opinion in pharmacology},
volume = {85},
number = {},
pages = {102582},
doi = {10.1016/j.coph.2025.102582},
pmid = {41202468},
issn = {1471-4973},
mesh = {Humans ; *Leukemia, Myeloid, Acute/genetics/therapy/diagnosis ; *CRISPR-Cas Systems ; *Gene Editing/methods ; Animals ; *Genetic Therapy/methods ; },
abstract = {CRISPR-Cas9 gene editing technology has gained attention as a new, reliable and manageable tool for the treatment of previously incurable monogenic diseases. Besides exciting results in this setting, ethical, safety and crucial technical issues have not been fully clarified. More importantly, the role of this potent editing tool in the context of a genetically complex and heterogeneous hematologic malignancy such as acute myeloid leukemia (AML) has not yet been defined to date. In this review we aim to summarize and exploring the ultimate CRISPR-cas9 based strategies for diagnosis, risk stratification and treatment in the context of AML.},
}
@article {pmid41177337,
year = {2025},
author = {Liu, B and Li, Y and Yang, Z and Wu, J and Jiang, Y and Zhao, L and Ge, J},
title = {A rapid and visual detection for canine Adenovirus-2 using CRISPR-Cas13a-based SHERLOCK technology.},
journal = {Journal of microbiological methods},
volume = {239},
number = {},
pages = {107314},
doi = {10.1016/j.mimet.2025.107314},
pmid = {41177337},
issn = {1872-8359},
mesh = {Animals ; Dogs ; *CRISPR-Cas Systems/genetics ; *Adenoviridae Infections/diagnosis/veterinary/virology ; *Adenoviruses, Canine/isolation &amp; purification/genetics ; Sensitivity and Specificity ; *Nucleic Acid Amplification Techniques/methods ; *Dog Diseases/diagnosis/virology ; *Molecular Diagnostic Techniques/methods ; },
abstract = {Canine adenovirus type 2 (CAdV-2) is an important pathogen causing infectious tracheobronchitis (ITB) and viral enteritis in puppies, often exacerbating clinical symptoms through co-infection with other viruses. However, existing diagnostic methods for CAdV-2 exhibit notable limitations. Specifically, they are time-consuming, require additional nucleic acid purification steps, depend on expensive detection equipment, and necessitate operation by professional personnel. Collectively, these limitations prevent the achievement of rapid and accurate CAdV-2 detection in resource-limited settings. In this study, we established a novel CAdV-2 detection method by integrating CRISPR/Cas13a collateral cleavage activity with HUDSON rapid nucleic acid extraction, recombinase-aided amplification (RAA), and a lateral flow strip. This isothermal assay allows for visual, naked-eye result interpretation and achieves a sensitivity of 10[2] copies/μL as read by lateral flow strips (corresponding to approximately 750 copies per reaction). It showed excellent specificity with no cross-reactivity observed against five other major canine viruses. When tested on 20 clinical samples, the assay demonstrated a 95 % concordance rate with the conventional simplex PCR results. The entire detection process is simple to perform, requires only basic equipment, and delivers results within 90 min. The developed CRISPR/Cas13a-based detection method exhibits significant application potential for CAdV-2 detection. This study develops a CRISPR/Cas13a-based point-of-care diagnostic tool for CAdV-2, delivering rapid, sensitive, and visual detection that significantly facilitates field-based pathogen surveillance and control efforts, while advancing the application of CRISPR diagnostics in veterinary infectious diseases.},
}
@article {pmid41167358,
year = {2025},
author = {Rahangdale, S and Vishwakarma, A and Chauhan, R and Singh, S and Singh, PK},
title = {Engineered sgRNA captures single-stranded donor template and delivers at the DSB site to enhance HDR.},
journal = {International journal of biological macromolecules},
volume = {332},
number = {Pt 2},
pages = {148614},
doi = {10.1016/j.ijbiomac.2025.148614},
pmid = {41167358},
issn = {1879-0003},
mesh = {Saccharomyces cerevisiae/genetics ; *Gene Editing/methods ; *RNA, Guide, CRISPR-Cas Systems/genetics ; CRISPR-Cas Systems/genetics ; *DNA Breaks, Double-Stranded ; *Recombinational DNA Repair ; *DNA, Single-Stranded/genetics ; Streptococcus pyogenes/genetics ; },
abstract = {The CRISPR-Cas9 system from Streptococcus pyogenes has revolutionized genome modification through precise editing across a wide range of organisms. Yeast supports efficient genome editing via plasmid-based Cas9-gRNA expression, while higher eukaryotes often require genome-integrated cassettes or RNP delivery. In this study, we engineered CRISPR components to enhance nuclear targeting and editing efficiency. We demonstrated the proof of concept in Saccharomyces cerevisiae using its CAN1 locus. We developed a dual-host compatible vector, encoding Cas9 nuclease fused with three nuclear localization signals (Cas9-3xNLS). The recombinant protein, expressed in E. coli and purified on a Ni-NTA column, showed DNA cleavage in an in vitro assay. Genome editing efficacy of Cas9-3xNLS was demonstrated in S. cerevisiae AH109 strain. Further, we engineered sgRNAs by extending their ends to facilitate the annealing to ssODN. We synthesized ssODNs having a complementary sequence either at 3' or 5' to anchor with sgRNAs. sgRNAs (unmodified and end extended) and ssODNs were introduced into yeast in various combinations. sgRNA with a 3' ssDNA-anchoring motif and ssODN representing the antisense strand of the target gene with sgRNA complementary motif at the 5' end (free homology arm at 3') improved HDR efficiency significantly. This combination yielded about a 1.64-fold increase in canavanine-resistant colonies as compared to the control via precise insertion of a stop codon. In contrast, extension of sgRNA at the 5' end did not show any advantage. This approach is flexible and easy to use and has the potential to enhance homology-directed repair in diverse organisms.},
}
@article {pmid41093192,
year = {2025},
author = {Chou, SJ and Wang, CH and Chang, YL and Fang, WC and Hwang, DK and Hsiao, YJ and Luo, YH and Lo, WL and Viet, NQ and Tang, KY and Lan, YT and Hsu, CC and Chen, SJ and Lin, TC and Yang, YP and Chiou, SH},
title = {Dual delivery of supramolecular nanoparticle-carried minicircle donor DNA with Cas9/gRNA improved HITI knock-in efficiency in X-linked juvenile retinoschisis.},
journal = {International journal of biological macromolecules},
volume = {332},
number = {Pt 2},
pages = {148300},
doi = {10.1016/j.ijbiomac.2025.148300},
pmid = {41093192},
issn = {1879-0003},
mesh = {Humans ; *Nanoparticles/chemistry ; *CRISPR-Cas Systems/genetics ; *Retinoschisis/genetics/therapy ; *RNA, Guide, CRISPR-Cas Systems/genetics ; *DNA, Circular/genetics ; Gene Editing/methods ; *Gene Knock-In Techniques/methods ; Gene Transfer Techniques ; CRISPR-Associated Protein 9/genetics ; Genetic Therapy/methods ; HEK293 Cells ; Eye Proteins/genetics ; Plasmids/genetics ; },
abstract = {X-linked retinoschisis (XLRS) is a hereditary mutation of the RS1 gene and is characterized by early-onset maculopathy with severe visual impairment. Current gene therapy utilizing CRISPR-associated protein 9 (Cas9) is ongoing; however, the optimization of nonviral/nanoparticle CRISPR/Cas9-based therapeutics for gene delivery into nondivided retinal neurons remains undetermined. Minicircles DNA, a circular DNA molecule lacking bacterial backbone sequences, has gained recognition for improving transfection efficiency and biosafety. Here, we developed a supramolecular nanoparticle (SMNP)-editing platform in which SMNPs carrying CRISPR/Cas9 integrated minicircle donor DNA (mc dDNA) to achieve highly efficient and precise gene knock-in. To increase the efficiency of RS1 gene knock-in, we replaced the conventional mc dDNA with a single flanking-Cas9/cut site. Furthermore, using homology-independent targeted integration (HITI) as an editing-nondivided cell strategy, SMNP-carried CRISPR/Cas9 could effectively facilitate the dual delivery of mc-RS1/GFP dDNA and Cas9/gRNA plasmids. Compared with the delivery of the Cas9/gRNA plasmid alone (~20 %), the delivery of mc-RS1/GFP dDNA via SMNPs had significantly higher transfection efficiency (90 %). Further flow cytometry analysis revealed that 5.99 % of the FACS-positive cells were detected in the mc-RS1/GFP dDNA group, markedly exceeding the 2.21 % with long-term expression in the conventional dDNA group. Moreover, when XLRS/iPSC-derived retinal neuron organoids were used as a patient-based disease model, compared with conventional plasmid-based delivery, robust RS1 expression with integration sustained transgene expression in XLRS/iPSC-derived retinal organoids. Collectively, these findings indicated that SMNP-mediated dual delivery of the Cas9/gRNA plasmid and mc-RS1/GFP dDNA substantially enhanced RS1-targeted integration with long-term transgene expression, providing safer and effective gene therapy for the treatment of XLRS.},
}
@article {pmid41270157,
year = {2025},
author = {Li, Y and Wu, Y and Zheng, Z and Wu, Y and Zhang, Y and Zhang, J and Quan, F and Zhao, W and Xu, R and Li, Y and Gao, H and Zhang, K},
title = {Renal clearable CRISPR nanosensor targeting mitochondrial DNA mutation for noninvasive monitoring of tumor progression and metastasis.},
journal = {Science advances},
volume = {11},
number = {47},
pages = {eadz4594},
pmid = {41270157},
issn = {2375-2548},
mesh = {*DNA, Mitochondrial/genetics ; *Mutation ; Humans ; *CRISPR-Cas Systems ; Animals ; Disease Progression ; Neoplasm Metastasis ; Mice ; Biomarkers, Tumor/genetics/urine ; Cell Line, Tumor ; *Biosensing Techniques/methods ; Mitochondria/genetics ; Lung Neoplasms/genetics ; },
abstract = {Mitochondrial DNA (mtDNA) mutations are emerging as important molecular features of tumorigenesis. Liquid biopsies, involving analysis of cell-free mtDNA, enable early cancer detection but suffer from low sensitivity due to scarce analytes. Here, we developed a CRISPR/Cas12a-mediated urinary biomarker, termed CasUber, for in vivo monitoring of tumor progression and metastasis. Our results demonstrate that CasUber can deliver a CRISPR detection system into tumor cell mitochondria, leverage the single-nucleotide variant recognition ability and trans-cleavage activity of Cas12a to convert tumor-specific mtDNA mutations into renal-clearable fluorescent biomarkers, and exocytosed along with the natural efflux pathway of damaged mtDNA. As a result, CasUber enables discrimination of ultrasmall tumor lesions (~1 cubic millimeter) and detection of lung tumor nodules earlier than bioluminescence imaging in a blood-lung metastasis model. This renal clearable nanosensor allows in situ recognition of specific gene mutation to generate amplified signals, overcoming the limitation of low mtDNA abundance and enabling noninvasive and ultrasensitive monitoring of tumor progression and metastasis via a simple urine test.},
}
@article {pmid41266743,
year = {2025},
author = {Zhang, W and Shi, J and Wang, B and Qu, H and Wu, X and Wang, X},
title = {An integrated Aptamer-CRISPR-Cas12a method for rapid and sensitive detection of carbendazim.},
journal = {Scientific reports},
volume = {15},
number = {1},
pages = {41119},
pmid = {41266743},
issn = {2045-2322},
support = {22A310016//Henan Province Higher Education Key Scientific Research Project/ ; },
mesh = {*Carbamates/analysis ; *Aptamers, Nucleotide/genetics/chemistry ; *CRISPR-Cas Systems ; *Benzimidazoles/analysis ; Limit of Detection ; *Biosensing Techniques/methods ; *Pesticide Residues/analysis ; Reproducibility of Results ; },
abstract = {Concerns over carbendazim (CBZ) pesticide residues in agricultural products and medicinal herbs have intensified due to their potential health and environmental risks. While existing detection techniques offer distinct advantages, they are often limited by complex procedures, specialized equipment, and high costs. To address these challenges, we developed a novel Aptamer-CRISPR/Cas12a assay, which combines the specificity of aptamers with the high sensitivity and precision of the CRISPR/Cas12a system. This assay achieves a linear detection range of 10-5000 ng/mL (R[2] = 0.9639 at 15 min and R[2] = 0.9774 at 30 min) and a limit of detection (LOD) of 10 ng/mL. In real samples, the average recovery rate of CBZ ranges from 92.10% to 102.86%, demonstrating robust accuracy and reliability. Notably, the method is user-friendly, requires minimal equipment, and delivers results in about 40 min, making it suitable for field applications. Furthermore, the crRNA serves as a universal sequence, enabling the detection of different targets by simply replacing the aptamer and complementary strand, while keeping the CRISPR/Cas12a system intact. This streamlined approach enhances flexibility and broad applicability. In conclusion, the Aptamer-CRISPR/Cas12a assay offers a practical solution for monitoring agricultural products, Chinese herbal medicine, and environmental safety.},
}
@article {pmid41266199,
year = {2025},
author = {Wu, X and Wang, M and Luo, S and Zhou, Z and Wang, Y and Du, G and Chen, J and Liu, X},
title = {Dual enhancement of mycoprotein nutrition and sustainability via CRISPR-mediated metabolic engineering of Fusarium venenatum.},
journal = {Trends in biotechnology},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.tibtech.2025.09.016},
pmid = {41266199},
issn = {1879-3096},
abstract = {Mycoprotein (MP) production represents a promising environmentally sustainable strategy to address global protein deficit. To enhance the nutritional profile and production efficiency of MP, we employed CRISPR/Cas9-mediated scarless gene knockout and obtained a Fusarium venenatum strain (designated FCPD), which exhibited a 32.9% increase in essential amino acid index (EAAI) through targeted truncation of competitive metabolic pathways and regulation of amino acid metabolism or biosynthesis. FCPD achieved a 44.3% reduction in substrate consumption while improving MP production rate by 88.4% compared with the wild type (WT) strain. The cradle-to-gate life cycle assessment (LCA) shows that FCPD could reduce environmental impacts such as global warming potential (GWP) by 4-61.3% under production scenarios in six representative countries. Comparative environmental performance demonstrated the superiority of FCPD-MP over cell-cultured meat and chicken meat. These findings establish CRISPR/Cas technology and metabolic engineering as the dual-purpose tool for both nutritional enhancement and environmental impact mitigation in alternative protein production.},
}
@article {pmid41265250,
year = {2025},
author = {Ramachandran, H and Becker, A and Dobner, J and Hildebrandt, B and Distelmaier, F and Rossi, A and Anand, R},
title = {CRISPR/Cas9-mediated editing of MIC13 in human induced pluripotent stem cells: A model for mitochondrial hepato-encephalopathy.},
journal = {Stem cell research},
volume = {89},
number = {},
pages = {103870},
doi = {10.1016/j.scr.2025.103870},
pmid = {41265250},
issn = {1876-7753},
abstract = {MIC13 is essential for cristae formation and functions as a key component of the large mitochondrial multi subunit MICOS complex. Mutations in MIC13 causes severe mitochondrial disease called mitochondrial hepato-encephalopathy. In this study, we describe the generation of a human induced pluripotent stem cell (iPSC) line carrying a patient-specific MIC13 mutation, introduced using a CRISPR/Cas knock-in approach. The resulting iPSC line will provide a valuable model to study the pediatric severe mitochondrial disease and to determine the pathological mechanisms as well as to facilitate the identification of potential therapeutic targets in the future.},
}
@article {pmid41264989,
year = {2025},
author = {Liu, X and Yang, M and Sun, D and Lu, C and Ma, Y and Jiang, Y and Ouyang, R and Miao, Y},
title = {Integrating amplification strategies and functional nanomaterials for advanced electrochemical biosensing of MicroRNA.},
journal = {Bioelectrochemistry (Amsterdam, Netherlands)},
volume = {168},
number = {},
pages = {109170},
doi = {10.1016/j.bioelechem.2025.109170},
pmid = {41264989},
issn = {1878-562X},
abstract = {MicroRNAs (miRNAs) are crucial disease biomarkers, yet their short length, low abundance, and high sequence homology pose significant challenges for sensitive detection. Electrochemical biosensing presents a promising alternative, though effective signal amplification remains essential. This review summarizes recent advances in amplification strategies for electrochemical miRNA detection, covering nucleic acid-based techniques-such as hybridization chain reaction (HCR), rolling circle amplification (RCA), and catalytic hairpin assembly (CHA)-as well as nanomaterial-assisted approaches using metal-organic frameworks and transition metal dichalcogenides. Key mechanisms, advantages, and limitations of each method are discussed, along with performance metrics (e.g., detection limit and linear range) and emerging hybrid systems like RCA-CRISPR/Cas. Current challenges, including probe complexity and nanomaterial aggregation, are also addressed. Finally, the review highlights future directions involving multi-mechanism integration and clinical translation, offering insights for the development of highly sensitive and reliable electrochemical biosensors to advance precision medicine.},
}
@article {pmid41233602,
year = {2025},
author = {Strefeler, A and Baker, ZN and Chollet, S and Foged, MM and Guerra, RM and Ivanisevic, J and Gallart-Ayala, H and Pagliarini, DJ and Jourdain, AA},
title = {Uridine-sensitized screening identifies demethoxy-coenzyme Q and NUDT5 as regulators of nucleotide synthesis.},
journal = {Nature metabolism},
volume = {7},
number = {11},
pages = {2221-2235},
pmid = {41233602},
issn = {2522-5812},
support = {310030_200796//Schweizerischer Nationalfonds zur F&#xf6;rderung der Wissenschaftlichen Forschung (Swiss National Science Foundation)/ ; },
mesh = {*Pyrophosphatases/metabolism/genetics ; Humans ; *Nucleotides/biosynthesis ; *Uridine/metabolism/pharmacology ; Phosphoribosyl Pyrophosphate/metabolism ; CRISPR-Cas Systems ; Pyrimidines/biosynthesis ; },
abstract = {Rapidly proliferating cells require large amounts of nucleotides, making nucleotide metabolism a widely exploited therapeutic target against cancer, autoinflammatory disorders and viral infections. However, regulation of nucleotide metabolism remains incompletely understood. Here, we reveal regulators of de novo pyrimidine synthesis. Using uridine-sensitized CRISPR-Cas9 screening, we show that coenzyme Q (CoQ) is dispensable for pyrimidine synthesis, in the presence of the demethoxy-CoQ intermediate as alternative electron acceptor. We further report that the ADP-ribose pyrophosphatase NUDT5 directly binds PPAT, the rate-limiting enzyme in purine synthesis, which inhibits its activity and preserves the phosphoribosyl pyrophosphate (PRPP) pool. In the absence of NUDT5, hyperactive purine synthesis exhausts the PRPP pool at the expense of pyrimidine synthesis, which promotes resistance to purine and pyrimidine nucleobase analogues. Of note, the interaction between NUDT5 and PPAT is disrupted by PRPP, highlighting an intricate allosteric regulation. Overall, our findings reveal a fundamental mechanism of nucleotide balance and position NUDT5 as a regulator of nucleobase analogue metabolism.},
}
@article {pmid40992601,
year = {2026},
author = {Demirayak, PS and Akay Sazaklioglu, S},
title = {CRISPR for detection of drug resistance genes.},
journal = {Clinica chimica acta; international journal of clinical chemistry},
volume = {579},
number = {},
pages = {120626},
doi = {10.1016/j.cca.2025.120626},
pmid = {40992601},
issn = {1873-3492},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Drug Resistance/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; },
abstract = {Resistance to antibiotics, anticancer, antiviral, and antiparasitic drugs has become one of the greatest threats to modern medicine, seriously straining global health systems. Antimicrobial resistance threatens the integrity of the health system by reducing the effectiveness of treatment protocols such as chemotherapy, organ transplantation, and major surgical interventions. In this case, not only the development of new drugs but also the rapid, sensitive, and specific detection of resistant microorganisms and genetic markers is of vital importance. Therefore, the need for more innovative diagnostic approaches suitable for field applications is increasing. CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)-based molecular diagnostic systems developed in recent years stand out as strong candidates that can fill the gap in this area. Thanks to their ability to recognize and target specific DNA or RNA sequences with high specificity, CRISPR systems enable rapid and sensitive detection of drug resistance genes. Various CRISPR effector proteins, such as Cas9, Cas12, and Cas13, have the potential to revolutionize diagnostic technologies due to their ability to both target-specifically cut and generate signals. This review will focus on the application of CRISPR technology for detecting drug resistance genes. In addition, the sensitivity, specificity, application areas, and technical challenges of the systems will be discussed through literature examples of current applications. The review aims to synthesize scientific developments in this field by examining how CRISPR-based diagnostic approaches can play a role in the global fight against drug resistance and to provide a guiding resource for future research.},
}
@article {pmid40992599,
year = {2026},
author = {Jiang, T and Zhang, C and Wang, D and Guo, Z and Guo, Y and Liu, H and Wang, Z},
title = {Rapid molecular diagnostic method for Gardnerella vaginalis based on CRISPR-Cas12a and recombinase-aided amplification (RAA).},
journal = {Clinica chimica acta; international journal of clinical chemistry},
volume = {579},
number = {},
pages = {120625},
doi = {10.1016/j.cca.2025.120625},
pmid = {40992599},
issn = {1873-3492},
mesh = {*Gardnerella vaginalis/genetics/isolation &amp; purification ; Humans ; Female ; *CRISPR-Cas Systems/genetics ; *Nucleic Acid Amplification Techniques/methods ; *Recombinases/metabolism ; *Molecular Diagnostic Techniques/methods ; *Vaginosis, Bacterial/diagnosis/microbiology ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {Imbalance of the vaginal microbiota, particularly the overgrowth of Gardnerella vaginalis, is the primary cause of bacterial vaginosis (BV), which poses a significant threat to women's reproductive health. Therefore, early and rapid diagnosis of BV is crucial. Current laboratory diagnostic methods for BV mainly rely on Amsel's clinical criteria, bacterial culture, and PCR techniques. However, these methods have notable limitations: Amsel's criteria are subject to operator subjectivity, culture methods are time-consuming and require specialized expertise, while PCR necessitates expensive instrumentation. These constraints hinder their widespread clinical application. To address this issue, developing a highly accurate and low-cost molecular diagnostic method holds significant clinical value for BV detection. In recent years, recombinase-aided amplification (RAA) and CRISPR-Cas12a gene-editing technologies have achieved groundbreaking progress in nucleic acid detection. This study innovatively integrates RAA isothermal amplification with CRISPR-Cas12a detection to successfully establish a rapid nucleic acid detection platform for Gardnerella vaginalis. Experimental results demonstrate that this platform achieves a detection sensitivity of 10 copies/mL for Gardnerella vaginalis genomic DNA, with no cross-reactivity against other common reproductive tract pathogens. In validation tests using 44 clinical vaginal swab samples, the platform showed a 100.00 % positive agreement rate compared to qPCR. These findings confirm that the CRISPR-Cas12a-based detection platform exhibits excellent specificity, sensitivity, and reliability, serving as an effective tool for monitoring Gardnerella vaginalis colonization levels. This approach provides a novel molecular diagnostic solution for early BV screening and prevention.},
}
@article {pmid41264695,
year = {2025},
author = {Rönspies, M and Khosravi, S and Helia, O and Valisi, A and Fajkus, J and Fojtová, M and Houben, A and Puchta, H},
title = {CRISPR-Cas-mediated heritable chromosome fusions in Arabidopsis.},
journal = {Science (New York, N.Y.)},
volume = {390},
number = {6775},
pages = {843-848},
doi = {10.1126/science.adz8505},
pmid = {41264695},
issn = {1095-9203},
mesh = {*Arabidopsis/genetics ; *Chromosomes, Plant/genetics ; *CRISPR-Cas Systems ; Gene Editing ; *Genome, Plant ; Karyotype ; Meiosis ; Recombination, Genetic ; Gene Fusion ; },
abstract = {The genome of Arabidopsis thaliana consists of 10 chromosomes. By inducing CRISPR-Cas-mediated breaks at subcentromeric and subtelomeric sequences, we fused entire chromosome arms, obtaining two eight-chromosome lines. In one line, both arms of chromosome 3 were fused to chromosome 1. In another line, the arms were transferred to chromosomes 1 and 5. Both chromosome number-reduced lines were fertile. Phenotypic and transcriptional analyses revealed no differences compared with wild-type plants. After crossing with the wild type, the progeny showed reduced fertility. The meiotic recombination patterns of the transferred chromosome arms were substantially changed. Directed chromosome number changes in plants may enable new breeding strategies, redefining linkage groups and establishing genetic barriers. Moreover, our data indicate that plants are highly robust to engineered karyotype changes.},
}
@article {pmid41263111,
year = {2025},
author = {Feng, X and Li, Y and Zheng, J and Chen, X and Yang, S and Chen, Y and Li, SC},
title = {MicrobialScope: an integrated genomic resource with rich annotations across bacteria, archaea, fungi, and viruses.},
journal = {Nucleic acids research},
volume = {},
number = {},
pages = {},
doi = {10.1093/nar/gkaf1234},
pmid = {41263111},
issn = {1362-4962},
support = {C2004-23Y//Young Collaborative Research/ ; JCYJ20220818101201004//Shenzhen Science and Technology Program/ ; 32300527//National Natural Science Foundation of China/ ; 32470695//National Natural Science Foundation of China/ ; 2022A1515110784//Guangdong Basic and Applied Basic Research Foundation/ ; 2023B0303040004//Key-Area Research and Development Program of Guangdong Province/ ; TC2024JC43//Basic Research Programs of Taicang, 2024/ ; //Shenzhen-Hong Kong Institute of Brain Science/ ; //SIAT-HKUST Joint Laboratory of Brain Science/ ; },
abstract = {Microorganisms, including bacteria, archaea, fungi, and viruses, are the most taxonomically diverse and ecologically dominant life forms on Earth, playing critical roles in ecosystems, human health, and industrial applications. While existing microbial databases such as BV-BRC and IMG archive both monoisolate and metagenome-assembled genomes (MAGs) across domains, challenges remain in standardized, multi-level annotations and interactive tools for all microbial groups. Here, we present MicrobialScope (https://microbial.deepomics.org/), a comprehensive microbial genomic platform that integrates large-scale genome collections, multilevel annotations, and interactive visualizations. MicrobialScope harbors 2 411 503 bacterial, 24 472 archaeal, 20 203 fungal, and 188 267 viral genomes derived from both monoisolate assemblies and MAGs. Integrating 15 state-of-the-art bioinformatics tools and 10 specialized databases, MicrobialScope provides extensive annotations encompassing basic genomic features, genomic element prediction (e.g., genes, tRNAs, tmRNAs, CRISPR-Cas and anti-CRISPR elements, secondary metabolite biosynthetic clusters, signal peptides, and transmembrane proteins), and functional and structural annotations. This includes 1 072 114 935 proteins with diverse annotations, 24 640 186 tRNAs and tmRNAs, 140 888 CRISPR-Cas systems, 173 256 anti-CRISPR elements, 105 121 secondary metabolite biosynthetic clusters, 13 235 096 signal peptides, and 50 811 729 transmembrane proteins. In addition, MicrobialScope offers unrestricted access to all data resources, interactive visualization tools, and built-in online analytical modules for intuitive exploration and comparative analysis. With its extensive genome collection, comprehensive annotations, and user-friendly interface, MicrobialScope serves as a scalable platform to advance genome research across diverse microbial domains.},
}
@article {pmid41262458,
year = {2025},
author = {Gao, Z and Gao, Y and Wang, S and Li, X and Cao, W and Deng, W and Yao, L and Wei, X and Zhang, Z and Wang, S and Zhang, Y and Li, M and Xie, Y},
title = {Application progress and biosafety challenges of gene editing and synthetic biotechnology in diagnosis, treatment and prevention of infectious diseases.},
journal = {Biosafety and health},
volume = {7},
number = {5},
pages = {312-322},
pmid = {41262458},
issn = {2590-0536},
abstract = {Global infectious disease prevention faces escalating challenges due to the continual emergence of novel pathogens and rapid viral mutations. Synthetic biology has revolutionized this field by enabling precise diagnostics, innovative vaccine platforms, and targeted therapeutics, yet it simultaneously raises concerns regarding dual-use potential, biosafety, and ethical governance. This systematic review (2015-2025, PubMed, Web of Science, Scopus) focuses on CRISPR-based diagnostics, synthetic vaccines, and engineered probiotics. CRISPR/Cas systems such as DETECTR (Cas12a) and SHERLOCK (Cas13a) demonstrate high sensitivity and rapid pathogen detection (e.g., SARS-CoV-2, Ebola), but their misuse could enhance pathogen virulence or enable bioweapon development. mRNA and viral vector vaccines offer flexible and rapid responses to emerging infections but encounter limitations in molecular stability, delivery system toxicity, and ecological safety. Engineered probiotics, designed as "living therapeutics," can detect pathogens and modulate immune responses, yet pose potential risks of horizontal gene transfer and host-specific variability. Overall, while synthetic biology provides transformative tools for infectious disease control, it necessitates robust global regulatory frameworks, standardized biosafety practices, and ethical oversight to ensure responsible and sustainable application.},
}
@article {pmid41262327,
year = {2025},
author = {Jia, Y and Horvath, K and Rananaware, SR and Jain, PK and Sampath, J},
title = {Exploring the temperature stability of CRISPR-Cas12b using molecular dynamics simulations.},
journal = {Molecular systems design &amp; engineering},
volume = {},
number = {},
pages = {},
pmid = {41262327},
issn = {2058-9689},
abstract = {The thermal stability of CRISPR-Cas nucleases is a critical factor for their successful application in 'one-pot' diagnostic assays that utilize high-temperature isothermal amplification. To understand the atomistic mechanism of stabilization in a previously engineered variant of the thermostable BrCas12b protein, we performed all-atom molecular dynamics (MD) simulations on the wild-type and mutant forms of apo BrCas12b. High-temperature simulations reveal a small structural change along with greater flexibility in the PAM-interacting domain of the mutant BrCas12b, with marginal structural and flexibility changes in the other mutated domains. Comparative essential dynamics analysis between the wild-type and mutant BrCas12b at both ambient and elevated temperatures provides insights into the stabilizing effects of the mutations. Our findings offer comprehensive insights into the important protein motions induced by these mutations. These results provide insights into thermal stability mechanisms in BrCas12b that may inform the future design of CRISPR-based tools.},
}
@article {pmid41262251,
year = {2025},
author = {Stelcer, E and Wozniak, A and Magner, D and Zeyland, J},
title = {Genetically modified pigs with α1,3-galactosyltransferase knockout and beyond: a comprehensive review of xenotransplantation strategies.},
journal = {Frontiers in immunology},
volume = {16},
number = {},
pages = {1663246},
pmid = {41262251},
issn = {1664-3224},
mesh = {Animals ; *Transplantation, Heterologous/methods ; *Galactosyltransferases/genetics/deficiency ; *Animals, Genetically Modified ; Swine ; *Graft Rejection/immunology/genetics/prevention &amp; control ; Gene Knockout Techniques ; Humans ; Heterografts/immunology ; },
abstract = {Xenotransplantation holds promise to eliminate the shortage of organs intended for humans in need. Pigs constitute the most suitable organ xenograft donor due to the fact that their organ anatomy physiological metabolism and immune system resemble those of humans. However, swine organs rapidly cause hyperacute rejection (HAR) and acute humoral xenograft rejection (AHXR) after transplantation. HAR and AHXR are caused by the presence of xenoreactive natural immunoglobulins directed toward a galactose alpha1-3-galactose (alpha-Gal) epitope on porcine vascular endothelium. In order to suppress both types of rejection, pigs with alpha1,3-galactosyltransferase gene knockout (GT-KO) and other genetic modifications (like simultaneous expression of the human complementary regulatory proteins) are intensively investigated. This review highlights the usefulness of GT-KO pig - derived organs such as kidney, heart, corneal, and lung in xenotransplantation. To obtain transgenic pigs researchers can use several techniques based on pronuclear and cytoplasmic microinjection, somatic cell nuclear transfer (SCNT), viral transduction of DNA and DNA transposable element -based technology, site specific nucleases and modifications of the CRISPR/Cas bacterial immune system. Some additional strategies like targeted immunosuppression or tolerance induction of B and T cells will be essential for sustained survival of xenografts. Although xenotransplantation with the use of pigs is a very rapidly evolving field, more research is needed to create perfectly compatible with the human immune system organs.},
}
@article {pmid41261856,
year = {2025},
author = {Xiao, Y and Zhao, R and Bao, Y and Lu, B and Jiang, Y and Tang, Y and Li, B},
title = {Cas12a-assisted split crRNA complex for analysis and detection of diverse entities.},
journal = {Nucleic acids research},
volume = {53},
number = {21},
pages = {},
pmid = {41261856},
issn = {1362-4962},
support = {SKL202402017//The Science and Technology Development Plan Project of Jilin Province/ ; SKL202302030//The Science and Technology Development Plan Project of Jilin Province/ ; 20240101005JJ//Jilin Province Science Fund for Distinguished Young Scholars/ ; 23GZZ03//Major Project of Changchun State Key Laboratory/ ; 20230203193SF//Key R&amp;D Program of Jilin Province/ ; 22525405//National Nature Science Foundation of China/ ; 22504138//National Nature Science Foundation of China/ ; 22474135//National Nature Science Foundation of China/ ; 22374142//National Nature Science Foundation of China/ ; },
mesh = {*CRISPR-Associated Proteins/metabolism/genetics ; *Endodeoxyribonucleases/metabolism/genetics ; *CRISPR-Cas Systems ; MicroRNAs/genetics/analysis ; DNA, Single-Stranded/genetics ; DNA/genetics ; *Bacterial Proteins/genetics/metabolism ; *Biosensing Techniques/methods ; },
abstract = {The Cas12a-crRNA system possesses inherent sequence-specific recognition of double-stranded DNA/single-stranded DNA (dsDNA/ssDNA) coupled with trans-cleavage activity toward ssDNA, making it a powerful tool for nucleic acid diagnostics. However, its application beyond nucleic acid targets remains challenging, limiting its potential as a universal detection platform. In this study, we systematically explore the key parameters governing the activation of a Cas12a-split crRNA system and established a comprehensive set of design guidelines. Building on these findings, we developed CASCADE (Cas12a-Assisted Split crRNA Complex for Analysis and Detection of Diverse Entities), an adaptable detection platform that extends Cas12a's application beyond nucleic acids. Using microRNA as a model, we validated the system's sensitivity, specificity, and mismatch discrimination capability. Additionally, we successfully demonstrated its capability for non-nucleic acid target detection by detecting tobramycin, kanamycin, biotin, and tetracycline repressor protein, confirming its sensitivity and specificity. Finally, by integrating a lateral flow assay (LFA), we enhanced the portability of CASCADE, enabling user-friendly, on-site detection. This work expands the application scope of the Cas12a system and offers a promising strategy for point-of-care diagnostics or environmental monitoring.},
}
@article {pmid41261174,
year = {2025},
author = {Seker-Polat, F and Rogozinska, M and Ban, Y and Abdula, F and Buyukcelebi, K and Xie, P and Fan, J and Abbaszadeh, N and Kingham, Y and Paylakhi, SZ and Zhang, B and Adli, M},
title = {Druggable genome CRISPR screening identifies the KEAP1/NRF2 axis as a mediator of PD-L1 expression.},
journal = {Communications biology},
volume = {8},
number = {1},
pages = {1610},
pmid = {41261174},
issn = {2399-3642},
support = {R01CA267544//U.S. Department of Health &amp; Human Services | National Institutes of Health (NIH)/ ; U54CA268084//U.S. Department of Health &amp; Human Services | National Institutes of Health (NIH)/ ; },
mesh = {*B7-H1 Antigen/genetics/metabolism ; Humans ; *Kelch-Like ECH-Associated Protein 1/metabolism/genetics ; *NF-E2-Related Factor 2/metabolism/genetics ; Cell Line, Tumor ; *Gene Expression Regulation, Neoplastic ; Animals ; Female ; Mice ; CRISPR-Cas Systems ; Pancreatic Neoplasms/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats ; Signal Transduction ; Ovarian Neoplasms/genetics ; },
abstract = {Cancer cells rapidly induce PD-L1 expression in response to inflammatory cytokines such as IFNγ from cytotoxic T cells. Increased surface PD-L1 is a primary mechanism of cancer cells evading cytotoxic T-cell-mediated immune clearance. Identifying how cancer cells increase PD-L1 expression may yield clinically relevant immune checkpoint regulators. However, the key regulators and molecular mechanisms mediating rapid PD-L1 induction are yet to be understood entirely. To identify targetable mechanisms controlling cytokine-induced PD-L1 expression, we performed functional CRISPR gene KO screening with a custom-designed sgRNA library that targets "druggable" genes. We performed the screening in 6 different cancer lines: 3 ovarian (OVCAR4, CaOV3, and SKOV3) and three pancreatic cancer (MiaPaca2, ASPC1 and KP4) cell lines. The screening recovered the known regulators of PD-L1 expression and uncovered several novel regulators of PD-L1 that control its expression in all cell lines or in a cancer-type-specific fashion. For example, while genetic or pharmacological depletion of CSNK1A1 results in reduced PD-L1 expression in ovarian cancer cells, CDK1 depletion modulates PD-L1 in pancreatic cancer cell lines. Significantly, we discovered that KEAP1 depletion or pharmacological inhibition diminishes PD-L1 in all cell lines tested (n = 6). Mechanistically, KEAP1 depletion-mediated reduced PD-L1 is due to transcriptional repression of the PD-L1 gene by NRF2 activation. As such, depletion of NRF2 restores PD-L1 expression, while its overexpression leads to diminished PD-L1 expression. Supporting this, pharmacological NRF2 activation resulted in significant antitumor immunity with increased cytotoxic effector T cell infiltration and reduced exhausted T cells, resulting in smaller xenografted tumors. These findings establish the KEAP1/NRF2 axis as a novel and potentially druggable mechanism of IFNγ-meditated PD-L1 expression in cancer cells.},
}
@article {pmid41260544,
year = {2025},
author = {Gupta, A and Beg, MA and Badhwar, S and Srivastava, S and Srivastava, R and Puri, N and Saxena, A and Abdin, MZ and Selvapandiyan, A},
title = {Nucleoside diphosphate kinase (LdNDK2): A metacyclogenesis-regulating kinase essential for Leishmania parasite survival within eukaryotic host cells.},
journal = {Microbial pathogenesis},
volume = {210},
number = {},
pages = {108192},
doi = {10.1016/j.micpath.2025.108192},
pmid = {41260544},
issn = {1096-1208},
abstract = {Nucleoside diphosphate kinase (NDK) transfers phosphate from nucleoside triphosphates (NTPs) to nucleoside diphosphates (NDPs) via a ping-pong mechanism, benefiting both prokaryotes and eukaryotes. In Leishmania donovani, we identified a putative NDK2 (LdNDK2), hypothesized to play a crucial role in nucleotide metabolism and cellular energy regulation. To investigate its function and enzymatic properties, we cloned, expressed, and purified recombinant LdNDK2, confirming enzymatic activity via the ADP-Glo assay. The secondary structure and thermal stability were analyzed using circular dichroism spectroscopy, while intrinsic tryptophan fluorescence assays revealed that ATP's gamma phosphate is first transferred to rLdNDK2, forming an intermediate phospho-enzyme complex. We further evaluated the role of rLdNDK2 in ATP-mediated cytolysis of LPS-activated THP-1 cells by measuring lactate dehydrogenase release. Using the CRISPR-Cas9 method, we fluorescently tagged LdNDK2, localizing it to distinct regions of the endomembrane system. In vitro growth studies of LdNDK2 deleted (LdNDK2[-/-]) procyclic promastigotes, through CRISPR-Cas9, revealed that these mutants exhibit a longer flagellum, a reduced cell body, an altered cell division cycle, and increased growth compared to wild-type parasites. LdNDK2[-/-] parasites were arrested at the metacyclic stage, contrasting with continuous differentiation in parental counterparts. Morphological alterations and growth defects were confirmed by re-expressing LdNDK2 in knockouts. Infection with LdNDK2[-/-] parasites significantly reduced the viability and recovery of mast cells and THP-1 macrophages and flow cytometry indicated increased apoptosis in host cells, likely due to parasites inability to utilize NDK enzyme for preventing cytolysis. Our findings highlight LdNDK2's crucial role in metacyclogenesis and immune evasion, underscoring its therapeutic potential for leishmaniasis.},
}
@article {pmid41205603,
year = {2025},
author = {Regan, SB and Medhi, D and Xu, Y and White, TB and Jiang, YZ and Kim, JE and Wang, SC and Deng, Q and Jia, S and Baasan, D and Connelly, JP and Chang, TC and Pruett-Miller, SM and Jasin, M},
title = {Megabase-scale loss of heterozygosity provoked by CRISPR-Cas9 DNA double-strand breaks.},
journal = {Molecular cell},
volume = {85},
number = {22},
pages = {4119-4137.e10},
doi = {10.1016/j.molcel.2025.10.015},
pmid = {41205603},
issn = {1097-4164},
mesh = {*DNA Breaks, Double-Stranded ; Humans ; *CRISPR-Cas Systems ; Animals ; *Loss of Heterozygosity ; Mice ; DNA End-Joining Repair ; Gene Editing/methods ; Flow Cytometry ; CRISPR-Associated Protein 9/genetics/metabolism ; Epithelial Cells/metabolism ; },
abstract = {Harnessing DNA double-strand breaks (DSBs) is a powerful approach for gene editing, but it may provoke loss of heterozygosity (LOH), a common feature of tumor genomes. To interrogate this risk, we developed a flow cytometry-based system (Flo-LOH), detecting LOH in ∼5% of mouse embryonic and human epithelial cells following a DSB. Inhibition of both non-homologous end joining (NHEJ) and microhomology-mediated end joining (MMEJ) massively increases LOH, although the dependence on individual pathways differs in the two cell types. Multiple mechanisms lead to LOH, including chromosome truncations with de novo telomere addition and whole chromosome loss. LOH spans megabases distal from the DSB but also frequently tens of megabases centromere-proximal, which can arise from breakage-fusion-bridge events. Unlike DSBs, Cas9 nicks and adenine base editing did not noticeably impact LOH. The capacity for large-scale LOH must therefore be considered when using DSB-based gene editing, especially in conjunction with end-joining inhibition.},
}
@article {pmid41194386,
year = {2025},
author = {Sanchez-Quirante, T and Kužmová, E and Riopedre-Fernandez, M and Golojuch, S and Vopálenský, P and Raindlová, V and El-Sagheer, AH and Brown, T and Hocek, M},
title = {Enzymatic Synthesis of Modified RNA Containing 5-Methyl- or 5-Ethylpyrimidines or Substituted 7-Deazapurines and Influence of the Modifications on Stability, Translation, and CRISPR-Cas9 Cleavage.},
journal = {ACS chemical biology},
volume = {20},
number = {11},
pages = {2755-2767},
doi = {10.1021/acschembio.5c00692},
pmid = {41194386},
issn = {1554-8937},
mesh = {*CRISPR-Cas Systems ; *Pyrimidines/chemistry/metabolism ; *RNA/chemistry/metabolism ; Humans ; *Purines/chemistry/metabolism ; Protein Biosynthesis ; DNA-Directed RNA Polymerases/metabolism ; Viral Proteins ; },
abstract = {A set of modified 5-methyl- and 5-ethylpyrimidine (uracil and cytosine) and 7-methyl-, 7-ethyl-, and 7-unsubstituted 7-deazapurine (deazaadenine and deazaguanine) ribonucleoside triphosphates was synthesized and used for enzymatic synthesis of base-modified RNA using in vitro transcription (IVT). They all were good substrates for T7 RNA polymerase in the IVT synthesis of model 70-mer RNA, mRNA encoding Renilla luciferase, and 99-mer single-guide RNA (sgRNA). The effect of modifications in the particular RNA on the stability and efficiency in in vitro and in cellulo translation as well as in CRISPR-Cas9 gene cleavage was quantified. In the in vitro translation assay, we observed moderately enhanced luciferase production with 5-methyluracil and -cytosine, while any 7-deazaadenines completely inhibited the translation. Surprisingly, in cellulo experiments showed a significant enhancement of translation with mRNA containing 7-deazaguanine and moderate enhancement with 5-methyl- or 5-ethylcytosine. Most of the modifications had a minimal effect on the efficiency of the gene cleavage in CRISPR-Cas9 except for 7-alkyl-7-deazaadenines that completely inhibited the cleavage. The results are important for further design of potential base-modified RNA therapeutics.},
}
@article {pmid41131871,
year = {2025},
author = {Campbell, KB and Ouye, RB and Wong, BL and Jiang, A and Okada, K and McKenney, RJ and Fisher, AJ and Beal, PA},
title = {Control of ADAR2 Dimerization and RNA Editing Efficiency by Site-Specific 2'-Fluoro Modification of Guide RNAs.},
journal = {ACS chemical biology},
volume = {20},
number = {11},
pages = {2637-2648},
doi = {10.1021/acschembio.5c00493},
pmid = {41131871},
issn = {1554-8937},
mesh = {*Adenosine Deaminase/metabolism/chemistry/genetics ; *RNA Editing ; *RNA-Binding Proteins/metabolism/chemistry/genetics ; Humans ; *RNA, Guide, CRISPR-Cas Systems/chemistry/metabolism/genetics ; RNA, Double-Stranded/metabolism/chemistry ; Protein Multimerization ; Binding Sites ; },
abstract = {Adenosine Deaminases Acting on RNA (ADARs) are an important class of RNA editing enzymes that catalyze the deamination of adenosine (A) to inosine (I) in double-stranded RNA (dsRNA). Since inosine is typically read as guanosine (G) during translation, ADARs can produce A to G transitions in dsRNA. Site-directed RNA editing (SDRE) is a promising therapeutic tool wherein guide RNAs can be used to direct endogenous human ADARs to reverse disease-causing mutations in specific RNA transcripts. Guide RNA (gRNA) modifications at locations that contact the ADAR active site are often used to improve editing efficiency. However, little is known about rate-enhancing chemical modifications in the gRNA at the dsRNA binding domain (dsRBD)-RNA interface. Analysis of published crystal structures of ADAR2 bound to dsRNA suggested positions at this interface would be sensitive to gRNA modification. In this work, gRNAs bearing 2'-modifications in the dsRBD binding site were synthesized and subsequently tested to determine their effects on the editing rate of therapeutically relevant ADAR targets. We found that replacing a single 2'-OH at specific positions on the gRNA with a 2'-F substantially increased the rate of in vitro ADAR2-catalyzed adenosine deamination for two different sequences, whereas 2'-OMe at these positions was inhibitory. This effect was also validated in cellulo. The rate of ADAR1-catalyzed deamination is not stimulated by these 2'-F modifications. A crystal structure of an ADAR2 fragment bound to duplex RNA bearing a single 2'-F at guide position +13 suggested a favorable interaction between the side chain of N241 of the auxiliary ADAR2 monomer and the 2'-F modification. Furthermore, electrophoretic mobility shift assays and mass photometry indicate 2'-F at position +13 facilitates ADAR2 dimerization on the RNA substrate. This work advances our understanding of the RNA features that define superior ADAR substrates and inform the design of gRNAs for therapeutic RNA editing.},
}
@article {pmid41099664,
year = {2025},
author = {Spavieri, JM and Inacio, TG and Seguchi, G and de Souza, BC and Pereira, GAG and de Mello, F},
title = {Impact of CRISPRi-Mediated Titration of GPD Genes on the Fermentative Performance of S. cerevisiae.},
journal = {ACS synthetic biology},
volume = {14},
number = {11},
pages = {4412-4423},
doi = {10.1021/acssynbio.5c00316},
pmid = {41099664},
issn = {2161-5063},
mesh = {*Saccharomyces cerevisiae/genetics/metabolism ; Fermentation/genetics ; Ethanol/metabolism ; *Saccharomyces cerevisiae Proteins/genetics/metabolism ; Glycerol/metabolism ; *CRISPR-Cas Systems/genetics ; *Glycerol-3-Phosphate Dehydrogenase (NAD+)/genetics/metabolism ; Gene Editing/methods ; },
abstract = {Glycerol is one of the main byproducts in ethanol fermentation due to its importance in redox balance and response to osmotic stress in Saccharomyces cerevisiae. Since its production diverts carbon from alcohol production, traditional gene-editing methods have been applied to the glycerol synthesis pathway. However, such approaches generate undesirable phenotypes for industrial applications. In the present study, we employed the CRISPR-dCas9 system to moderately downregulate the expression of GPD1 and GPD2, the two main genes involved in this metabolism. GPD2 gene expression downregulation and a graded reduction in glycerol production after repression of four different target sites in each paralogue were achieved. Employment of the CRISPRi approach for GPD gene modulation resulted in higher specific ethanol productivity (SEP) than that of single knockout cells. Targeted modulation in a region -140 basepairs upstream of the transcription start site (TSS) of GPD1 resulted in a 3% increase in ethanol production compared to the wild type and gpd Δ strains. Such regulation, combined with GPD2 deletion, revealed the higher SEP among all tested strains. Furthermore, a GPD1-modulated strain maintained tolerance to high osmolarity in very high-gravity (VHG) fermentation while maintaining its ethanol production levels above those observed in the control strain.},
}
@article {pmid41072420,
year = {2025},
author = {Zhao, G and Liu, Y and Zhang, G and Wang, J and Zou, Z and Wang, Y and Xu, S and Han, D and Xu, Z and Chen, Z and Yang, M and Zeng, Z and Wu, Y and Zhou, X and Huang, J},
title = {Engineered hypercompact Fanzor-ωRNA system with enhanced genome editing activity.},
journal = {Molecular cell},
volume = {85},
number = {22},
pages = {4138-4151.e4},
doi = {10.1016/j.molcel.2025.09.031},
pmid = {41072420},
issn = {1097-4164},
mesh = {Humans ; *Gene Editing/methods ; HEK293 Cells ; Animals ; Dependovirus/genetics ; *RNA, Guide, CRISPR-Cas Systems/genetics/metabolism ; *Genome, Human ; CRISPR-Cas Systems ; Mice ; },
abstract = {As programmable RNA-guided DNA nucleases found in eukaryotes, Fanzors have shown promise in genome editing with their compact size. A wide variety of RuvC-containing Fanzor nucleases have been identified in various eukaryotes and their viruses. However, low editing efficiency limits the application of Fanzor in mammalian genome editing. In this study, we introduce SpuFz1 V4, an engineered RNA-guided DNA endonuclease with vigorous editing activity in the human genome. Furthermore, we demonstrate the substantial potential of the Fanzor system as a base editor. SpuFz1 V4 is currently the most active RNA-guided DNA nuclease of eukaryotic origin, belonging to the Fanzor1 family. Due to its compact size, SpuFz1 V4 can be efficiently delivered via a single adeno-associated virus (AAV) into the retina, achieving robust in vivo genome editing, which has the potential to be applied in both basic research and disease treatment applications.},
}
@article {pmid41017740,
year = {2025},
author = {Cao, X and Zeng, Z and Cao, X and He, Y and Wang, L and Li, D and Zhang, X},
title = {Generation and Phenotypic Analysis of the IL-10RAR104W/R104W Mouse Model.},
journal = {Inflammatory bowel diseases},
volume = {31},
number = {11},
pages = {3149-3159},
doi = {10.1093/ibd/izaf099},
pmid = {41017740},
issn = {1536-4844},
support = {32025023//National Natural Science Foundation of China/ ; 22YF1437700//Shanghai Science and Technology Program/ ; },
mesh = {Animals ; Mice ; *Disease Models, Animal ; Phenotype ; *Inflammatory Bowel Diseases/genetics/pathology ; *Interleukin-10 Receptor alpha Subunit/genetics ; *Colitis/pathology/genetics ; *Point Mutation ; Humans ; Macrophages ; CRISPR-Cas Systems ; Mice, Inbred C57BL ; },
abstract = {BACKGROUND: Very-early-onset inflammatory bowel disease (VEO-IBD) is a form of IBD that manifests in infants and young children, with a significant proportion of them carrying interleukin 10 receptor alpha (IL-10RA) mutations. Despite the increasing incidence rate, the pathogenesis of VEO-IBD remains elusive, and treatment options are limited. The utilization of a humanized mouse model holds promise for further investigation into VEO-IBD. Previous study has revealed that VEO-IBD patients had a homozygous C > T mutation at IL-10RA position 301, which can be pathogenic.<br><br>METHODS: We generated the corresponding point mutation mouse model via CRISPR/Cas9 technology. Subsequently, we performed various experiments to assess the colitis phenotype in mice and conducted a preliminary exploration of the model's utility.<br><br>RESULTS: The mouse model progressively developed spontaneous colitis between 6 and 12 weeks. Hematoxylin and eosin (H&amp;E) staining revealed abnormal colonic structure and massive local immune cell infiltration. The mouse model has abnormal levels of inflammatory cytokines in the colonic tissue, with an expansion of F4/80+ macrophages, CD4+ T cells, and B220+ B cells. Among the macrophages, the level of tissue-resident macrophages associated with anti-inflammation was reduced in IL-10RAR104W/R104W mice, while the level of immature macrophages associated with pro-inflammation was increased. Furthermore, we found that bone marrow transplantation can alter the composition of intestinal macrophage populations and treat intestinal inflammation in mutant mice. Finally, the result of subcutaneous tumor-bearing experiments indicated a faster tumor growth rate in the mutant mice.<br><br>CONCLUSIONS: In summary, we have successfully constructed a humanized mouse model with a stable spontaneous colitis phenotype, which is a valuable model for the therapeutic exploration of VEO-IBD.},
}
@article {pmid40954215,
year = {2025},
author = {Romero-Moya, D and Torralba-Sales, E and Calvo, C and Marin-Bejar, O and Magallon-Mosella, M and Distefano, M and Pera, J and Castaño, J and De Giorgio, F and Gonzalez, J and Iglesias, A and Berenguer-Balaguer, C and Schilling, M and Plass, M and Pasquali, L and Català, A and Molina, O and Wlodarski, MW and Bigas, A and Giorgetti, A},
title = {CRISPR-engineered human GATA2 deficiency model uncovers mitotic dysfunction and premature aging in HSPCs, impairing hematopoietic fitness.},
journal = {Leukemia},
volume = {39},
number = {12},
pages = {3015-3025},
pmid = {40954215},
issn = {1476-5551},
support = {PID2020-15591RB-100//Ministry of Economy and Competitiveness | Agencia Estatal de Investigaci&#xf3;n (Spanish Agencia Estatal de Investigaci&#xf3;n)/ ; FORT23/00032//Ministry of Economy and Competitiveness | Instituto de Salud Carlos III (Institute of Health Carlos III)/ ; AC23_2/00040//Ministry of Economy and Competitiveness | Instituto de Salud Carlos III (Institute of Health Carlos III)/ ; AC23_2/00014//Ministry of Economy and Competitiveness | Instituto de Salud Carlos III (Institute of Health Carlos III)/ ; KOG-202109-01162//European Hematology Association (EHA)/ ; 101029927//EC | EU Framework Programme for Research and Innovation H2020 | H2020 Priority Excellent Science | H2020 Marie Sk&#x142;odowska-Curie Actions (H2020 Excellent Science - Marie Sk&#x142;odowska-Curie Actions)/ ; LCF-PR-HR24-00150//"la Caixa" Foundation (Caixa Foundation)/ ; PID2023-151556OB-I00//"la Caixa" Foundation (Caixa Foundation)/ ; PID2022-142966OB-I00//Ministerio de Econom&#xed;a y Competitividad (Ministry of Economy and Competitiveness)/ ; },
mesh = {Humans ; *Hematopoietic Stem Cells/metabolism/pathology ; *GATA2 Transcription Factor/genetics/deficiency ; *CRISPR-Cas Systems ; *Mitosis/genetics ; *GATA2 Deficiency/genetics/pathology ; Animals ; Mutation ; Mice ; Cell Proliferation ; *Cellular Senescence/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {GATA2 deficiency is a monogenic transcriptopathy disorder characterized by bone marrow failure (BMF), immunodeficiency, and a high risk of developing myelodysplastic neoplasms (MDS) and acute myeloid leukemia (AML). Although informative mouse models have been developed, the mechanisms by which GATA2 haploinsufficiency drives disease initiation in humans remain incompletely understood. To address this, we developed a novel humanized model using CRISPR/Cas9 technology to knock-in GATA2-R398W variant in primary cord blood CD34[+] cells. Additionally, we introduced specific mutations in SETBP1 and ASXL1 to model distinct premalignant stages of GATA2 deficiency. Through clonal competition and serial transplantation assays, we demonstrated that human CD34[+] cells harboring the GATA2 mutation exhibit significantly reduced fitness in vivo when compete with wild-type cells. Notably, this fitness disadvantage persists even when GATA2 mutations are combined with oncogenic SETBP1 and ASXL1 drivers, underscoring the dominant, deleterious effect of GATA2 deficiency on hematopoietic stem cell function. Functional in vitro analyses revealed that GATA2-R398W mutation impairs cell proliferation, disrupts cell cycle progression, and induces mitotic defects, which may contribute to hematopoietic stem/progenitor cell loss and impaired self-renewal. Transcriptomic profiles of GATA2-mutant cells revealed that these functional defects are associated with reduced HSC self-renewal capacity and upregulation of the pre-aging phenotype. Our work highlights the feasibility of generating a human GATA2 deficiency model suitable for studying the biological consequences of various GATA2 variants and the generation of a platform to test potential phenotype-rescuing therapeutics.},
}
@article {pmid40753279,
year = {2025},
author = {Tan, GZH and Maurya, KS and Krishnamoorthi, S and Boonyaves, K and Urano, D},
title = {Regulatory helix deletion in glutamate decarboxylase reduces GABA and enhances Agrobacterium-mediated transient expression in lettuce.},
journal = {Journal of plant research},
volume = {138},
number = {6},
pages = {1033-1044},
pmid = {40753279},
issn = {1618-0860},
support = {A19E4a0101//Agency for Science, Technology and Research/ ; },
mesh = {*gamma-Aminobutyric Acid/metabolism ; *Glutamate Decarboxylase/genetics/metabolism ; *Lactuca/genetics/metabolism/enzymology ; *Agrobacterium/genetics ; Gene Expression Regulation, Plant ; Plants, Genetically Modified ; *Plant Proteins/metabolism/genetics ; CRISPR-Cas Systems ; },
abstract = {Gamma-aminobutyric acid (GABA) is a metabolite involved in plant growth and stress responses, with its synthesis regulated by glutamate decarboxylase (GAD). Plant GAD enzymes have an autoinhibitory α-helix at the C-terminus, which calmodulin (CaM) binding typically relieves. Eliminating this C-terminal motif usually increases GABA levels in crops. In this case study, we generated a CRISPR/Cas9-edited lettuce line with a 14-amino acid deletion in the C-terminal helix of LsGAD2, the isozyme primarily expressed in most tissues. This targeted truncation removes CaM-binding residues while retaining the key Lys cluster (Lys489, Lys490, Lys491) responsible for autoinhibition, resulting in a significant reduction in GABA content without affecting growth. The LsGAD1/2-ΔC line showed a transcriptomic profile resembling stress responses in the wildtype under unstressed conditions. Reduced GABA levels appeared to upregulate genes involved in stress perception, signalling, and defense-related metabolic and hormonal changes, potentially mediated by WRKY-family transcription factors. Likely due to lower GABA levels and altered defense responses, LsGAD1/2-ΔC plants showed increased Agrobacterium-mediated transient expression of β-glucuronidase. Overall, our study suggests that targeted genetic manipulation of the C-terminal helix of GAD enzymes can reduce GABA levels while enhancing transformation efficiency in lettuce, thus presenting a means for engineering for such purposes.},
}
@article {pmid41259334,
year = {2025},
author = {Siroosi, M and Ghasemi, F and Jabalameli, F and Emaneini, M and Salehi, M and Beigverdi, R and Amoozegar, MA},
title = {Investigating the impact of type I-E CRISPR-Cas systems and acrEI10 on multidrug-resistance in clinical isolates of Klebsiella pneumoniae.},
journal = {PloS one},
volume = {20},
number = {11},
pages = {e0335756},
doi = {10.1371/journal.pone.0335756},
pmid = {41259334},
issn = {1932-6203},
mesh = {*Klebsiella pneumoniae/genetics/drug effects/isolation &amp; purification ; *CRISPR-Cas Systems/genetics ; *Drug Resistance, Multiple, Bacterial/genetics ; Humans ; *Bacterial Proteins/genetics ; Anti-Bacterial Agents/pharmacology ; Klebsiella Infections/microbiology/drug therapy ; Microbial Sensitivity Tests ; beta-Lactamases/genetics ; },
abstract = {Klebsiella pneumoniae is a pathogen related to nosocomial infections with a high rate of antibiotic resistance. The aim of this study was to understand the impact of the presence of CRISPR-Cas systems and an anti-CRISPR gene on multidrug-resistance in K. pneumoniae isolates. The study analyzed 100 clinical K. pneumoniae isolates obtained from a hospital setting. The investigation involved determining antibiotic resistance profiles, including ESBL production, identifying specific carbapenemase and aminoglycoside resistance genes, detecting the presence of CRISPR-Cas systems, identifying the anti-CRISPR gene acrEI10, and sequencing CRISPR arrays. Correlation analysis between resistance genes and CRISPR-Cas systems was also performed. All isolates in this study were determined to be multidrug-resistant (MDR), with resistance rates exceeding 70% for the majority of antibiotics tested. The most prevalent carbapenemase genes were blaOXA-48 and blaNDM, while aminoglycoside resistance was primarily mediated by aac(6´)-Ia and ant(2")-Ia. Only 7% of the isolates harbored CRISPR-Cas systems and the gene acrEI10, which encodes an anti-CRISPR protein, was detected in one of the CRISPR-Cas positive isolates. Sequencing of the CRISPR array from this isolate showed similarities between the spacers and sequences found in plasmids and K. pneumoniae chromosome. No strong correlation was identified between the antibiotic resistance genes and CRISPR-Cas systems. Findings from this study suggest a complex interplay between these factors in MDR isolates of K. pneumoniae and show that further investigations are needed to have a better understanding of the mechanisms related to the coexistence of these elements and their impact on dissemination of antibiotic resistance genes.},
}
@article {pmid41257956,
year = {2025},
author = {Feng, C and Wang, Y and Liu, C and Dong, F and Guo, M and Liu, F and Li, Y and Zhang, L},
title = {Programmable no-nonspecific genetic analytical system via dual-circle-based rolling circle amplification with an efficient CRISPR/Cas12a biosensing strategy.},
journal = {Scientific reports},
volume = {15},
number = {1},
pages = {40723},
pmid = {41257956},
issn = {2045-2322},
support = {CSTB2024NSCQ-MSX0785//Natural Science Foundation Project of Chongqing, Chongqing Science and Technology Commission/ ; 2023YQB061//Young Ph.D. Talent Incubation Program of the Second Affiliated Hospital of Army Medical University/ ; CQYC20220303658//Chongqing Science and Technology Innovation Leading Talent Support Program/ ; 2024ZDXM012//Chongqing Municipal Science and Health Joint Medical Research Project Key Project/ ; 82472384//National Natural Science Foundation of China/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; *Nucleic Acid Amplification Techniques/methods ; *Biosensing Techniques/methods ; Humans ; DNA, Single-Stranded/genetics ; *Endodeoxyribonucleases/metabolism/genetics ; DNA, Circular/genetics ; Limit of Detection ; Bacterial Proteins ; CRISPR-Associated Proteins ; },
abstract = {Ultrasensitive and specific detection of DNA is highly important for early cancer screening and diagnosis. Nucleic acid amplification technology is the most commonly used method for oncogene detection, but nonspecific amplification may occur. We designed a nicking endonuclease (NEase)-mediated exponential rolling circle amplification (RCA) that avoids nonspecific amplification for the CRISPR/Cas12a preamplification process. The purpose was to construct a NEase-assisted target recycling (NATR)-triggered no-nonspecific exponential RCA (NER) reaction integrated with a CRISPR/Cas12a (NATR-NER/Cas12a) system, enabling ultrasensitive and high-fidelity target detection. Innovatively, two circular single-stranded DNAs (ssDNAs) with NEase recognition sites were designed as the preprimer and template for RCA. In the presence of the target, the endonuclease Nt.BstNBI cleaves the circular preprimers into linear fragments, triggering the NER reaction. This generates many short ssDNA fragments, which are recognized by CRISPR/Cas12a and generates a fluorescence signal. The proposed strategy exhibited a wide linear range (10 fM-1 nM), a low detection limit (0.77 fM), and specifically recognized single mismatched DNA. In serum samples, this method exhibited good agreement with real-time quantitative polymerase chain reaction (qPCR) results at lower cost. The developed NATR-NER/Cas12a system provides a promising tool for the early screening and clinical diagnosis of cancer in resource-limited areas.},
}
@article {pmid41256794,
year = {2025},
author = {Zhou, X and Ye, C and Xie, M and Wei, Y and Zhao, Y and Liu, X and Ma, J and Qing, J and Chen, Z},
title = {Advances in the application of CRISPR technology in pathogen detection: amplification-based and amplification-free strategies.},
journal = {Frontiers in cellular and infection microbiology},
volume = {15},
number = {},
pages = {1645699},
pmid = {41256794},
issn = {2235-2988},
mesh = {Humans ; *Molecular Diagnostic Techniques/methods ; *Nucleic Acid Amplification Techniques/methods ; *CRISPR-Cas Systems ; Sensitivity and Specificity ; *Clustered Regularly Interspaced Short Palindromic Repeats ; *Bacteria/genetics/isolation &amp; purification ; *Communicable Diseases/diagnosis ; },
abstract = {CRISPR technology, with its high specificity and programmability, has become an important tool for the detection of human pathogens. The timely and accurate detection of pathogens is crucial for public health. In recent years, significant progress has been made in the application of CRISPR technology for pathogen detection. However, several challenges remain, including detection sensitivity, specificity, and operational convenience. This review summarizes the latest advances in CRISPR technology for pathogen detection, with a focus on the principles and performance comparisons of amplification-based CRISPR (such as those combined with isothermal amplification techniques like RPA and LAMP) and amplification-free CRISPR (such as cascade CRISPR, sensor technologies, and digital droplet CRISPR). It also discusses their applications in pathogen detection. In addition, the article analyzes the advantages and limitations of CRISPR detection technology and looks forward to future development trends, providing a theoretical basis for the optimization of rapid diagnostic techniques for pathogens.},
}
@article {pmid41255389,
year = {2025},
author = {Pizzoccheri, R and Falchi, FA and Alloni, A and Caldarulo, M and Camboni, T and Zambelli, F and Pavesi, G and Visentin, C and Camilloni, C and Sertic, S and Briani, F},
title = {Pathological PNPase variants with altered RNA binding and degradation activity affect the phenotype of bacterial and human cell models.},
journal = {NAR molecular medicine},
volume = {2},
number = {1},
pages = {ugae028},
pmid = {41255389},
issn = {2976-856X},
abstract = {Human PNPase (hPNPase) is an essential RNA exonuclease located in mitochondria, where it contributes to RNA import from the cytoplasm, degradation of mitochondrial RNA and R-loop homeostasis. Biallelic mutations in the hPNPase PNPT1 gene cause different genetic diseases, ranging from hereditary hearing loss to Leigh syndrome. In this work, we used an Escherichia coli model to test the effects of four pathological PNPT1 mutations associated with diseases of different severity. Moreover, we generated a new human cell model by introducing PNPT1 mutations into 293T cells via CRISPR-Cas editing. Notably, the bacterial cells expressing the different mutant alleles exhibited similar phenotypes consistent with hPNPase loss of function. In contrast, the human cell model responded differently to the two mutations tested, with responses correlating with the severity of the respective pathologies. We interpreted the data derived from both models in the light of the in vitro RNA binding and degradation activity of the wild-type and mutated hPNPase variants. We found that all pathogenic mutations tested caused defects in protein assembly and affected the degradation and RNA binding efficiency to varying degrees. However, the severity of the conditions caused by different mutations did not correlate with the catalytic activity of the mutant proteins.},
}
@article {pmid41254867,
year = {2025},
author = {Podkowik, M and Tillman, A and Takats, C and Carion, H and Putzel, G and McWilliams, J and See, B and Wang, G and Munoz-Gomez, S and Otto, C and Drlica, K and Marraffini, L and Pironti, A and Hochman, S and Kerantzas, C and Shopsin, B},
title = {CRISPR-Cas-associated SCCmec variants in methicillin-resistant Staphylococcus aureus evade rapid diagnostic detection.},
journal = {The Journal of infectious diseases},
volume = {},
number = {},
pages = {},
doi = {10.1093/infdis/jiaf575},
pmid = {41254867},
issn = {1537-6613},
abstract = {Rapid molecular assays guiding treatment of methicillin-resistant Staphylococcus aureus (MRSA) detect SCCmec (Xpert) or the SCCmec-orfX junction (BCID2). Sequence variation in this region can disrupt primer binding, yielding false-negative results. Investigation of a missed bloodstream infection linked escape to a CRISPR-Cas-associated SCCmec variant, leading to identification of 64 variants from 45 patients-2% of 2,432 screened. Misdiagnosis was restricted to clonal complex 5, a hospital-associated lineage; 11 of 40 SCCmec/junctions evaded detection by BCID2 or Xpert. Variants had mecA instability and circulated in healthcare settings. Our findings reveal a unique escape mechanism and underscore a threat to diagnostic accuracy.},
}
@article {pmid41254256,
year = {2025},
author = {Wang, Q and Wang, Z and Liu, H and Lv, Y and Zhou, C and Li, C and Fan, H and Ouyang, X and Tao, L and Pei, X and Xie, T},
title = {PAM-readID is a rapid, simple, and accurate PAM determination method for CRISPR-Cas enzymes in mammalian cells.},
journal = {Communications biology},
volume = {8},
number = {1},
pages = {1601},
pmid = {41254256},
issn = {2399-3642},
mesh = {*CRISPR-Cas Systems/genetics ; Humans ; *Gene Editing/methods ; HEK293 Cells ; *CRISPR-Associated Protein 9/metabolism/genetics ; Animals ; DNA Breaks, Double-Stranded ; },
abstract = {One CRISPR-Cas enzyme's recognized protospacer adjacent motif (PAM) profile always shows intrinsic differences between assays with different working environments, such as in vitro, in bacterial cells, or in mammalian cells. The developed methods in mammalian cells are technically complex and not readily amenable to be broadly adopted, highlighting the urgent need for a well-established PAM-determining method in mammalian cells. In this study, we construct a rapid, simple, and accurate method for determining the PAM recognition profile of CRISPR-Cas nucleases in mammalian cells. The developed method is termed PAM-readID, PAM REcognition-profile-determining Achieved by Double-stranded oligodeoxynucleotides Integration in DNA double-stranded breaks. Using PAM-readID, the PAM recognition profiles of SaCas9, SaHyCas9, Nme1Cas9, SpCas9, SpG, SpRY, and AsCas12a in mammalian cells are well produced. An accurate PAM preference for SpCas9 can be identified by analysis with extremely low sequence depth (500 reads). PAM-readID can also define a PAM recognition profile of Cas9 based on Sanger sequencing with a significantly lower cost of time and price than that of high-throughput sequencing. We present an easy-to-use method for comprehensively revealing functional PAM of CRISPR-Cas nucleases in mammalian cells, which can contribute towards accelerating the advancement of exploiting novel genome editing nucleases.},
}
@article {pmid41253931,
year = {2025},
author = {Ni, J and Gong, J and Ran, Y and Bai, R and Huang, P and Zheng, Z and Zhou, M and Lan, F and Gu, W and Liu, X},
title = {Optimization of gene knockout approaches and sgRNA selection in hPSCs with inducible Cas9 expression.},
journal = {Scientific reports},
volume = {15},
number = {1},
pages = {40461},
pmid = {41253931},
issn = {2045-2322},
support = {JCYJ20220531091615034//Shenzhen Fundamental Research Program/ ; },
mesh = {Humans ; *RNA, Guide, CRISPR-Cas Systems/genetics ; *CRISPR-Cas Systems/genetics ; *Gene Knockout Techniques/methods ; Gene Editing/methods ; *Pluripotent Stem Cells/metabolism/cytology ; *CRISPR-Associated Protein 9/genetics/metabolism ; Cell Line ; INDEL Mutation ; },
abstract = {CRISPR/Cas9 has been extensively used for gene knockout, enabling functional studies of genetic loss-of-function in human pluripotent stem cells (hPSCs). However, commonly used Cas9 systems usually exhibit limited and variable efficiencies, and identifying single-guide RNAs (sgRNAs) with high cleavage activity-while avoiding ineffective ones-remains a major challenge. To address these issues, we generated a doxycycline-inducible spCas9-expressing hPSCs (hPSCs-iCas9) line and developed it into an optimized gene knockout system through systematically refining critical parameters. Through this optimization, the system achieved stable INDELs (Insertions and Deletions) efficiencies of 82-93% for single-gene knockouts, over 80% for double-genes knockouts, and up to 37.5% homozygous knockout efficiency for large DNA fragment deletions. Moreover, using this optimized system, we precisely evaluated three widely used gRNA scoring algorithms and integrated Western blotting to rapidly identify the ineffective sgRNA. As a result, among the tested algorithms, Benchling provided the most accurate predictions. Notably, we identified an ineffective sgRNA targeting exon 2 of ACE2, where the edited cell pool exhibited 80% INDELs but retained ACE2 protein expression. Together, these findings provide a robust framework for improving gene knockout efficiency in hPSCs and offer practical guidance for reliable sgRNA selection in gene editing experiments.},
}
@article {pmid41252463,
year = {2025},
author = {Fehrenbach, A and Mitrofanov, A and Alkhnbashi, OS and Backofen, R and Baumdicker, F},
title = {An evolutionary approach to predict the orientation of CRISPR arrays.},
journal = {PLoS computational biology},
volume = {21},
number = {11},
pages = {e1013706},
doi = {10.1371/journal.pcbi.1013706},
pmid = {41252463},
issn = {1553-7358},
abstract = {CRISPR-Cas is a defense system of bacteria and archaea against phages. Parts of the foreign DNA, called spacers, are incorporated into the CRISPR array which constitutes the immune memory. The orientation of CRISPR arrays is crucial for analyzing and understanding the functionality of CRISPR systems and their targets. Several methods have been developed to identify the orientation of a CRISPR array. To predict the orientation, different methods use different features such as the repeat sequences between the spacers, the location of the leader sequence, the Cas genes, or PAMs. However, those features are often not sufficient to predict the orientation with certainty, or different methods disagree. Remarkably, almost all CRISPR systems have been found to insert spacers in a polarized manner at the leader end of the array. We introduce CRISPR-evOr, a method that leverages the resulting patterns to predict the acquisition orientation for (a group of) CRISPR arrays by reconstructing and comparing the likelihood of their evolutionary history with respect to both possible acquisition orientations. The new method is independent of Cas type, leader existence and location, and transcription orientation. CRISPR-evOr is thus particularly useful for arrays that other CRISPR orientation tools cannot predict confidently and to verify or resolve conflicting predictions from existing tools. CRISPR-evOr currently confidently predicts the orientation of 28.3% of the arrays in the considered subset of CRISPRCasdb, which other tools like CRISPRDirection and CRISPRstrand cannot reliably orient. As our tool leverages evolutionary information we expect this percentage to grow in the future when more closely related arrays will be available. Additionally, CRISPR-evOr provides confident decisions for rare subtypes of CRISPR arrays, where knowledge about repeats and leaders and their orientation is limited.},
}
@article {pmid41252186,
year = {2025},
author = {Calhoun, CCS and Capps, MES and Muya, K and Gannaway, WC and Martina, V and Conklin, CL and Klein, MC and Webster, JM and Torija-Olson, EG and Thyme, SB},
title = {Removal of developmentally regulated microexons has a minimal impact on larval zebrafish brain morphology and function.},
journal = {eLife},
volume = {13},
number = {},
pages = {},
pmid = {41252186},
issn = {2050-084X},
mesh = {Animals ; *Zebrafish/genetics/growth &amp; development ; *Brain/anatomy &amp; histology/growth &amp; development/physiology ; Larva/growth &amp; development/genetics ; *Exons/genetics ; *Zebrafish Proteins/genetics/metabolism ; CRISPR-Cas Systems ; *Gene Expression Regulation, Developmental ; *Alternative Splicing ; Phenotype ; },
abstract = {Microexon splicing is a vertebrate-conserved process through which small, often in-frame, exons are differentially included during brain development and across neuron types. Although the protein sequences encoded by these exons are highly conserved and can mediate interactions, the neurobiological functions of only a small number have been characterized. To establish a more generalized understanding of their roles in brain development, we used CRISPR/Cas9 to remove 45 microexons in zebrafish and assessed larval brain activity, morphology, and behavior. Most mutants had minimal or no phenotypes at this developmental stage. Among previously studied microexons, we uncovered baseline and stimulus-driven phenotypes for two microexons (meA and meB) in ptprd and reduced activity in the telencephalon in the tenm3 B0 isoform. Although mild neural phenotypes were discovered for several microexons that have not been previously characterized, including in ppp6r3, sptan1, dop1a, rapgef2, dctn4, vti1a, and meaf6. This study establishes a general approach for investigating conserved alternative splicing events and prioritizes microexons for downstream analysis.},
}
@article {pmid41232532,
year = {2025},
author = {Yang, Y},
title = {What we can learn from the first personalized CRISPR-treated baby to tackle genetic brain disorders.},
journal = {Neuron},
volume = {113},
number = {22},
pages = {3697-3702},
doi = {10.1016/j.neuron.2025.10.023},
pmid = {41232532},
issn = {1097-4199},
mesh = {Humans ; *Gene Editing/methods ; *Genetic Therapy/methods ; *CRISPR-Cas Systems/genetics ; *Brain Diseases/genetics/therapy ; *Precision Medicine/methods ; Infant ; },
abstract = {The landmark report on personalized CRISPR genome editing to treat an infant (baby KJ) with a life-threatening liver disease sparked widespread attention,[1] ushering in a new era of precision genetic intervention. This piece discusses the key challenges and opportunities in translating this milestone into treatments for genetic brain disorders.},
}
@article {pmid41222482,
year = {2025},
author = {Chen, W and He, Y and Yuan, J and Yang, F},
title = {Catalytic hairpin assembly cascade-initiated proximity with self-priming amplification for CRISPR-enhanced ultrasensitive detection of coronary heart disease-associated microRNAs.},
journal = {Analytical methods : advancing methods and applications},
volume = {17},
number = {45},
pages = {9254-9261},
doi = {10.1039/d5ay01456e},
pmid = {41222482},
issn = {1759-9679},
mesh = {*MicroRNAs/genetics/analysis/blood ; Humans ; *CRISPR-Cas Systems/genetics ; *Nucleic Acid Amplification Techniques/methods ; Limit of Detection ; *Coronary Disease/genetics/diagnosis ; *Biosensing Techniques/methods ; },
abstract = {Accurate detection of specific microRNAs (miRNAs) is essential for the early diagnosis of coronary heart disease. Emerging technologies, including functional nuclease-mediated target amplification and DNA nanotechnology, offer substantial potential for precise miRNA identification in clinical diagnostics. This study presents a highly sensitive and specific biosensing platform that integrates catalytic hairpin assembly (CHA) cascade-initiated proximity based self-priming amplification and CRISPR/Cas12a-mediated signal generation for miRNA quantification. Target miRNA initiates the CHA cascade, yielding a toehold-bearing CHA product. This toehold subsequently enables "Variable primer" extension, transcribing double-stranded DNA (dsDNA). The resultant dsDNA activates CRISPR/Cas12a, triggering collateral cleavage and signal amplification. Leveraging this dual-amplification strategy (CHA and CRISPR/Cas12a), the assay achieves a sub-femtomolar detection limit (0.36 fM). Dual-sequence verification, including CHA and CRISPR/Cas12a recognition, ensures exceptional specificity. Validation using spiked serum samples confirmed precise miRNA quantification. Collectively, this biosensor demonstrates significant promise for clinical molecular diagnostics.},
}
@article {pmid41217062,
year = {2025},
author = {Vieyra, F and Pindi, C and Lisi, GP and Morzan, UN and Palermo, G},
title = {Design Rules for Expanding PAM Compatibility in CRISPR-Cas9 from the VQR, VRER and EQR variants.},
journal = {The journal of physical chemistry. B},
volume = {129},
number = {46},
pages = {11949-11958},
doi = {10.1021/acs.jpcb.5c06153},
pmid = {41217062},
issn = {1520-5207},
mesh = {Molecular Dynamics Simulation ; *CRISPR-Cas Systems ; DNA/chemistry/metabolism/genetics ; *CRISPR-Associated Protein 9/chemistry/metabolism/genetics ; Gene Editing ; },
abstract = {Expanding the range of Protospacer Adjacent Motifs (PAMs) recognized by CRISPR-Cas9 is essential for broadening genome-editing applications. Here, we combine molecular dynamics simulations with graph-theory and centrality analyses to dissect the principles of PAM recognition in three Cas9 variants - VQR, VRER, and EQR - that target noncanonical PAMs. We show that efficient recognition is not dictated solely by direct contacts between PAM-interacting residues and DNA but also by a distal network that stabilizes the PAM-binding domain and preserves long-range communication with REC3, a hub that relays signals to the HNH nuclease. A key role emerges for the D1135 V/E substitution, which enables stable DNA binding by K1107 and preserves key DNA phosphate locking interactions via S1109, securing stable PAM engagement. In contrast, variants carrying only R-to-Q substitutions at PAM-contacting residues, though predicted to enhance adenine recognition, destabilize the PAM-binding cleft, perturb REC3 dynamics, and disrupt allosteric coupling to HNH. Together, these findings establish that PAM recognition requires local stabilization, distal coupling, and entropic tuning, rather than a simple consequence of base-specific contacts. This framework provides guiding principles for engineering Cas9 variants with expanded PAM compatibility and improved editing efficiency.},
}
@article {pmid41183414,
year = {2026},
author = {Luo, Q and Zheng, C and Huang, Y and Liang, L and Gong, Y and Zhang, J and Tang, Q and Zhang, K and Liao, X},
title = {Polydopamine-stabilized CsPbBr3 enables toxicity-reduced ECL detection of MMP-2 via CRISPR/Cas12a trans-cleavage.},
journal = {Biosensors &amp; bioelectronics},
volume = {293},
number = {},
pages = {118193},
doi = {10.1016/j.bios.2025.118193},
pmid = {41183414},
issn = {1873-4235},
mesh = {*Biosensing Techniques/methods ; *Indoles/chemistry ; *Polymers/chemistry ; *Matrix Metalloproteinase 2/isolation &amp; purification/chemistry/blood ; Gold/chemistry ; Electrochemical Techniques/methods ; CRISPR-Cas Systems ; Humans ; Limit of Detection ; Luminescent Measurements/methods ; Lead/chemistry ; Titanium/chemistry ; },
abstract = {Lead-halide perovskites offer bright electrochemiluminescence (ECL) but suffer from aqueous instability and Pb[2+] safety concerns. We construct a CsPbBr3@PDA-Au nanointerface that couples polydopamine (PDA) passivation with Au-assisted charge transfer, integrated into a peptide-to-CRISPR/Cas12a amplification scheme for ultrasensitive detection of matrix metalloproteinase-2 (MMP-2). PDA conformally coats CsPbBr3, suppressing trap-mediated quenching and mitigating Pb[2+] release, while providing catechol/amine anchors for a tetrahedral DNA scaffold bearing a ferrocene-labeled hairpin. Upon MMP-2 cleavage of a GPLG↓VRG peptide-DNA chimera, the released strand activates Cas12a trans-cleavage, removing the proximal quencher and switching the interface from "off" to "on." Under unified processing (baseline subtraction and renormalization), the platform achieves a 5.6 aM detection limit with high selectivity over non-target proteases and excellent inter-device reproducibility (triplicate error bars reported). TEM/HRTEM visualize uniform nanocubes; XPS/FTIR evidence PDA catechol/quinone/amine coordination; XRD confirms phase-pure CsPbBr3; and corrected CV/EIS analyses verify accelerated interfacial kinetics after Au decoration. The sensor retains ∼98 % intra-day and ≥91 % 7-day signal, supporting operational robustness. By integrating interfacial stabilization, catalytic electron transfer, and molecular amplification, this modular design advances eco-conscious, perovskite-based ECL diagnostics for low-abundance protease monitoring and broader clinical bioanalysis.},
}
@article {pmid41183412,
year = {2026},
author = {Liu, Y and Li, H and Wang, J and Liu, J and Zhou, X},
title = {Dual-acting CRISPR/Cas12a system enhanced hydrogel fluorescent aptasensor for one-pot detection of tetracycline in water.},
journal = {Biosensors &amp; bioelectronics},
volume = {293},
number = {},
pages = {118181},
doi = {10.1016/j.bios.2025.118181},
pmid = {41183412},
issn = {1873-4235},
mesh = {*Biosensing Techniques/methods ; *Tetracycline/analysis/isolation &amp; purification ; *Aptamers, Nucleotide/chemistry ; *CRISPR-Cas Systems/genetics ; Hydrogels/chemistry ; Limit of Detection ; *Water Pollutants, Chemical/analysis/isolation &amp; purification ; *Anti-Bacterial Agents/analysis ; Water/chemistry ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {Rapid yet ultrasensitive and accurate detection of tetracycline (TC) in aquatic environments subjected to composite pollution remains challenging. CRISPR-mediated biosensors have been extensively studied to achieve ultrasensitive detection. However, these biosensors have always been coupled with a time-consuming nucleic acid amplification process to improve the sensitivity, which may cause erratic signal due to the sophisticated biochemical reaction cascades, hence leading to inaccurate results. To address this issue, we proposed a novel amplification-free fluorescent aptasensor based on polyacrylamide-deoxyribonucleic acid hydrogel integrated with a dual-acting CRISPR/Cas12a system for ultrasensitive and rapid detection of TC in water. With this well-designed dual-acting CRISPR/Cas12a system, a single aptamer-target specific molecular binding event can synchronously initiate pairing between two distinct CRISPR RNA and their target nucleic acid modified on the hydrogel, enabling dual activation of Cas12a. The aptasensor instantly emitted quantifiable strong fluorescence due to the efficient cleavage of reporter probes by the twofold activated Cas12a, demonstrating a TC detection limit of 0.035 μg/L, with approximately 10.6-fold and 5.6-fold sensitivity improvement over the two corresponding single-crRNA systems. The entire detection process can be accomplished in one pot within 10 min. The one-step hydrogel aptasensor shows superior resistance to matrix interference over the conventional solution-phase system, achieving satisfactory recovery percentages (92 %-105 %) for TC in different water matrices. This study offers a new perspective on CRISPR/Cas12a biosensor design and advances the environmental antibiotic monitoring field.},
}
@article {pmid41175692,
year = {2026},
author = {Hao, X and Qin, C and He, C and Yuan, Q and Yang, P and Wen, Y and Wu, Y and Cui, R and Zhang, Y and Chen, W and Liu, Y and Ramadan, S and Ying, Y and Li, D and Xu, L},
title = {In situ detection of bacteria from skin interstitial fluid via CRISPR microneedles: An amplification-free platform for point-of-care diagnostics.},
journal = {Biosensors &amp; bioelectronics},
volume = {293},
number = {},
pages = {118123},
doi = {10.1016/j.bios.2025.118123},
pmid = {41175692},
issn = {1873-4235},
mesh = {*Biosensing Techniques/instrumentation ; *Staphylococcus aureus/isolation &amp; purification/genetics ; Humans ; *Skin/microbiology ; *Extracellular Fluid/microbiology ; CRISPR-Cas Systems ; Gold/chemistry ; Graphite/chemistry ; Limit of Detection ; *DNA, Bacterial/genetics/isolation &amp; purification ; Wearable Electronic Devices ; Needles ; Point-of-Care Systems ; *Staphylococcal Infections/microbiology/diagnosis ; },
abstract = {We report a CRISPR/Cas12a-functionalized microneedle (MN) biosensor for in situ, amplification-free detection of pathogenic bacteria directly from skin interstitial fluid. The platform integrates conductive poly(styrene)/gold/graphene oxide MNs with a minimized electrochemical signal transducer, enabling real-time and highly specific sensing of bacterial DNA through CRISPR-mediated cleavage of ferrocene-labeled ssDNA reporters. Using Staphylococcus aureus as a model target, this strategy achieves direct detection limits of 0.69 pM in ex situ assays and 6.3 pM in situ, without the need for target amplification. The sensor also detects bacterial loads as low as 4.27 × 10[5] CFU/mL in interstitial fluid, which is below typical clinical thresholds. Engineered for biocompatibility and minimal invasiveness, the MNs effectively access interstitial fluid, capture targets and produce quantifiable electrochemical signals within 1 h. Paired with a portable reader and smartphone interface, this wearable system offers a user-friendly tool for point-of-care diagnostics and personalized infectious disease monitoring.},
}
@article {pmid41161233,
year = {2026},
author = {Shan, J and Wang, W and Sheng, Y and Hong, B and Luo, L and Liu, X and Ma, Y and Wang, J},
title = {Ultra-fast one-pot isothermal detection of respiratory virus: ADNA-initiated CRISPR/Cas12a-mediated RCA cycle.},
journal = {Biosensors &amp; bioelectronics},
volume = {293},
number = {},
pages = {118141},
doi = {10.1016/j.bios.2025.118141},
pmid = {41161233},
issn = {1873-4235},
mesh = {*CRISPR-Cas Systems/genetics ; *Nucleic Acid Amplification Techniques/methods ; *SARS-CoV-2/isolation &amp; purification/genetics ; *Biosensing Techniques/methods ; Humans ; Limit of Detection ; *Molecular Diagnostic Techniques/methods ; *COVID-19/diagnosis/virology ; Endodeoxyribonucleases/genetics ; Bacterial Proteins ; CRISPR-Associated Proteins ; },
abstract = {Respiratory virus detection is a prominent area of molecular diagnostics, yet current clinical diagnostic methods lack ultra-rapid and highly sensitive detection capabilities. Here, we reported an ultra-rapid, one-pot isothermal assay called "ADNA-initiated CRISPR-Cas12a-mediated RCA cycle" (ACRE). ACRE was developed through computational studying, engineered design of nucleic acid, and enzyme kinetics analysis, combining rolling circle amplification (RCA) with Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas12a system for respiratory virus detection. The assay involved an engineered assistant DNA (ADNA) initiation reaction, followed by leveraging the cis-cleavage activity of Cas12a coupled with an engineered Padlock, which converted the linear RCA into the RCA cycle. When the RCA cycle is continuously in operation, the trans-cleavage activity of Cas12a facilitates both signal output and amplification. The limit of detection (LOD) for three respiratory viruses (SARS-CoV-2, Inf A, and Inf B) was as low as several hundred attomoles (751 aM, 3.7 fM, and 863 aM), with single-nucleotide specificity. Remarkably, the current assay can detect targets with concentrations above 10 pM within 2.5 min, without the reverse transcription step or specialized instrumentation. Given its exceptional speed, sensitivity, and specificity, ACRE could serve as a robust assay for detecting respiratory virus, enabling molecular diagnostics in clinical settings.},
}
@article {pmid41138445,
year = {2026},
author = {Hu, M and Liu, F and Zhang, J and Yang, L and Yan, Y and Wang, Y and Liu, M and An, H and Zhu, L and Ai, Y and Jiang, X},
title = {Integrated CRISPR/Cas12a-activated liposomal SERS amplification in microfluidic chips for ultrasensitive ampicillin detection.},
journal = {Biosensors &amp; bioelectronics},
volume = {293},
number = {},
pages = {118131},
doi = {10.1016/j.bios.2025.118131},
pmid = {41138445},
issn = {1873-4235},
mesh = {*Ampicillin/analysis/isolation &amp; purification ; Liposomes/chemistry ; CRISPR-Cas Systems/genetics ; *Spectrum Analysis, Raman/methods ; *Biosensing Techniques ; Limit of Detection ; *Anti-Bacterial Agents/analysis ; Animals ; Gold/chemistry ; Lab-On-A-Chip Devices ; Milk/chemistry ; Silver/chemistry ; Humans ; Metal Nanoparticles/chemistry ; },
abstract = {The misuse of antibiotics has created a vicious cycle: from excessive use in livestock breeding to elevated residue levels in food products, ultimately leading to heightened antibiotic resistance in humans, a phenomenon with significant public health consequences. Food-based antibiotic testing is crucial for preventing antibiotic misuse and protecting human health. However, traditional analytical methods exhibit high equipment dependency, restricting on-site detection in hills, plateaus, and other regions with inconvenient transportation. This study developed an integrated CRISPR/Cas12a-activated liposomal Surface-enhanced Raman scattering (SERS) amplification system within a microfluidic chip for ultrasensitive ampicillin detection. The method achieves primary signal amplification by converting ampicillin concentration into a DNA signal through catalytic hairpin assembly (CHA), followed by secondary signal amplification using an Au@Ag substrate coupled with SERS technology for 4-MPBA@liposome marker detection. The platform demonstrated a linear detection range of 1 fM-1 nM with a limit of detection as low as 740 aM. Notably, the method exhibited excellent stability, selectivity, and sensitivity, successfully detecting ampicillin residues in environmental water and milk samples. It provides a technological approach for environmental monitoring and food safety, demonstrating significant practical application value.},
}
@article {pmid41108536,
year = {2025},
author = {Siddiqui, S and Siddiqui, H and Riguene, E and Nomikos, M},
title = {Zebrafish: A Versatile and Powerful Model for Biomedical Research.},
journal = {BioEssays : news and reviews in molecular, cellular and developmental biology},
volume = {47},
number = {12},
pages = {e70080},
pmid = {41108536},
issn = {1521-1878},
support = {//NPRP-Blue Skies Research Award/ ; NPRP-BSRA1-0504-210082//Qatar Research Development and Innovation Council (QRDI), Doha, Qatar/ ; },
mesh = {*Zebrafish/genetics ; Animals ; Humans ; *Biomedical Research/methods ; *Disease Models, Animal ; Precision Medicine/methods ; CRISPR-Cas Systems ; Gene Editing ; },
abstract = {Zebrafish (Danio rerio) have become a versatile model in precision medicine, bridging fundamental biology with translational applications. Their optical transparency, rapid development, and high genetic conservation with humans enable real-time imaging and cost-efficient high-throughput screening. Advances in CRISPR/Cas9, prime editing, and morpholino approaches have expanded their utility for modeling diverse human diseases. In addition to well-established roles in cardiovascular, neurological, metabolic, oncological, and infectious disease research, emerging applications include non-invasive larval urine assays, functional validation of rare human variants, host-microbiome interactions, and automated behavioral profiling for neuropsychiatric conditions. Limitations such as species-specific lipid metabolism and limited antibody availability remain, yet recent integration of single-cell transcriptomics, computational modeling, and machine learning is enhancing translational relevance. Collectively, these innovations position zebrafish as a scalable and powerful platform for disease modeling and personalized therapeutic strategies, underscoring their growing impact in the evolving landscape of precision medicine.},
}
@article {pmid41101316,
year = {2025},
author = {Chitboonthavisuk, C and Martin, C and Huss, P and Peters, JM and Anantharaman, K and Raman, S},
title = {Systematic genome-wide mapping of host determinants of bacteriophage infectivity.},
journal = {Cell systems},
volume = {16},
number = {11},
pages = {101427},
doi = {10.1016/j.cels.2025.101427},
pmid = {41101316},
issn = {2405-4720},
mesh = {*Escherichia coli/virology/genetics ; *Bacteriophages/genetics/pathogenicity ; *Host-Pathogen Interactions/genetics ; CRISPR-Cas Systems/genetics ; Genome, Viral/genetics ; Bacteriophage T7/genetics/pathogenicity ; Host Microbial Interactions/genetics ; },
abstract = {Bacterial host factors regulate the infection cycle of bacteriophages. Except for some well-studied host factors (e.g., receptors or restriction-modification systems), the contribution of the rest of the host genome on phage infection remains poorly understood. We developed phage-host analysis using genome-wide CRISPR interference and phage packaging ("PHAGEPACK"), a pooled assay that systematically and comprehensively measures each host gene's impact on phage fitness. PHAGEPACK combines CRISPR interference with phage packaging to link host perturbation to phage fitness during active infection. Using PHAGEPACK, we constructed a genome-wide map of genes impacting T7 phage fitness in permissive E. coli, revealing pathways that affect phage packaging. When applied to the non-permissive E. coli O121, PHAGEPACK identified pathways leading to host resistance; their removal increased phage susceptibility up to a billion-fold. Bioinformatic analysis indicates that phage genomes carry homologs or truncations of key host factors, potentially for fitness advantage. In summary, PHAGEPACK offers insights into phage-host interactions, phage evolution, and bacterial resistance.},
}
@article {pmid41101079,
year = {2026},
author = {Ma, Q and Rong, Z and Shen, J and Yang, M and Han, Y},
title = {Integrated one-pot RPA-CRISPR/Cas13a platform enables ultrasensitive and field-deployable JAK2 V617F detection for myeloproliferative neoplasm diagnosis.},
journal = {Journal of pharmaceutical and biomedical analysis},
volume = {268},
number = {},
pages = {117197},
doi = {10.1016/j.jpba.2025.117197},
pmid = {41101079},
issn = {1873-264X},
mesh = {Humans ; *Janus Kinase 2/genetics ; *CRISPR-Cas Systems/genetics ; *Myeloproliferative Disorders/genetics/diagnosis ; Mutation ; Sensitivity and Specificity ; Recombinases ; Real-Time Polymerase Chain Reaction/methods ; Nucleic Acid Amplification Techniques/methods ; },
abstract = {Myeloproliferative neoplasms (MPNs) are clonal hematopoietic malignancies driven by mutations like JAK2 V617F, a constitutively activating variant diagnostic for polycythemia vera and subsets of essential thrombocythemia and primary myelofibrosis. Sanger sequencing is the gold standard for detecting JAK2 mutations, but it faces limitations in terms of cost, sensitivity, and portability. CRISPR diagnostics generally requires preamplification, which increases the risk of contamination. There remains an unmet need for field-deployable, rapid MPNs diagnostics in resource-limited settings. We developed ONE-CASPR, a one-pot RPA-CRISPR/Cas13a system enabling ultrasensitive (0.1 % mutant allele frequency), rapid (30 min), portable JAK2 V617F detection. The platform synergizes recombinase polymerase amplification (RPA) with Cas13a trans-cleavage activity in a single-tube reaction at 37 °C, executed via a miniaturized wireless analysis device (CPod) to eliminate cross-contamination while preserving accuracy. Clinical validation across 36 patient samples demonstrated 100 % concordance with real-time quantitative PCR (qPCR) and droplet digital PCR (ddPCR) in both sensitivity and specificity. ONE-CASPR provides a rapid, sensitive, field-deployable, and user-friendly solution for MPNs point-of-care diagnosis in resource-limited settings, with broad molecular screening application potential.},
}
@article {pmid41047505,
year = {2025},
author = {Rajalekshmi, S and Sathyan, KM},
title = {Direct Protein Degradation: Emerging Tools to Probe Biological Complexity in Mammalian Systems.},
journal = {BioEssays : news and reviews in molecular, cellular and developmental biology},
volume = {47},
number = {12},
pages = {e70075},
doi = {10.1002/bies.70075},
pmid = {41047505},
issn = {1521-1878},
support = {//School of Medicine, Southern Illinois University/ ; },
mesh = {Animals ; *Proteolysis ; Humans ; Gene Editing/methods ; Mammals/metabolism ; *Proteins/metabolism/genetics ; CRISPR-Cas Systems ; },
abstract = {Conditional degron approaches for acute and reversible protein depletion have become standard tools for studying gene function in cells and model organisms. Traditional gene perturbation methods have advanced gene function studies but are limited by slow kinetics, potential irreversibility, and lethality when targeting essential genes. To overcome these limitations, tag-based and antibody-based direct protein degradation technologies have been developed. These direct protein degradation systems utilize endogenous protein degradation pathways to achieve rapid and reversible protein depletion. When combined with genome editing, these systems provide precise temporal-and in some cases, spatial-control over endogenous protein expression. In this review, we will discuss the current status of tag-based and antibody-based direct protein degron technologies. We aim to provide a comprehensive guide for selecting these tools, highlighting their context-dependent applications and potential improvements to enhance efficiency and reliability.},
}
@article {pmid41045937,
year = {2025},
author = {Koo, BM and Todor, H and Sun, J and van Gestel, J and Hawkins, JS and Hearne, CC and Banta, AB and Huang, KC and Peters, JM and Gross, CA},
title = {Comprehensive genetic interaction analysis of the Bacillus subtilis envelope using double-CRISPRi.},
journal = {Cell systems},
volume = {16},
number = {11},
pages = {101406},
doi = {10.1016/j.cels.2025.101406},
pmid = {41045937},
issn = {2405-4720},
mesh = {*Bacillus subtilis/genetics/metabolism ; Bacterial Proteins/genetics/metabolism ; Gene Regulatory Networks/genetics ; *Cell Wall/genetics/metabolism ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Peptidoglycan ; CRISPR-Cas Systems/genetics ; },
abstract = {Understanding bacterial gene function remains a major challenge. Double-mutant genetic interaction analysis addresses this challenge by uncovering the functional partners of targeted genes, enabling association of genes of unknown function with known pathways and unraveling of connections among well-studied pathways, but such approaches are difficult to implement at the genome scale. Here, we use double-CRISPR interference (CRISPRi) to systematically quantify genetic interactions at scale for the Bacillus subtilis cell envelope, including essential genes. We discover >1,000 genetic interactions, some known and others novel. Our analysis pipeline and experimental follow-ups reveal the shared and distinct roles of paralogous genes such as mreB and mbl in peptidoglycan and teichoic acid synthesis and identify additional genes involved in the well-studied process of cell division. Overall, our study provides valuable insights into gene function and demonstrates the utility of double-CRISPRi for high-throughput dissection of bacterial gene networks, providing a blueprint for future studies in diverse species. A record of this paper's transparent peer review process is included in the supplemental information.},
}
@article {pmid41045936,
year = {2025},
author = {Dénéréaz, J and Eray, E and Jana, B and de Bakker, V and Todor, H and van Opijnen, T and Liu, X and Veening, JW},
title = {Dual CRISPRi-seq for genome-wide genetic interaction studies identifies key genes involved in the pneumococcal cell cycle.},
journal = {Cell systems},
volume = {16},
number = {11},
pages = {101408},
doi = {10.1016/j.cels.2025.101408},
pmid = {41045936},
issn = {2405-4720},
mesh = {*Streptococcus pneumoniae/genetics ; *Cell Cycle/genetics ; *CRISPR-Cas Systems/genetics ; Genome, Bacterial/genetics ; Computational Biology/methods ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Gene Regulatory Networks/genetics ; Genome-Wide Association Study/methods ; },
abstract = {Uncovering genotype-phenotype relationships is hampered by genetic redundancy. For example, most genes in Streptococcus pneumoniae are non-essential under laboratory conditions. A powerful approach to unravel genetic redundancy is by identifying gene-gene interactions. We developed a broadly applicable dual CRISPRi-seq method and analysis pipeline to probe genetic interactions (GIs) genome-wide. A library of 869 dual single-guide RNAs (sgRNAs) targeting high-confidence operons was created, covering over 70% of the genetic elements in the pneumococcal genome. Testing these 378,015 unique combinations, 4,026 significant GIs were identified. Besides known GIs, we found previously unknown positive and negative interactions involving genes in fundamental cellular processes such as division and chromosome segregation. The presented methods and bioinformatic approaches can serve as a roadmap for genome-wide gene interaction studies in other organisms. All interactions are available for exploration via the Pneumococcal Genetic Interaction Network (PneumoGIN), which can serve as a starting point for new biological discoveries. A record of this paper's transparent peer review process is included in the supplemental information.},
}
@article {pmid40991130,
year = {2025},
author = {Kapoor, SA and Choudhary, P and Kasana, RC},
title = {Exploring CRISPR/Cas9-Mediated Gene Editing Advances in Conventional and Non-conventional Yeast Species.},
journal = {Applied biochemistry and biotechnology},
volume = {197},
number = {11},
pages = {7083-7122},
pmid = {40991130},
issn = {1559-0291},
mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems ; *Yeasts/genetics ; *Saccharomyces cerevisiae/genetics ; },
abstract = {In recent years, using modern technologies, researchers have harnessed the potential of yeast species for various industrial uses, such as the bioproduction of biopharmaceuticals, food additives, industrial biocatalysts, and biofuels. To improve the efficiency and potential of yeast species for industrial uses, genetic modification is carried out. Various genome engineering techniques, including Cre-loxP, homing endonucleases, zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9), have been employed by different research groups for the genetic manipulation of yeast species. Among different genome engineering techniques, CRISPR/Cas9 has become popular because of its precise editing at targeted loci with increased efficiency. The ease of use, effectiveness, and adaptability of CRISPR/Cas9 make multiplexing possible for simultaneously targeting multiple genes, which was earlier very challenging through traditional methods. Moreover, the ability to perform marker-free editing is the significant advantage offered by CRISPR/Cas9. This review focuses on the applications of the CRISPR/Cas9 system in both conventional and non-conventional yeast species. Further, we discussed the advancements of CRISPR/Cas9, including the regulation of gene transcription-activation/repression and other genome engineering aspects. Additionally, innovations in CRISPR/Cas9, such as cloning-free CRISPR/Cas9 assembly, CRISPR-targeted in vivo editing (ACtive), CRISPR/Cas9-induced gene conversion, and selective ploidy ablation (CRI-SPA) are also discussed for enhancing the potential applications of CRISPR/Cas9 in diverse yeast species.},
}
@article {pmid41249581,
year = {2025},
author = {Turgeman-Grott, I and Golan, N and Neri, U and Naki, D and Altman-Price, N and Eizenshtein, K and Choudhary, DK and Levy, R and Navok, S and Cohen, L and Shalev, Y and Singla, H and Reshef, L and Gophna, U},
title = {A previously undescribed archaeal virus suppresses host immunity.},
journal = {EMBO reports},
volume = {},
number = {},
pages = {},
pmid = {41249581},
issn = {1469-3178},
support = {787514//EC | European Research Council (ERC)/ ; 1599/24//Israel Science Foundation (ISF)/ ; },
abstract = {Extremophilic archaea can have chronic viral infections that are well-tolerated by the hosts and may potentially protect against more lethal infections. Here we show that a natural Haloferax strain (48N), is chronically infected by a lemon-shaped virus. This viral infection is not cleared spontaneously, despite the multiple defense systems of the host. Curing 48N of its virus led to radical changes in the gene expression profile of 48N and a dramatic improvement in its growth rate. Remarkably, the cured 48N is the fastest-growing haloarchaeon reported to date, with a generation time of ~107 min at 45 °C, and faster than any known Haloferax species at this temperature. The virus subverts host defenses by reducing its transcription, including the CRISPR spacer acquisition machinery. Nonetheless, even in the virus-cured background, spacer acquisition is very low, indicating that another genetic element is disrupting CRISPR activity. Our results suggest that the slow growth of some halophilic archaea could be due to the effects of proviruses within their genomes that consume resources and alter the gene expression of their hosts.},
}
@article {pmid41249545,
year = {2025},
author = {Warkad, S and Kumar, A and Gampa, M and Goswami, S and T, V and Kumar, S and Dalal, M and Mishra, D and Jha, GK and C, V and Kumar, RR},
title = {Small RNAs big impact: a review on microRNA-mediated tolerance in wheat under terminal heat.},
journal = {Functional &amp; integrative genomics},
volume = {25},
number = {1},
pages = {245},
pmid = {41249545},
issn = {1438-7948},
support = {CABin//Indian Council of Agricultural Research/ ; NICRA//Indian Council of Agricultural Research/ ; },
mesh = {*Triticum/genetics/physiology ; *MicroRNAs/genetics/metabolism ; Gene Expression Regulation, Plant ; *Thermotolerance/genetics ; *Heat-Shock Response/genetics ; *RNA, Plant/genetics/metabolism ; CRISPR-Cas Systems ; },
abstract = {MicroRNAs (miRNAs) are key regulators of gene expression in plant responses to abiotic stresses, including heat stress. High temperatures during the critical developmental stages of wheat (Triticum aestivum L.) drastically limit growth and production. Recent research has found that specific miRNAs regulate molecular complexes and physiological responses by targeting transcription factors, heat shock proteins, and signaling components, thereby modulating heat stress tolerance pathways. This review highlights current knowledge about heat-responsive miRNAs in wheat, including their validated targets and functional involvement in thermotolerance. In addition, we summarized the potential CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats /CRISPR associated protein 9) genome editing tool for precise alteration of miRNA genes or their targets, degradome profiling, the secondary structure of miRNA, and how interplay of miRNAs with HSFs and HSPs in target gene regulation to improve heat resilience. A comprehensive understanding of miRNA-regulated networks presents novel possibilities for developing climate-resilient wheat varieties, thereby ensuring food security in the face of global warming.},
}
@article {pmid41249169,
year = {2025},
author = {Ramadoss, GN and Namaganda, SJ and Kumar, MM and Hamilton, JR and Sharma, R and Chow, KG and Workley, LA and Macklin, BL and Sun, M and Ha, AS and Liu, JC and Fellmann, C and Watry, HL and Dierks, PH and Bose, RS and Jin, J and Perez, BS and Sandoval Espinoza, CR and Matia, MP and Lu, SH and Judge, LM and Shy, BR and Nussenzweig, A and Adamson, B and Murthy, N and Doudna, JA and Kampmann, M and Conklin, BR},
title = {Characterizing and controlling CRISPR repair outcomes in nondividing human cells.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {9883},
pmid = {41249169},
issn = {2041-1723},
mesh = {Humans ; Induced Pluripotent Stem Cells/metabolism/cytology ; *Gene Editing/methods ; *DNA Repair/genetics ; *CRISPR-Cas Systems/genetics ; *Neurons/metabolism/cytology ; Myocytes, Cardiac/metabolism ; DNA Damage ; T-Lymphocytes/metabolism ; },
abstract = {Genome editing is poised to revolutionize treatment of genetic diseases, but poor understanding and control of DNA repair outcomes hinders its therapeutic potential. DNA repair is especially understudied in nondividing cells like neurons, limiting the efficiency and precision of genome editing in many clinically relevant tissues. Here, we address this barrier by using induced pluripotent stem cells (iPSCs) and iPSC-derived neurons to examine how postmitotic human neurons repair Cas9-induced DNA damage. CRISPR editing outcomes differ dramatically in neurons compared to genetically identical dividing cells: neurons take longer to fully resolve this damage, and upregulate non-canonical DNA repair factors in the process. Manipulating this response with chemical or genetic perturbations allows us to direct DNA repair toward desired editing outcomes in nondividing human neurons, cardiomyocytes, and primary T cells. By studying DNA repair in clinically relevant cells, we reveal unforeseen challenges and opportunities for precise therapeutic editing.},
}
@article {pmid41248011,
year = {2025},
author = {Dereki, I and Chondrou, V and Lagoumintzis, G and Sgourou, A},
title = {Epigenetic Engineering of K562 Cells: Dual-Vector Episomal Strategy for Stable Targeted DNA Methylation using dCas9-DNMT3A and -HDAC1 Fusion Proteins.},
journal = {Journal of visualized experiments : JoVE},
volume = {},
number = {224},
pages = {},
doi = {10.3791/69328},
pmid = {41248011},
issn = {1940-087X},
mesh = {Humans ; *DNA Methylation/genetics ; K562 Cells ; DNA Methyltransferase 3A ; *DNA (Cytosine-5-)-Methyltransferases/genetics/biosynthesis ; Plasmids/genetics ; *Histone Deacetylase 1/genetics/metabolism ; Genetic Vectors/genetics ; Recombinant Fusion Proteins/genetics/biosynthesis ; *Epigenesis, Genetic ; RNA, Guide, CRISPR-Cas Systems/genetics ; CRISPR-Associated Protein 9/genetics ; CRISPR-Cas Systems ; },
abstract = {Investigating the precise role of DNA methylation in gene transcriptional regulation and developing therapies targeting specific gene methylation patterns presents significant challenges due to the scarcity of versatile tools capable of inducing site-specific and long-term epigenetic modifications for modulating gene expression. The study aimed to develop and validate an innovative episomal-based system to facilitate stable DNA methylation at a targeted gene locus, potentially useful for both basic epigenetic research and therapeutic applications. To achieve this, the K562 cell line was co-transfected with two distinct episomal vectors. Both vector types were engineered to express guide RNAs (gRNAs) targeting a 367 bp unmethylated region within the CpG island 326, located upstream of the ZBTB7A gene. Each vector encoded a deactivated form of endonuclease Cas9 (dead or dCas9) fused to either the catalytic domain of DNA methyltransferase DNMT3A (dCas-DNMT3A-CD) or the full-length histone deacetylase HDAC1 (dCas-HDAC1). The dCas sequence included two Nuclear Localization Signals (NLS) to ensure the protein's nuclear import. This dual system expression cassette promotes a chromatin state potentially conducive to long-term epigenetic silencing, promising robust and durable epigenetic results. This intervening approach to the host epigenome via utilization of self-replicating episomal vectors offers several advantages: maintenance and expression of vectors at low copy numbers throughout multiple cell divisions without integration into the host genome, hence minimizing off-target effects and preserving genome integrity. We report the precise and significant increase of DNA methylation at the targeted ZBTB7A CpG island 326. The findings validate that the engineered episomal CRISPR/dCas systems can elicit durable, site-specific DNA methylation. Therefore, this system is a valuable research tool for evaluating the functional effects of targeted methylation changes and a promising platform for developing future epigenetic treatments.},
}
@article {pmid41243026,
year = {2025},
author = {Menelih, A and Girma, A and Assamere, A},
title = {Mechanisms and applications of epigenome editing in plants: current status, challenges and future perspectives.},
journal = {Functional &amp; integrative genomics},
volume = {25},
number = {1},
pages = {243},
pmid = {41243026},
issn = {1438-7948},
mesh = {*Gene Editing/methods ; *Epigenome ; CRISPR-Cas Systems ; *Epigenesis, Genetic ; *Plants/genetics ; DNA Methylation ; *Genome, Plant ; Epigenome Editing ; },
abstract = {Epigenome editing has become a leading-edge technology of programmable, heritable and reversible control of gene expression in plants without changing the DNA sequence. CRISPR/dCas9 systems along with transcription activator-like effectors (TALEs) and zinc finger systems have made it possible to manipulate DNA methylation, histone modifications, and RNA epigenetic marks in a precise and locus-specific fashion. These tools have been used on major regulatory genes of flowering time, stress adjustment, and yield maximization in model and crop plants. This review synthesizes the current status of plant epigenome editing advances and highlights mechanistic innovations including SunTag, CRISPRoff/on and RNA m6A editing. It also emphasizes new paradigm shifts in chromatin reprogramming, including transcription-resistive chromatin states, locus-specific H3K27me3 demethylation, and nanobody-mediated chromatin targeting. Furthermore, it considers the consequences of these shifts in the context of trait stability and epigenetic inheritance. Moreover, the relative evaluation of dCas9-, TALE-, and ZFP-based platforms indicated that there are still enduring problems in the performance of delivery, off-target effects, and transgenerational stability. The review concludes with a conceptual framework connecting epigenome editing to climate-smart crop improvement and outlines future research priorities focused on combinatorial multi-omics integration and the development of environmentally responsive editing platforms.},
}
@article {pmid41242874,
year = {2025},
author = {Akter, T and Baishnab, SM and Damtie, M and Dumpati, S and Eshete, TB and Irfan, S and Kumar, M and Sara, M and Urmi, UL and Willcox, M},
title = {A review of technological advances in the management of bacterial, fungal, viral, and Acanthamoeba keratitis.},
journal = {Clinical &amp; experimental optometry},
volume = {},
number = {},
pages = {1-11},
doi = {10.1080/08164622.2025.2579169},
pmid = {41242874},
issn = {1444-0938},
abstract = {Microbial keratitis is a major cause of monocular blindness worldwide. Trauma and contact lens wear are major risk factors. Bacteria are a major cause, but fungi, viruses, and Acanthamoeba can also cause the disease. Therapeutic options are dwindling with the rise of antimicrobial resistance in all of these microbes, but especially bacteria. Infection with resistant strains results in worse clinical outcomes. New antimicrobial therapies are being developed but most have yet to be either used to treat ocular infections or become commercially available. For bacterial keratitis, antimicrobial peptides (AMPs) are a potential new option, as are bacteriophages and even bacteria that eat other bacteria or maggot secretions. For fungal keratitis, ocular natamycin, with oral ketoconazole or voriconazole treatments, offer improved outcomes for severe cases. For viral keratitis, AMPs again offer hope as new therapeutics, as do RNA interference and CRISPR-Cas systems. The recent release of a commercially available polyhexamethylene biguanide eye drop is a step forward, but new therapies that can kill both Acanthamoeba trophozoites and cysts are still needed. Beyond antibiotics, photoactivated chromophores for keratitis-corneal collagen cross-linking and photodynamic therapy have been successfully used. Therapies for large ulcers or scarring include corneal gluing, tissue scaffolds such as amniotic membrane or conjunctival flaps, and tissue replacement through penetrating or lamellar keratoplasty.},
}
@article {pmid41242543,
year = {2025},
author = {Hillary, VE and Rajan, V and Ceasar, SA},
title = {CRISPR's impact on cancer: From fundamental models to clinical solutions.},
journal = {Life sciences},
volume = {},
number = {},
pages = {124087},
doi = {10.1016/j.lfs.2025.124087},
pmid = {41242543},
issn = {1879-0631},
abstract = {CRISPR is a programmable genome editing method that has transformed genome engineering and advanced cancer research. Various engineering iterations of CRISPR, including base and prime editing, facilitate more accurate and adaptable genomic manipulations, opening new frontiers in understanding and combating cancer. This review emphasizes CRISPR's significant influence in cancer biology, from its ability to accurately recreate somatic mutations in cellular and animal models to its application in developing precise diagnostic tools. We also examine advancements in cancer therapies, particularly through the ex vivo engineering of robust chimeric antigen receptor (CAR) T cells that perform effectively in restrictive tumor microenvironments. Lastly, we consider the existing limitations of CRISPR-Cas systems and outline future directions that could further enhance the application of this powerful technology against cancer.},
}
@article {pmid41213415,
year = {2026},
author = {Xu, YY and Zhou, SM and Wang, LY and Zhang, R and Li, K and Qian, ZY and Xiao, L},
title = {Methods for detecting off-target effects of CRISPR/Cas9.},
journal = {Biotechnology advances},
volume = {86},
number = {},
pages = {108750},
doi = {10.1016/j.biotechadv.2025.108750},
pmid = {41213415},
issn = {1873-1899},
mesh = {*CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; Humans ; Animals ; },
abstract = {The CRISPR/Cas9 system has emerged as a revolutionary tool for gene editing, widely used in the biomedical field due to its simplicity, efficiency, and cost-effectiveness. However, evidence suggests that CRISPR/Cas9 can induce off-target effects, leading to unintended mutations that may compromise the precision of gene modifications. Consequently, predicting，detecting and evaluating these off-target effects is crucial for optimizing the accuracy and reliability of CRISPR/Cas9 system. This paper provides an overview of the various methodologies and strategies, used or to be used for identifying off-target effects in CRISPR/Cas9-based genome editing, offering insights to improve the precision and safety of CRISPR applications in research and therapeutics.},
}
@article {pmid41208769,
year = {2025},
author = {Luo, D and Wang, N and Major-Monfried, H and Ralls, J and Rha, S and Maitland, SA and Ponnienselvan, K and Yamada, M and Bauer, DE and Wolfe, SA and Kentsis, A},
title = {Modular Platform for Therapeutic Drug Delivery Using Trifunctional Bio-Orthogonal Macromolecular Conjugates.},
journal = {Bioconjugate chemistry},
volume = {36},
number = {11},
pages = {2472-2486},
doi = {10.1021/acs.bioconjchem.5c00440},
pmid = {41208769},
issn = {1520-4812},
mesh = {Humans ; *Cell-Penetrating Peptides/chemistry ; *Drug Delivery Systems/methods ; *Macromolecular Substances/chemistry ; Gene Editing ; Cell Line, Tumor ; Peptidomimetics/chemistry/pharmacology ; Ligands ; CRISPR-Cas Systems ; },
abstract = {Targeted delivery of macromolecular therapeutics holds great promise for overcoming the limitations of conventional small molecules, enabling the modulation of protein-protein interactions and precise genome editing. However, efficient, safe, and cell type-specific delivery remains a major challenge. To address this, we developed a modular platform for synthesizing heterotrifunctional bio-orthogonal macromolecular conjugates (BMCs) by engineering diverse combinations of targeting ligands, cell-penetrating peptides (CPPs), and bioactive cargos. We optimized facile bioconjugation chemistries to generate BMCs with improved yields, structural integrity, and activity. Modular BMCs accommodate diverse components, including antibodies and receptor ligands for targeting, CPPs for intracellular trafficking, and optical probes, therapeutic peptidomimetics, and CRISPR-Cas9 nuclease as cargo to confer specific biological activities. We assayed their utility across multiple applications: BMCs with fluorescently labeled cargo revealed endosomal escape and intracellular accumulation; peptidomimetic MYB transcription factor inhibitor BMCs exhibited potent antileukemic activity against acute myeloid leukemia cells; and Cas9 BMCs achieved rapid delivery and cell type-specific gene editing in human cells. The BMC approach enables the customizable delivery of functional macromolecules, nominating BMCs as a broadly applicable platform for biomedical applications.},
}
@article {pmid41186998,
year = {2025},
author = {Wang, W and Liu, H and Yang, Z and Wang, L and Li, W and He, H and Chen, S and Xu, M and Lu, Y},
title = {Enhancement of Spinosyn Production by Integrating a Static and Dynamic CRISPRi-Mediated Metabolic Switch in Saccharopolyspora spinosa.},
journal = {Journal of agricultural and food chemistry},
volume = {73},
number = {46},
pages = {29717-29728},
doi = {10.1021/acs.jafc.5c08276},
pmid = {41186998},
issn = {1520-5118},
mesh = {*Saccharopolyspora/genetics/metabolism/growth &amp; development ; *Macrolides/metabolism ; *Bacterial Proteins/metabolism/genetics ; Metabolic Engineering/methods ; CRISPR-Cas Systems ; Multigene Family ; Gene Expression Regulation, Bacterial ; *Insecticides/metabolism ; },
abstract = {Spinosyns, polyketide-derived macrolide insecticides produced by Saccharopolyspora spinosa, have broad market prospects. However, their large-scale production is still challenged by the low titers of industrial strains. Here, we developed an integrated static and dynamic CRISPRi strategy to redirect metabolic flux toward spinosyn overproduction without affecting cell growth. Initially, static CRISPRi-mediated repression of four genes (gltA1, fabH3, fabH4, and glgC) in three key nodes of primary metabolism boosted spinosyn titers by 19-55% without growth impairment. Simultaneous repression of these genes led to a further increase of spinosyn production. However, static repression of two other genes (pyc or gltA2) severely hindered bacterial growth and reduced spinosyn biosynthesis. To address this, two growth phase-dependent promoters from the spinosyn biosynthetic gene cluster (BGC) were characterized and employed for dynamic repression of pyc and gltA2, resulting in increased spinosyn titers by 44-68% while maintaining robust growth. Finally, combined static and dynamic repression of these six targets simultaneously achieved over a 2-fold increase of spinosyn production. Collectively, integrated static and dynamic CRISPRi-based reprogramming of primary metabolic pathways in S. spinosa achieved a balance between growth and secondary metabolism, thereby leading to spinosyn overproduction. This strategy has broad potential applicability in other actinomycetes for natural product overproduction.},
}
@article {pmid41170688,
year = {2025},
author = {Wang, S and Guan, X and Sun, S},
title = {A CRISPR/Cas12a-based aptasensor enhanced by functionalized AuNPs for sensitive full-range C-reactive protein detection.},
journal = {Journal of materials chemistry. B},
volume = {13},
number = {45},
pages = {14749-14760},
doi = {10.1039/d5tb01727k},
pmid = {41170688},
issn = {2050-7518},
mesh = {*C-Reactive Protein/analysis ; *Gold/chemistry ; *Aptamers, Nucleotide/chemistry/metabolism ; Humans ; *Metal Nanoparticles/chemistry ; *CRISPR-Cas Systems ; *Biosensing Techniques/methods ; Limit of Detection ; Particle Size ; *Endodeoxyribonucleases/metabolism ; Bacterial Proteins ; CRISPR-Associated Proteins ; },
abstract = {C-reactive protein (CRP) is a well-established biomarker whose plasma levels increase significantly during inflammatory and infectious episodes. It plays a vital role in the diagnosis of bacterial infections and autoimmune diseases, and cardiovascular risk assessment. However, conventional detection techniques often struggle to balance high sensitivity with a broad dynamic range. Here, we developed and validated a novel fluorescence-based aptasensor named AuCA (AuNP-enhanced CRISPR Aptasensor), which integrates nucleic acid aptamers, magnetic beads (MBs), gold nanoparticles (AuNPs), and the CRISPR/Cas12a system for robust CRP quantification. In this system, MBs functionalized with aptamer 1 selectively captured the target protein, while AuNPs co-modified with aptamer 2 and a Cas12a activation sequence (Trigger) enabled efficient signal transduction. Upon target binding, the Trigger activated the Cas12a/crRNA complex, initiating trans-cleavage of fluorescent reporters and producing a markedly amplified signal. The optimized AuCA platform achieved a low detection limit of 60 ng mL[-1] and a quantifiable range of 0.1-150 μg mL[-1]. It exhibited excellent specificity and resistance to biological interference, ensuring reliable measurements even in complex sample matrices. When applied to clinical human plasma, AuCA demonstrated results that are in strong concordance with results from a commercial immunoturbidimetric assay. AuCA allowed the simultaneous detection of both standard and hypersensitive CRP (hsCRP), supporting comprehensive full-range CRP (frCRP) analysis with strong potential for clinical applications.},
}
@article {pmid41151122,
year = {2025},
author = {Cea Salazar, VI and Boender, AJ and Seelke, AMH and Gaard, L and Mederos, SL and Rogers, S and Gutierrez, XZ and Bales, KL and Young, LJ and Trainor, BC},
title = {CRISPR-mediated knockdown of oxytocin receptor in extended amygdala reduces stress-induced social avoidance in female California mice.},
journal = {Hormones and behavior},
volume = {176},
number = {},
pages = {105845},
doi = {10.1016/j.yhbeh.2025.105845},
pmid = {41151122},
issn = {1095-6867},
mesh = {Animals ; *Receptors, Oxytocin/genetics/metabolism ; Female ; *Stress, Psychological/genetics/metabolism ; Peromyscus ; Mice ; *Amygdala/metabolism ; *Social Behavior ; Gene Knockdown Techniques ; Nucleus Accumbens/metabolism ; CRISPR-Cas Systems ; Septal Nuclei/metabolism ; *Avoidance Learning/physiology ; Behavior, Animal/physiology ; },
abstract = {Oxytocin receptors (OTRs) within the extended amygdala and nucleus accumbens (NAc) have been implicated in modulating social behaviors, particularly following stress. The effects of OTR could be mediated by modulating the activity of pre-synaptic axon terminals or via receptors in post-synaptic neurons or glia. Using a viral-mediated CRISPR/Cas9 gene editing system in female California mice (Peromyscus californicus), we selectively knocked down OTR in the anteromedial bed nucleus of the stria terminalis (BNST) or NAc to examine their roles modulating social approach and vigilance behaviors. Knockdown of OTR in the BNST attenuated stress-induced decreases of social approach and had less robust effects on vigilance when interacting with a target mouse behind a wire barrier. In this large arena, where mice could control their proximity to a target mouse, BNST OTR knockdown also increased investigation of a non-social stimulus (empty cage). Behavioral effects of BNST OTR knockdown were weaker in the small arena where focal mice physically interacted with target mice. Interestingly, OTR knockdown in the NAc, reduced stress-induced social vigilance without affecting social approach. These effects could mediate altered encoding of socially aversive experiences, as knockdown manipulations were performed before stress exposure. Together, these results highlight effects of local OTR on social behavior that are region-specific.},
}
@article {pmid41123407,
year = {2025},
author = {Xu, Z and Huang, Y and Dong, Y and An, X and Tong, Y and Li, M},
title = {Engineering broad-spectrum phage-resistant Escherichia coli via adaptive and programmable defense strategies.},
journal = {Applied and environmental microbiology},
volume = {91},
number = {11},
pages = {e0159625},
doi = {10.1128/aem.01596-25},
pmid = {41123407},
issn = {1098-5336},
support = {2022FY101100//Science &amp; Technology Fundamental Resources Investigation Program/ ; 202423m10050004//Anhui Province Scientific and Technological Research Project/ ; 22322908D//S&amp;T Program of Hebei/ ; 82341119//National Natural Science Foundation of China/ ; XK2025-05//Interdisciplinary Research Center of Beijing University of Chemical Technology/ ; },
mesh = {*Escherichia coli/virology/genetics ; *Coliphages/genetics/physiology/isolation &amp; purification ; CRISPR-Cas Systems ; Genome, Viral ; Fermentation ; },
abstract = {UNLABELLED: Phage contamination, which impacts product quality and production efficiency, remains a major challenge in industrial fermentation. Although bacteria have evolved various defense systems to combat phage infection, these systems often suffer from narrow host specificity and limited efficacy. In this study, we isolated and characterized a novel lytic Escherichia coli phage, TR2, from a contaminated fermentation substrate. Its strong environmental stability, short latency period, and high lytic activity render it a significant threat to fermentation processes. Genomic sequencing revealed that phage TR2 has a linear, double-stranded DNA genome of 45,171 bp with a G+C content of 44% and 74 coding sequences. On the basis of the physiological characteristics and genomic features of this phage, we developed two strategies to generate phage-resistant E. coli strains: (i) selection of spontaneous mutations in bacterial surface receptors to prevent phage adsorption and infection and (ii) integration of an exogenous CRISPR/Cas9 system to confer sequence-specific immunity. Spontaneous mutation provides broad-spectrum resistance but at the cost of fitness and evolutionary stability, whereas CRISPR/Cas9 ensures long-term, programmable immunity with minimal growth defects. Importantly, both strategies successfully protected bacterial cultures from phage infection without compromising recombinant protein production, highlighting their potential for industrial application. Our findings provide a practical approach for mitigating phage contamination in industrial fermentation processes. This study also highlights the advantages and limitations of spontaneous mutations and natural phage defense systems, offering valuable insights for the design of more effective phage-resistant microbial platforms.<br><br>IMPORTANCE: Phage contamination is a significant challenge in industrial fermentation and severely impacts product quality and production efficiency. We systematically compared spontaneous mutation and CRISPR/Cas9-mediated immunity as two strategies for engineering phage-resistant E. coli strains. Both approaches effectively protected bacterial cultures from phage infection without compromising recombinant protein production, underscoring their potential for industrial applications. Notably, spontaneous mutation conferred broad-spectrum resistance but was associated with fitness costs and limited evolutionary stability. In contrast, CRISPR/Cas9-based immunity offered long-term, programmable protection with minimal growth impairment. By delineating the trade-offs between these two strategies, our work provides a framework for selecting tailored phage resistance solutions suited to diverse biomanufacturing scenarios.},
}
@article {pmid40962686,
year = {2025},
author = {Chen, H and Fan, S and Chen, K and Wang, F and Lu, M and Wu, Y and Lu, H and Li, J},
title = {CRISPR-edited iPSCs reveal BSN gene mutations induce neuronal hyperexcitability via astrocyte lipid accumulation.},
journal = {Neurotherapeutics : the journal of the American Society for Experimental NeuroTherapeutics},
volume = {22},
number = {6},
pages = {e00740},
doi = {10.1016/j.neurot.2025.e00740},
pmid = {40962686},
issn = {1878-7479},
mesh = {*Astrocytes/metabolism ; *Induced Pluripotent Stem Cells/metabolism ; Humans ; *Neurons/metabolism/physiology ; *Mutation/genetics ; *Lipid Metabolism/genetics/physiology ; Gene Editing/methods ; CRISPR-Cas Systems ; *Nerve Tissue Proteins/genetics ; Cells, Cultured ; Epilepsy/genetics ; },
abstract = {Mutations in the BSN gene, encoding the presynaptic protein Bassoon, are implicated in epilepsy, but their impact on astrocytes remains unclear. Using CRISPR/Cas9, we introduced patient-derived BSN mutations (p.M1903V and c.5672insCG) into human induced pluripotent stem cells (iPSCs) and differentiated them into astrocytes. We found that mutant astrocytes exhibited significant lipid accumulation, evidenced by elevated free cholesterol, reduced arginase activity, and increased lipid droplets. Proteomic analysis revealed upregulation of lipid metabolism regulators, such as APOE and FASN. Electrophysiological recordings showed impaired Kir4.1 potassium channel function, depolarized resting membrane potential, and increased capacitance in mutant astrocytes following kainic acid stimulation. Co-culture experiments with neurons demonstrated that BSN-mutant astrocytes led to reduced neurite outgrowth, elevated neuronal apoptosis, increased pro-inflammatory cytokines (IL-1β, TNF-α), and neuronal hyperexcitability. These findings demonstrate that BSN mutations disrupt astrocyte lipid homeostasis and impair neurosupportive functions, thereby driving neuronal hyperexcitability. This study establishes astrocytes as critical mediators of epilepsy pathogenesis in BSN-related disorders and highlights lipid metabolism as a potential therapeutic target.},
}
@article {pmid41242302,
year = {2025},
author = {Ishihara, K and Kitagawa, S and Adachi, N and Akutsu, M and Senda, T and Inanaga, H and Numata, T},
title = {Cryo-EM structure of Archaeoglobus fulgidus type III-B CRISPR-Cas effector and intermediate crRNA processing during effector assembly.},
journal = {Biochemical and biophysical research communications},
volume = {792},
number = {},
pages = {152978},
doi = {10.1016/j.bbrc.2025.152978},
pmid = {41242302},
issn = {1090-2104},
abstract = {Type III CRISPR-Cas effectors recognize target RNAs complementary to the crRNA guide, activating diverse downstream antiviral responses. In contrast to type III-A systems, the architecture of the type III-B effector (Cmr), comprising six proteins (Cmr1-Cmr6) and a crRNA, remains incompletely defined. Moreover, although maturation of the 3' region of type III crRNA has been attributed to polynucleotide phosphorylase (PNPase), an alternative maturation pathway has been suggested but remains to be elucidated. Here we determined the cryo-EM structure of the Cmr1-lacking Archaeoglobus fulgidus Cmr (AfCmrΔ1) bound to a target analog at 3.4 Å resolution. The complex forms a continuous basic channel that accommodates a crRNA-target heteroduplex. Comparative interface analysis explains why the previously reported cross-species Cmr assembly retains activity, revealing interface flexibility that enables compatible Cmr3-Cmr4 and Cmr2-Cmr5 interactions. Furthermore, we show the cooperative, site-specific processing of an intermediate crRNA that requires both AfCmrΔ1 and AfCmr1 and proceeds without divalent cations. In addition to identifying the cleavage site within the intermediate crRNA, mutational analysis of AfCmr1 reveals residues critical for the reaction. These findings suggest an alternative pathway for crRNA maturation during type III effector assembly that complements PNPase-mediated trimming of the intermediate crRNA, thereby expanding the mechanistic landscape of type III CRISPR-Cas systems.},
}
@article {pmid41242144,
year = {2025},
author = {Yu, ES and Jang, H and Kwon, JM and Jeong, H and Park, J and Kang, BH and Rho, D and Son, S and Kang, T and Jeong, KH},
title = {Nanoplasmonic real-time RT-RPA and CRISPR/Cas12a detection for rapid point-of-care molecular diagnostics.},
journal = {Biosensors &amp; bioelectronics},
volume = {294},
number = {},
pages = {118216},
doi = {10.1016/j.bios.2025.118216},
pmid = {41242144},
issn = {1873-4235},
abstract = {The rapid and precise detection of nucleic acids is crucial for effective disease diagnosis and management at the point-of-care (POC) level. Here we report a palm-sized plasmonic photothermal platform for real-time on-chip recombinase polymerase amplification (RPA) and CRISPR/Cas12a detection. An ultrathin photothermal nanoplasmonic cavity (PNC) of Au nanoislands (AuNIs) and an aluminum reflector delivers uniform and efficient photothermal heating under white LED illumination. The configuration drives isothermal amplification and CRISPR-mediated cleavage in a single microchamber while a fluorescence microlens array (FMLA) camera records real-time emission. The compact platform detects the SARS-CoV-2 E gene in 25 min at 25.7 copies per cartridge and achieves 100 % concordance with RT-qPCR across 42 clinical samples. This all-in-one platform can offer a robust and cost-effective solution for molecular diagnostics, facilitating scalable and real-time testing of infectious diseases in decentralized POC settings.},
}
@article {pmid41241907,
year = {2026},
author = {Treichel, AJ and Bazzini, AA and Tornini, VA},
title = {Functional Testing of Microproteins in a Vertebrate Model of Development.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2992},
number = {},
pages = {183-201},
pmid = {41241907},
issn = {1940-6029},
mesh = {Animals ; *Zebrafish/genetics/embryology/metabolism ; CRISPR-Cas Systems ; *Zebrafish Proteins/genetics/metabolism ; Ribosomes/metabolism/genetics ; Gene Expression Regulation, Developmental ; Embryonic Development/genetics ; Micropeptides ; },
abstract = {While thousands of putative microproteins have been identified through ribosome profiling, reporter assays, and mass spectrometry-based methods, their functional testing has remained challenging. Advances in genome sequencing and CRISPR/Cas technologies enable the prioritization and testing of candidate microprotein functions for roles in development, for example, in the maternal-to-zygotic transition or in neurodevelopment. Here, we describe the functional testing of microproteins in vivo using a vertebrate model of early development, Danio rerio (zebrafish).},
}
@article {pmid41241835,
year = {2025},
author = {Zhang, J and Li, L and Zhu, Y and Qian, K and Xu, Q and Qin, Y and Wu, L},
title = {Amplification-Free CRISPR-Cas System Integrated Centrifugal Digital Microfluidic Platform Developed for Multiplexed Respiratory Pathogen Nucleic Acid Analysis.},
journal = {Analytical chemistry},
volume = {},
number = {},
pages = {},
doi = {10.1021/acs.analchem.5c04524},
pmid = {41241835},
issn = {1520-6882},
abstract = {In response to the urgent demand for highly sensitive and rapid multiplex detection technologies in the prevention and control of respiratory infectious diseases, this study presents the development of an integrated CRISPR-Cas9/Cas13a detection platform based on a centrifugal digital microfluidic chip. It aims to overcome the reliance of traditional real-time fluorescence quantitative PCR on specialized equipment and trained personnel. Additionally, it addresses the issue of false positives commonly associated with existing isothermal amplification technologies, while also meeting the requirement for preamplification in sensitive CRISPR-based detection methods. In this study, Methicillin-resistant Staphylococcus aureus (MRSA) and influenza A virus subtype H1N1 were selected as model pathogens. The off-chip CRISPR-Cas9/Cas13a dual nucleic acid detection system was initially developed and optimized to enable highly specific detection of MRSA-mecA DNA at a concentration of 173 pM and H1N1-HA RNA at a concentration of 117 pM. Subsequently, the optimal centrifugal digital chip structure was designed and screened to achieve a droplet filling rate of 99.6%. The optimized CRISPR system was finally integrated into the digital chip, resulting in significantly improved sensitivity, reaching 0.7 copies/μL for MRSA DNA and 1.2 copies/μL for H1N1 RNA within a 20 min reaction time at 37 °C. Furthermore, both the negative and positive detection rates achieved 100% accuracy across all 20 simulated clinical samples. The platform integrates centrifugal digital droplet segmentation technology with the CRISPR-Cas system in an innovative manner, enabling subcopy sensitivity detection without the need for nucleic acid preamplification. Therefore, this convenient, cost-effective, and contamination-resistant method provides a reliable solution for the rapid detection of respiratory pathogens in resource-constrained scenarios.},
}
@article {pmid41241617,
year = {2025},
author = {Kalogeropoulos, K and van Beljouw, SPB and Feldmann, D and van den Berg, DF and Brouns, SJJ},
title = {Proteases in bacteriophage defense systems and their potential in bioengineering.},
journal = {Trends in biochemical sciences},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.tibs.2025.10.006},
pmid = {41241617},
issn = {0968-0004},
abstract = {Novel phage defense systems featuring diverse enzymatic activities are continually being discovered. Among these, defense systems employing proteolytic enzymes have been identified, revealing a previously unrecognized enzymatic activity in phage defense. These protease-associated defense systems represent an untapped reservoir for new biotechnological tools and may serve as a springboard for the development of proteome editors. This review outlines recent advancements in the discovery and characterization of protease-containing defense systems, proposes methods for further exploration and investigation of protease activity, and considers the prospect of protease defense systems for modulating protein processing and cell fate.},
}
@article {pmid41220317,
year = {2025},
author = {Rayad, N and Chowdhury, EA and Meno-Tetang, GML},
title = {The Impact of QSP Modeling on the Design and Optimization of Gene Therapy Approaches.},
journal = {CPT: pharmacometrics &amp; systems pharmacology},
volume = {14},
number = {11},
pages = {1760-1764},
doi = {10.1002/psp4.70131},
pmid = {41220317},
issn = {2163-8306},
mesh = {*Genetic Therapy/methods ; Humans ; Gene Editing/methods ; Dependovirus/genetics ; Genetic Vectors ; Animals ; RNA, Messenger/administration &amp; dosage/genetics ; Nanoparticles/administration &amp; dosage ; CRISPR-Cas Systems ; *Models, Biological ; Lipids/chemistry ; Tissue Distribution ; Liposomes ; },
abstract = {Quantitative Systems Pharmacology (QSP) is increasingly utilized to support the design and translation of gene therapies. This perspective outlines the application of QSP modeling across three domains of gene therapy: mRNA-based therapeutics, adeno-associated virus (AAV) vectors, and genome editing systems. We highlight opportunities for dose optimization, biomarker interpretation, and mechanistic understanding, while addressing current limitations in model generalizability, data sparsity, and translational relevance. Examples include QSP platforms for lipid nanoparticle (LNP)-delivered mRNA, physiologically based pharmacokinetics (PBPK)-informed AAV biodistribution models, and CRISPR-Cas9-based editing systems. These case studies demonstrate QSP's value in de-risking development and personalizing therapies for rare and complex diseases.},
}
@article {pmid41191401,
year = {2025},
author = {Guo, Y and Xiong, Y and Tong, W and Huang, X and Xiong, Y},
title = {CRISPR/Cas12a-Based Dynamic Light Scattering Assay for Nucleic Acid Amplification-Free and Ultrasensitive Detection of Listeria monocytogenes.},
journal = {Analytical chemistry},
volume = {97},
number = {45},
pages = {25232-25244},
doi = {10.1021/acs.analchem.5c04740},
pmid = {41191401},
issn = {1520-6882},
mesh = {*Listeria monocytogenes/isolation &amp; purification/genetics ; *CRISPR-Cas Systems ; *Dynamic Light Scattering/methods ; Limit of Detection ; Gold/chemistry ; Metal Nanoparticles/chemistry ; Nucleic Acid Amplification Techniques ; *Biosensing Techniques/methods ; Particle Size ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {Ultrasensitive signal transduction systems are essential for developing rapid, robust, and sensitive nucleic acid tests using the amplification-free CRISPR-Cas12a system. In this study, we introduce a novel platform termed CRISPR-DART (dynamic light scattering Assisted Rapid Test), which combines CRISPR-Cas12a with a dynamic light scattering (DLS) signal readout. We systematically evaluated the effects of nanoparticle size (20, 60, and 100 nm) and morphology (spherical, cubic, and flower-like) on DLS sensing performance. Results demonstrate that larger and more structurally complex nanoparticles significantly enhance scattering intensity, allowing stable DLS signals at lower concentrations and improving sensitivity for detecting low-abundance nucleic acid targets. By utilizing the high specificity and trans-cleavage activity of CRISPR-Cas12a, target-triggered cleavage of single-stranded DNA linkers modulates nanoparticle aggregation, enabling quantitative nucleic acid detection. Notably, CRISPR-DART based on 100 nm gold nanoflowers with large size and complex morphology achieves a detection limit (LOD) of 32 aM, an improvement of 5 orders of magnitude over conventional CRISPR-Cas12a assays using fluorophore-quencher as signal output. Furthermore, the amplification-free CRISPR-DART platform also achieves a LOD of 92 CFU/mL for Listeria monocytogenes in food samples, and after a brief preincubation step, successfully detects 1 CFU in 25 g of food sample. In summary, the CRISPR-DART platform provides a straightforward, highly sensitive, and specific tool for rapid on-site diagnostics and food safety monitoring.},
}
@article {pmid41052028,
year = {2025},
author = {Khan, S and Chen, L and Chou, CL and Khundmiri, SJ and Knepper, MA},
title = {Inducible Avp knockout mouse line.},
journal = {American journal of physiology. Renal physiology},
volume = {329},
number = {6},
pages = {F784-F795},
doi = {10.1152/ajprenal.00340.2025},
pmid = {41052028},
issn = {1522-1466},
support = {ZIA-HL001285//HHS | NIH | National Heart, Lung, and Blood Institute (NHLBI)/ ; ZIA-HL006129//HHS | NIH | National Heart, Lung, and Blood Institute (NHLBI)/ ; },
mesh = {Animals ; Mice, Knockout ; *Arginine Vasopressin/genetics/deficiency/metabolism ; Aquaporin 2/metabolism/genetics ; *Tamoxifen/pharmacology ; Mice ; Male ; *Kidney/metabolism/pathology ; Female ; Mice, Inbred C57BL ; CRISPR-Cas Systems ; },
abstract = {Arginine vasopressin (AVP) is a peptide hormone synthesized in the hypothalamus and secreted by the posterior pituitary. Previous studies toward understanding AVP physiology relied heavily on Brattleboro rats, which have a spontaneous mutation in the Avp gene and lack circulating AVP. However, these rats are difficult to breed due to high neonatal death and behavioral issues, causing commercial breeders to stop production. To address this, we developed a mouse line with tamoxifen-inducible deletion of Avp. We used CRISPR/Cas9 to insert loxP sites into the Avp gene. These mice were then bred with mice expressing a tamoxifen-inducible Cre recombinase. The resulting conditional knockout mice (Avp[flx/flx]Cre[+]) are viable, fertile, and healthy before induction. Administration of tamoxifen in 8-12-wk-old mice successfully deleted Avp, as confirmed by Sanger sequencing. This deletion caused a significant decrease in urine osmolality, a hallmark of AVP deficiency. The kidney structure remained normal, with no signs of medullary atrophy. In addition, these mice exhibited a substantially decreased expression of the aquaporin 2 water channel (AQP2), which is involved in water reabsorption in the kidney inner medulla. We illustrate the use of this model by using RNA-seq to profile the consequences of Avp deletion on gene expression in the kidney. The curated RNA-seq data can be browsed, searched, or downloaded at https://esbl.nhlbi.nih.gov/Databases/AVP-KO/. In conclusion, we successfully created an inducible Avp knockout mouse line that has been made available to the research community. This model will be valuable for studying water balance regulation, polycystic kidney disease, and the neural, vascular, and metabolic functions of vasopressin.NEW &amp; NOTEWORTHY We developed an inducible Avp knockout mouse line that will be shared with the research community and is likely to be useful for further study of the regulation of water balance and polycystic kidney disease, as well as neural, vascular, and metabolic roles of vasopressin.},
}
@article {pmid41241097,
year = {2025},
author = {Hołubowicz, R and Gao, F and Du, SW and Menezes, CR and Zhang, J and Hołubowicz, MW and Chen, PZ and Armbrust, N and Geilenkeuser, J and Liu, DR and Jeffery Truong, DJ and Westmeyer, GG and Palczewska, G and Palczewski, K},
title = {Scalable purification enables high-quality virus-like particles for therapeutic translation.},
journal = {The Journal of biological chemistry},
volume = {},
number = {},
pages = {110946},
doi = {10.1016/j.jbc.2025.110946},
pmid = {41241097},
issn = {1083-351X},
abstract = {Emerging molecular therapies introduce enzymatic activity into cells by delivering genes, transcripts, or proteins. Owing to their robust cell-entry capacity, virus-like particles (VLPs) represent a technology of choice in genome editing, where low doses of heterologous proteins and nucleic acids are essential. However, clinical translation of VLP vectors is hindered by inadequate purification methods. Current approaches, relying primarily on ultracentrifugation, suffer from inconsistent product quality and poor scalability. Here, we report the development of a broadly applicable purification strategy that improves the purity and therapeutic efficacy of genome-editing VLPs. Considering the characteristic properties of murine leukemia virus (MLV)-derived engineered VLPs (eVLPs) and HIV-derived engineered nucleocytosolic vehicles for loading of programmable editors (ENVLPEs+), we developed a workflow that involves single- and multi-modal chromatographic steps, effectively removing host cell proteins and cell-culture contaminants while improving VLP integrity and biological activity. Our purified VLPs displayed superior protein composition, consistency, and enhanced functional delivery compared to VLPs partially purified by conventional ultracentrifugation methods. Mass spectrometric analysis revealed a substantial decrease in contaminants, with VLP-specific proteins comprising >90% of the final product. In vivo studies confirmed improved therapeutic outcomes when chromatographically purified VLPs were used. Our scalable purification platform addresses critical manufacturing bottlenecks and constitutes a starting point for further development of VLP therapeutics, enabling robust production of pure VLPs for diverse applications such as genome editing, vaccine development, and other uses that require intracellular protein delivery.},
}
@article {pmid41240805,
year = {2025},
author = {Ghoreshi, ZA and Ali-Hassanzadeh, M and Mashayekhi-Sardoo, H and Askarpour, H and Arefinia, N},
title = {The identification of tuberculosis using CRISPR technique: A systematic review and meta-analysis.},
journal = {Diagnostic microbiology and infectious disease},
volume = {114},
number = {2},
pages = {117183},
doi = {10.1016/j.diagmicrobio.2025.117183},
pmid = {41240805},
issn = {1879-0070},
abstract = {BACKGROUND: Rapid and precise detection of Mycobacterium tuberculosis (MTB) is essential for effective management and control of tuberculosis. The diagnostic challenge is particularly acute for extrapulmonary TB, which accounts for approximately 15-20% of cases and often presents with paucibacillary samples. Clustered regularly interspaced short palindromic repeats (CRISPR) technology has emerged as a promising tool for pathogenic diagnosis across diverse sample types owing to its specificity and adaptability. This systematic review and meta-analysis aimed to appraise the diagnostic accuracy of CRISPR-based techniques in identifying MTB.<br><br>METHODS: A comprehensive search was conducted in Medline, Scopus, Embase, and ISI Web of Science to retrieve relevant studies, adhering to PRISMA guidelines. Quality was assessed using the Joanna Briggs comprehensive checklist. Data synthesis and analyses, including subgroup analyses, were performed with Meta-Disc 1.4, examining variables like CRISPR variants, gene targets, pre-amplification techniques, and signal readout methods.<br><br>RESULTS: From 341 identified studies, 13 met the inclusion criteria, encompassing 1,572 MTB strains. The pooled sensitivity and specificity of CRISPR-based techniques were 0.91 (95%CI: 0.89-0.92) and 0.97 (95%CI: 0.95-0.98), respectively. The pooled diagnostic odds ratio was 498.67 (95%CI: 255.1-974.7), with an AUC of 0.99 in the SROC curve, denoting excellent diagnostic accuracy. Subgroup analysis by sample type revealed that sputum samples achieved sensitivity and specificity of 0.92 (95%CI: 0.89-0.94) and 0.97 (95%CI: 0.94-0.99), while extrapulmonary samples (BALF, pus, CSF) showed comparable performance with sensitivity of 0.89 (95%CI: 0.84-0.93) and specificity of 0.98 (95%CI: 0.94-0.99).<br><br>CONCLUSION: CRISPR-based methods exhibit substantial diagnostic sensitivity and specificity for detecting MTB across both pulmonary and extrapulmonary samples, with notable variances across different CRISPR variants and methodological approaches.},
}
@article {pmid41240616,
year = {2025},
author = {Zong, W and Xie, S and Chu, H and Han, S and Zhang, X},
title = {Beyond traditional antibacterial agents: Novel approaches to combat resistant pathogens.},
journal = {European journal of medicinal chemistry},
volume = {302},
number = {Pt 2},
pages = {118362},
doi = {10.1016/j.ejmech.2025.118362},
pmid = {41240616},
issn = {1768-3254},
abstract = {As bacterial infections caused by antibiotic-resistant strains become increasingly prevalent, traditional antibacterial therapies face mounting challenges. These resilient pathogens not only complicate the treatment of common infections but also undermine the efficacy of therapies for major diseases. This growing threat underscores the urgent need for innovative therapeutic strategies. In recent years, the combinatorial use of antibacterial agents has emerged as a promising approach to enhance efficacy and combat resistant bacteria. This review first provides an overview of antibacterial classifications and their mechanisms of action against bacterial infections. It then explores two combined treatment strategies: antibacterial-antibacterial combinations and antibacterial-non-antibacterial pairings, alongside a drug delivery technology: antibacterial-loaded liposomes. Furthermore, we highlight emerging frontiers in antimicrobial strategies, including CRISPR-Cas technologies, AI-driven discovery platforms, nanomaterials beyond liposomes, microbiota-based therapies, and immunotherapeutic approaches. Finally, we offer a forward-looking perspective on the challenges and opportunities shaping the future of antibacterial development in the biomedical field.},
}
@article {pmid41178406,
year = {2025},
author = {Pei, C and Yan, B and Wang, Y and Chen, T and Du, K and Ma, L and Wang, J},
title = {TdT/Cas12a cascade amplification biosensor for sensitive ALP activity detection.},
journal = {The Analyst},
volume = {150},
number = {23},
pages = {5330-5337},
doi = {10.1039/d5an00938c},
pmid = {41178406},
issn = {1364-5528},
mesh = {Humans ; *Biosensing Techniques/methods ; *Alkaline Phosphatase/metabolism/analysis ; HeLa Cells ; CRISPR-Cas Systems ; Limit of Detection ; *DNA Nucleotidylexotransferase/metabolism/chemistry ; *Endodeoxyribonucleases/metabolism/genetics ; *Bacterial Proteins/metabolism/genetics ; *CRISPR-Associated Proteins/metabolism/genetics ; },
abstract = {This study presents a novel biosensor based on TdT and CRISPR-Cas12a, which integrates the catalytic activity of terminal deoxynucleotidyl transferase (TdT) with the trans-cleavage property of CRISPR-Cas12a to achieve ultra-sensitive biomolecular detection. The biosensor exhibited a broad linear detection range from 0 to 0.2 U L[-1] and a remarkably low detection limit of 1.7 × 10[-3] U L[-1], demonstrating high specificity and sensitivity. In practical validation, the biosensor successfully quantified alkaline phosphatase (ALP) activity in both cervical cancer cells and HeLa cell lysates, even at a dilution factor of up to 10[6]-fold. Its sensitivity allowed precise detection at the single-cell level. This technology offers a robust, simple, and cost-effective platform for cancer diagnosis, treatment monitoring, and enzyme inhibitor screening, while maintaining excellent detection performance in complex biological samples. This breakthrough establishes a foundation for serological tumor screening and early disease diagnosis, while also opening new avenues for enhanced cancer management and clinical translation, indicating significant potential in translational medicine.},
}
@article {pmid41171167,
year = {2025},
author = {Tharmatt, A and Guha, S and Kumeria, T and Yadav, S and Mittal, A and Chitkara, D},
title = {Lipopolymeric Nanoplex-Mediated CRISPR/Cas9 Delivery for VEGF-A Knockdown in Psoriatic Angiogenesis.},
journal = {ACS applied bio materials},
volume = {8},
number = {11},
pages = {10137-10157},
doi = {10.1021/acsabm.5c01478},
pmid = {41171167},
issn = {2576-6422},
mesh = {*CRISPR-Cas Systems ; *Psoriasis/drug therapy/metabolism/genetics/pathology ; Humans ; *Vascular Endothelial Growth Factor A/genetics/metabolism/antagonists &amp; inhibitors ; Animals ; *Biocompatible Materials/chemistry/pharmacology/chemical synthesis ; Particle Size ; *Neovascularization, Pathologic/drug therapy/genetics ; Mice ; Materials Testing ; *Polymers/chemistry ; Gene Knockdown Techniques ; *Nanoparticles/chemistry ; Angiogenesis ; },
abstract = {Psoriasis is a chronic, incurable inflammatory skin disease characterized by immune cell infiltration, aberrant keratinocyte differentiation, and enhanced angiogenesis. Overexpression of the vascular endothelial growth factor-A (VEGF-A) gene promotes angiogenesis and is essential for endothelial cell migration, adhesion, and proliferation. Therefore, downregulating VEGF-A represents a promising therapeutic strategy for angiogenesis-related disorders. We investigated the application of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) ribonucleoprotein complexes (sgRNA/eGFP-Cas9 RNPs) targeting VEGF-A in psoriasis. To enable efficient delivery in vitro and in vivo, we developed lipopolymeric nanoplexes (NPXs) encapsulating sgRNA/eGFP-Cas9 RNPs. These NPXs exhibited a particle size of 142.2 nm (polydispersity index: 0.144), a zeta potential of +4.27 mV, and achieved >70% transfection efficiency in HaCaT (human immortalized keratinocyte) cells. Ex vivo skin permeation studies demonstrated 66% of permeation after 24 h. The optimized NPX formulation was incorporated into a Carbopol-based gel, which displayed non-Newtonian, shear-thinning behavior with variable thixotropy and achieved 48% of skin permeation after 24 h. In vivo efficacy assessment in an imiquimod-induced psoriasis model in Swiss albino mice showed significantly improved Psoriasis Area and Severity Index (PASI) scores, reduced epidermal damage, and suppressed keratinocyte proliferation compared to naked RNPs and blank gel controls. Gene editing analysis revealed an indel frequency of 40.7% by T7 endonuclease I assay and 14% by Sanger sequencing. Enhanced cellular uptake, efficient skin permeation and retention, and improved therapeutic efficacy collectively highlight the potential of NPX-mediated CRISPR/Cas9 delivery as a noninvasive strategy for psoriasis treatment.},
}
@article {pmid40982615,
year = {2025},
author = {Kanwal, F and Aslam, A and Torriero, AAJ},
title = {Microalgae-based biodiesel: integrating AI, CRISPR and nanotechnology for sustainable biofuel development.},
journal = {Emerging topics in life sciences},
volume = {8},
number = {3},
pages = {131-143},
pmid = {40982615},
issn = {2397-8554},
mesh = {*Microalgae/metabolism/genetics ; *Biofuels ; *Nanotechnology/methods ; *Artificial Intelligence ; *Clustered Regularly Interspaced Short Palindromic Repeats ; *CRISPR-Cas Systems ; },
abstract = {Microalgae are a promising feedstock for biodiesel due to their rapid growth, high lipid content and ability to use non-arable land and wastewater. This review synthesises recent advances in artificial intelligence (AI)-driven strain optimisation, engineering, nanotechnology-assisted processing, and life cycle and technoeconomic insights to evaluate pathways for industrialisation. Over the past decade (2015-2024), genetic engineering and, more recently, AI-guided strain selection have improved lipid productivity by up to 40%. Cultivation advances, including hybrid photobioreactor-open pond systems and precision pH/CO2 control, have enhanced biomass yields while reducing costs. Innovation in lipid extraction, such as supercritical CO2 and microwave-assisted methods, now achieves >90% yields with lower toxicity, while magnetic nanoparticle-assisted harvesting and electroflocculation have reduced energy inputs by 20-30%. Life cycle analyses (net energy ratio ~2.5) and integration of high-value co-products (e.g. pigments and proteins) underscore the need to align biological innovations with techno-economic feasibility. This review uniquely integrates advances in AI, CRISPR and nanotechnology with life cycle and techno-economic perspectives, providing a comprehensive framework that links laboratory-scale innovation to industrial feasibility and positions microalgal biodiesel as a viable contributor to global decarbonisation strategies.},
}
@article {pmid40447760,
year = {2025},
author = {Hebert, JD and Xu, H and Tang, YJ and Ruiz, PA and Detrick, CR and Wang, J and Hughes, NW and Donosa, O and Siah, VP and Andrejka, L and Karmakar, S and Aboiralor, I and Tang, R and Sotillo, R and Sage, J and Cong, L and Petrov, DA and Winslow, MM},
title = {Efficient and multiplexed somatic genome editing with Cas12a mice.},
journal = {Nature biomedical engineering},
volume = {9},
number = {11},
pages = {1982-1997},
pmid = {40447760},
issn = {2157-846X},
support = {T34FT8013//Tobacco-Related Disease Research Program (TRDRP)/ ; K00 CA234962/CA/NCI NIH HHS/United States ; DGE-2146755//National Science Foundation (NSF)/ ; R01 CA231253/CA/NCI NIH HHS/United States ; R35 HG011316/HG/NHGRI NIH HHS/United States ; R35-CA231997//U.S. Department of Health &amp; Human Services | National Institutes of Health (NIH)/ ; R01 CA234349/CA/NCI NIH HHS/United States ; R01-GM141627//U.S. Department of Health &amp; Human Services | National Institutes of Health (NIH)/ ; R01-CA231253//U.S. Department of Health &amp; Human Services | National Institutes of Health (NIH)/ ; K00CA234962//U.S. Department of Health &amp; Human Services | NIH | National Cancer Institute (NCI)/ ; PF-21-112-01-MM//American Cancer Society (American Cancer Society, Inc.)/ ; P01 CA244114/CA/NCI NIH HHS/United States ; R35-HG011316//U.S. Department of Health &amp; Human Services | National Institutes of Health (NIH)/ ; P30-CA124435//U.S. Department of Health &amp; Human Services | National Institutes of Health (NIH)/ ; R01-CA234349//U.S. Department of Health &amp; Human Services | National Institutes of Health (NIH)/ ; R01 CA230025/CA/NCI NIH HHS/United States ; R01 GM141627/GM/NIGMS NIH HHS/United States ; R35 CA231997/CA/NCI NIH HHS/United States ; P30 CA124435/CA/NCI NIH HHS/United States ; P01-CA244114//U.S. Department of Health &amp; Human Services | National Institutes of Health (NIH)/ ; },
mesh = {Animals ; *Gene Editing/methods ; Mice ; Mice, Transgenic ; *CRISPR-Cas Systems/genetics ; *CRISPR-Associated Proteins/genetics/metabolism ; RNA, Guide, CRISPR-Cas Systems/genetics ; Acidaminococcus/genetics/enzymology ; *Bacterial Proteins/genetics ; Genome ; *Endodeoxyribonucleases/genetics ; Genotype ; },
abstract = {Somatic genome editing in mouse models has increased our understanding of the in vivo effects of genetic alterations. However, existing models have a limited ability to create multiple targeted edits, hindering our understanding of complex genetic interactions. Here we generate transgenic mice with Cre-regulated and constitutive expression of enhanced Acidaminococcus sp. Cas12a (enAsCas12a), which robustly generates compound genotypes, including diverse cancers driven by inactivation of trios of tumour suppressor genes or an oncogenic translocation. We integrate these modular CRISPR RNA (crRNA) arrays with clonal barcoding to quantify the size and number of tumours with each array, as well as the impact of varying the guide number and position within a four-guide array. Finally, we generate tumours with inactivation of all combinations of nine tumour suppressor genes and find that the fitness of triple-knockout genotypes is largely explainable by one- and two-gene effects. These Cas12a alleles will enable further rapid creation of disease models and high-throughput investigation of coincident genomic alterations in vivo.},
}
@article {pmid41240306,
year = {2026},
author = {Harada, A},
title = {Golgi Units as Modules in the Ribbon.},
journal = {Sub-cellular biochemistry},
volume = {110},
number = {},
pages = {35-42},
pmid = {41240306},
issn = {0306-0225},
mesh = {*Golgi Apparatus/metabolism/ultrastructure/genetics ; Glycosylation ; Humans ; Glycosaminoglycans/metabolism/biosynthesis ; CRISPR-Cas Systems ; HeLa Cells ; Animals ; },
abstract = {Using super-resolution microscopies, SCLIM and STORM, and CRISPR/Cas9 knockin technology, we show that the Golgi complex is assembled by a number of small "Golgi units" that are 1-3 μm in diameter. Each Golgi unit can be considered a functional and morphological module because it contains all sets of glycosylation enzymes that occupy small domains named "zones." Golgi units change shape dynamically. They attach, detach, fuse, and separate from each other. The zones of glycosylation enzymes rapidly move near the rim of the unit. However, the behaviors of N- and O-linked glycosylation enzyme zones differ from those of glycosaminoglycan (GAG)-synthesizing enzyme zones in localization and dynamics. Since depletion of giantin dissociates the Golgi units, it is responsible for their attachment. Giantin depletion also prevents the movement of GAG-synthesizing enzymes between units, leading to incomplete GAG synthesis. Here, we present the model of the Golgi and its glycosylation enzymes that can explain a number of characteristics of the glycosylation processes.},
}
@article {pmid41240134,
year = {2025},
author = {Gong, J and Soleimani Samarkhazan, H and Siavashi, M and Servatian, N and Pirsavabi, F},
title = {CRISPR-Cas9 in leukemia immunotherapy: precision engineering of CAR-T cells and tumor-microenvironment modulation.},
journal = {Molecular biology reports},
volume = {53},
number = {1},
pages = {90},
pmid = {41240134},
issn = {1573-4978},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Leukemia/therapy/immunology/genetics ; Gene Editing/methods ; Receptors, Chimeric Antigen/genetics/immunology ; *Tumor Microenvironment/immunology/genetics ; *Immunotherapy, Adoptive/methods ; Precision Medicine/methods ; *Immunotherapy/methods ; T-Lymphocytes/immunology ; Animals ; },
abstract = {Together with base editors, prime editors, and alternative nuclease platforms, CRISPR-Cas9 technology has transformed the field of genetic engineering by providing unprecedented precision in genome editing and creating new opportunities for therapeutic interventions. This technology comprises a versatile genome-editing toolkit for achieving a variety of therapeutic objectives. In the context of leukemia, a group of life-threatening hematologic malignancies, CRISPR-Cas9 has emerged as a transformative tool for immunotherapy. By enabling targeted modifications of immune cells, such as T cells, this technology enhances their ability to recognize and eradicate leukemic cells. CRISPR-Cas9 facilitates the disruption of immune checkpoint inhibitors, the insertion of chimeric antigen receptors (CARs), and the correction of genetic mutations that drive leukemia progression. These advancements have led to the development of more potent and personalized immunotherapies, such as CAR-T cell therapies, with improved efficacy and reduced off-target effects. Moreover, CRISPR-Cas9 allows researchers to model leukemia in vitro, providing deeper insights into disease mechanisms and accelerating the discovery of novel therapeutic targets. Despite challenges such as delivery efficiency and potential immunogenicity, the integration of CRISPR-Cas9 into leukemia immunotherapy represents a paradigm shift, offering hope for durable remissions and potentially curative outcomes. As clinical trials progress, this technology promises to redefine the standard of care for leukemia patients, ushering in a new era of precision medicine. This narrative review explores the revolutionary applications of CRISPR-Cas9 in redefining therapeutic strategies for leukemia.},
}
@article {pmid41240131,
year = {2025},
author = {Behera, L and Samal, KC and Mishra, A and Sahoo, JP and Dash, M and Mishra, A},
title = {CRISPR-driven innovations in rice (Oryza sativa L.) breeding: precision development of male sterile lines.},
journal = {Molecular biology reports},
volume = {53},
number = {1},
pages = {89},
pmid = {41240131},
issn = {1573-4978},
mesh = {*Oryza/genetics ; *Plant Infertility/genetics ; *Plant Breeding/methods ; Gene Editing/methods ; Pollen/genetics ; *CRISPR-Cas Systems/genetics ; },
abstract = {Male sterility is a vital trait exploited in hybrid seed production to boost crop yield and improve quality. In rice, different male sterility systems have been developed, significantly advancing the production of high-yielding hybrids. The three main types of male sterility in rice are cytoplasmic male sterility (CMS), photoperiod-sensitive genic male sterility (PGMS), and genic male sterility (GMS). Among these, CMS is the most widely used, arising from interactions between mitochondrial (cytoplasmic) and nuclear genes, resulting in pollen dysfunction. PGMS, on the other hand, is influenced by environmental cues such as day length and temperature, while GMS is attributed to mutations in specific nuclear genes affecting anther or pollen development. A thorough understanding of the genetic and molecular mechanisms underlying these systems is essential for efficient hybrid rice breeding. CMS lines are typically crossed with maintainer and restorer lines carrying fertility-restoring genes to produce fertile F1 hybrids. Recent advancements in molecular biology, genomics, and genome editing technologies have accelerated the development of novel male-sterile and fertility-restoring lines, thereby enhancing the precision and scalability of hybrid breeding programs. These innovations are not only expanding the genetic base of hybrid rice but also making the production process more sustainable. As global food demand rises alongside climate uncertainties, the strategic use of male sterility in rice breeding holds immense potential for improving agricultural productivity.},
}
@article {pmid41239469,
year = {2025},
author = {Ma, J and Zhang, J and Guo, X},
title = {Harnessing CRISPR-Cas9 for Lactobacillus improvement in silage production: current knowledge and future perspectives.},
journal = {Journal of animal science and biotechnology},
volume = {16},
number = {1},
pages = {150},
pmid = {41239469},
issn = {1674-9782},
support = {U20A2002//National Natural Science Foundation of China/ ; },
abstract = {High-quality silage is the cornerstone to sustainable livestock development and animal food production. As the core fermentation bacteria of silage, Lactobacillus directly regulates silage fermentation by producing lactic acid, enzymes, and other bioactive molecules. However, traditional screening methods for functional strains are labor-intensive and time-consuming. Recent advances in synthetic biology, particularly the development of CRISPR-Cas genome editing technology, offer a revolutionary approach to designing Lactobacillus strains with customized traits. This review systematically reviewed the importance of silage in sustainable agricultural development and the limitations of current silage preparation and promotion. It also discussed the application of strain engineering approaches in optimizing the phenotypic performance of Lactobacillus for better silage. Building on this, we reviewed the research progress of CRISPR-Cas9 gene editing in Lactobacillus and discussed how to leverage its high efficiency and precision to optimize the strain's traits for improved silage quality and functionality. CRISPR-Cas9 toolkits are expected to achieve directed evolution of strain performance, ultimately yielding next-generation silage microbial inoculants with multiple functions, adaptability to multiple substrates, and eco-friendly characteristics. The use of such innovative biotechnologies would facilitate resource-efficient utilization, promote animal performance and health for sustainable development in livestock production.},
}
@article {pmid41239088,
year = {2025},
author = {Xu, H and Chen, H and Li, Y and Wang, Y and Zeng, H and Xu, Z and Chen, T and Kong, D and Huang, W and Cheng, H and Zhou, H and Jiang, X and Feng, J},
title = {CRISPR/Cas12a-SERS biosensor based on sea urchin-like AuNPs for the detection of β-thalassemia mutant gene CD31.},
journal = {Mikrochimica acta},
volume = {192},
number = {12},
pages = {810},
pmid = {41239088},
issn = {1436-5073},
mesh = {*Biosensing Techniques/methods ; *beta-Thalassemia/genetics/diagnosis ; *CRISPR-Cas Systems ; Animals ; *Spectrum Analysis, Raman/methods ; *Gold/chemistry ; *Metal Nanoparticles/chemistry ; Mutation ; Sea Urchins/chemistry ; Limit of Detection ; Humans ; *Endodeoxyribonucleases/genetics/metabolism ; DNA, Single-Stranded/chemistry/genetics ; *CRISPR-Associated Proteins/metabolism/genetics ; Sulfhydryl Compounds/chemistry ; Bacterial Proteins ; Benzoates ; },
abstract = {Beta-thalassemia is a single-gene recessive disorder caused by mutations in the HBB gene, and approximately 1.5% of the global population are carriers of β-thalassemia. It is therefore vital to establish a rapid and sensitive method to detect the mutant genes of β-thalassemia. In this study, a CRISPR/Cas12a-mediated amplification-free surface-enhanced Raman spectroscopy (SERS) biosensor was developed. This biosensor uses sea urchin-shaped gold nanoparticles (SUGNPs) as the SERS enhancement substrate and 4-mercaptobenzoic acid (4-MBA) as the Raman reporter. It couples the SUGNPs/4-MBA with magnetic beads through single-strand DNA (ssDNA) to form an SERS probe with magnetic responsiveness. The presence of the β-thalassemia target mutation gene CD31 activates the cleavage activity of Cas12a, leading to non-specific cleavage of single-stranded DNA (ssDNA) on the probe. This results in a significant reduction in SERS intensity. This signal change enables quantitative detection of the target gene, thereby significantly enhancing the sensitivity of nucleic acid testing. We employed magnetic separation technology to enrich target nucleic acids in serum while eliminating matrix interference, enabling specific recognition and quantitative detection of the mutated CD31 gene. This method exhibits excellent linearity over a concentration range 0.1 fM to 10 pM, with a detection limit of 0.1 fM and a detection time of only 40 min. Compared to traditional qPCR and other CRISPR methods, this approach is simple, rapid, and offers advantages such as high sensitivity, high specificity, and cost-effectiveness. By simply replacing the crRNA, it can detect multiple β-thalassemia and other disease genes, demonstrating broad clinical application potential.},
}
@article {pmid41236708,
year = {2025},
author = {Zahedi, S},
title = {CRISPR-Based Functional Genomics in Pluripotent Stem Cells.},
journal = {Stem cell reviews and reports},
volume = {},
number = {},
pages = {},
pmid = {41236708},
issn = {2629-3277},
abstract = {The integration of CRISPR-based functional genomics with pluripotent stem cell (PSC) technologies has been recognized as a transformative approach for investigating gene function, modeling human disease, and advancing regenerative medicine. The aim of this review is to provide a comprehensive evaluation of recent developments in CRISPR-Cas platforms, including gene knockouts, base and prime editing, and CRISPR activation or interference (CRISPRa/i), as applied to PSC systems. Studies employing human PSCs, including embryonic stem cells and induced pluripotent stem cells, have been examined to summarize methodologies for genome-wide screening, lineage tracing, and therapeutic engineering. Advances in editing efficiency, delivery strategies, and genomic safety have been reported, while limitations persist in the form of off-target modifications, epigenetic variability, and cell-type-specific responses. Notable applications include the generation of immune-evasive PSC lines, the development of organoid models for physiological and pathological studies, and the implementation of phenotypic screening for disease-relevant traits. Collectively, these technological and methodological advancements have established functional genomics of PSC-CRISPRSPR as a versatile and powerful framework for elucidating fundamental aspects of human biology, dissecting complex traits, and accelerating the translation of discoveries from experimental research to clinical implementation.},
}
@article {pmid41130353,
year = {2026},
author = {Lee, SY and Kim, HS and Kim, YG and Shin, S and Kweon, S and Lee, JJ and Lee, GM},
title = {Genome-wide CRISPR screening identifies genes in recombinant human embryonic kidney 293 cells for increased ammonia resistance.},
journal = {Metabolic engineering},
volume = {93},
number = {},
pages = {184-193},
doi = {10.1016/j.ymben.2025.10.008},
pmid = {41130353},
issn = {1096-7184},
mesh = {Humans ; *Ammonia/pharmacology/metabolism ; HEK293 Cells ; *CRISPR-Cas Systems ; *Antibodies, Monoclonal/genetics/biosynthesis ; Gene Knockout Techniques ; *Clustered Regularly Interspaced Short Palindromic Repeats ; *Genome, Human ; },
abstract = {Ammonia, a byproduct of glutamine metabolism, inhibits cell growth and reduces product yield and quality in mammalian cell culture. To identify novel genes associated with ammonia resistance, a genome-wide CRISPR knockout screening was conducted in monoclonal antibody (mAb)-producing human embryonic kidney 293 (HEK-mAb) cells using a virus-free, recombinase-mediated cassette exchange-based gRNA interrogation method. The knockout cell library was subcultured for five consecutive passages under 20 mM NH4Cl, enriching cells with a sgRNA that conferred a proliferation advantage under high-ammonia conditions. Next-generation sequencing analysis of the enriched population identified three target genes -WNT3, TSPAN1, and CYHR1-among 19,114 genes. Knockout of these genes in HEK-mAb cells resulted in a 1.33- to 1.56-fold increase in maximum viable cell concentration and a 1.28- to 1.58-fold increase in maximum mAb concentration under 20 mM NH4Cl. Notably, WNT3 knockout maintained N-glycan galactosylation proportions of mAb despite ammonia stress. These findings highlight the effectiveness of genome-wide CRISPR knockout screening in identifying novel gene targets for ammonia-resistant HEK293 cell, offering a promising strategy for improving mAb production.},
}
@article {pmid41110569,
year = {2025},
author = {Cui, Z and Liang, W and Li, J and Bai, Z},
title = {CRISPR/Cas9 gene editing strategy for cancer therapy: non-viral nanocarrier-mediated delivery of plasmids, RNA and ribonucleoprotein complexes.},
journal = {International journal of biological macromolecules},
volume = {331},
number = {Pt 1},
pages = {148389},
doi = {10.1016/j.ijbiomac.2025.148389},
pmid = {41110569},
issn = {1879-0003},
mesh = {*CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; Humans ; *Ribonucleoproteins/genetics ; *Neoplasms/therapy/genetics ; *Plasmids/genetics ; Animals ; *RNA/genetics ; *Gene Transfer Techniques ; Genetic Therapy/methods ; Nanoparticles/chemistry ; RNA, Guide, CRISPR-Cas Systems/genetics ; },
abstract = {Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR-associated protein 9 (Cas9) gene editing systems are among the most prevalent tools in modern genetic research and have been extensively studied in the context of cancer therapy. CRISPR/Cas9 systems can be categorized into three main delivery forms: plasmid DNA (pDNA) containing both Cas9 and guide RNA (sgRNA), messenger RNA (mRNA) of Cas9 along with sgRNA, and ribonucleoprotein (RNP) complexes consisting of Cas9 protein bound to sgRNA. This article will review the various CRISPR/Cas9 systems, focusing on the non-viral especially nanocarrier delivery strategies and common protocols used for their delivery and the challenges encountered during this process. This review will offer guidance on the potential applications of CRISPR/Cas9 gene editing technology for the treatment of malignant tumors in the future.},
}
@article {pmid41093194,
year = {2025},
author = {Yang, G and Rao, Q and Dong, W and Yin, X and Shen, R and Wang, R and Deng, X and Peng, X and Tao, Y and Li, S and Wang, X and Tang, Y and Du, D},
title = {Rapid and sensitive one-pot CRISPR-Cas12a detection platform for common fungal species in ocular infections using multiplex crRNAs.},
journal = {International journal of biological macromolecules},
volume = {331},
number = {Pt 1},
pages = {148279},
doi = {10.1016/j.ijbiomac.2025.148279},
pmid = {41093194},
issn = {1879-0003},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Fungi/genetics/isolation &amp; purification ; *Eye Infections, Fungal/microbiology/diagnosis/genetics ; DNA, Fungal/genetics ; },
abstract = {Corneal fungal infection is a major cause of visual impairment and can cause blindness if not diagnosed promptly. Traditional diagnostic methods are often time-consuming and require specialized expertise, UNDERSCORING the urgent need for rapid, sensitive, and user-friendly diagnostic tools. In this study, we utilized multiplex crRNAs in the Cas12a system to develop a highly sensitive and specific platform for the molecular detection of common fungal species causing corneal infections in a single reaction. We enhanced detection sensitivity through recombinase-aided amplification (RAA) and designed a streamlined tube with insert one-pot protocol to minimize aerosol contamination risk. Remarkably, our platform achieved a limit of detection of just 4 copies of fungal genomic DNA per reaction in only 32 min. It allows for direct detection from simulated tear samples, providing a potential for convenient and non-invasive testing option. Furthermore, our platform effectively tests clinical samples such as aqueous humor (AH) and vitreous humor (VH), demonstrating superior sensitivity compared to traditional PCR-based methods. These results highlight the potential of our platform as an on-site diagnostic solution for both clinical and non-clinical settings, contributing to the preservation of visual function.},
}
@article {pmid41043753,
year = {2025},
author = {Yang, Q and Zhao, H and Zhai, Y and Wang, Q and Zhu, Z and Rui, C and Yuan, H and Cui, L},
title = {Ryanodine receptor I4734M mutation confers diamide resistance but reduces invasiveness in Spodoptera frugiperda.},
journal = {International journal of biological macromolecules},
volume = {331},
number = {Pt 2},
pages = {148059},
doi = {10.1016/j.ijbiomac.2025.148059},
pmid = {41043753},
issn = {1879-0003},
mesh = {Animals ; *Spodoptera/genetics/drug effects ; *Ryanodine Receptor Calcium Release Channel/genetics ; *Mutation ; *Diamide/pharmacology ; *Insecticide Resistance/genetics ; ortho-Aminobenzoates/pharmacology ; Insecticides/pharmacology ; CRISPR-Cas Systems ; Gene Editing ; Pyrazoles ; },
abstract = {Although Spodoptera frugiperda populations with the ryanodine receptor (RyR) I4734M mutation occurred in their native regions, this mutation has not been detected in the Eastern Hemisphere. In order to clarify the functional role of this mutation, the first HDR-mediated RyR[I4734M] mutation was introduced into S. frugiperda using CRISPR/Cas9 technology and a homozygous SfRyR[I4734M] strain was successfully established. Meanwhile, a novel, non-invasive genotyping method based on insect fecal DNA to efficiently identify genome-edited individuals was developed. Compared with the wild type, the genome-edited SfRyR[I4734M] strain showing high-level resistance to chlorantraniliprole (396.7-fold) and tetraniliprole (149.1-fold), and moderate resistance to cyantraniliprole (32.3-fold) and flubendiamide (29.5-fold). Reciprocal crossing experiments indicated that resistance to chlorantraniliprole was inherited in an autosomally incompletely recessive mode. Furthermore, the SfRyR[I4734M] substitution adversely reduced the fitness and flight ability of S. frugiperda. The mutant strain exhibited significantly decreased fecundity and severely impaired flight distance and velocity. These findings provided in vivo genetic validation of SfRyR[I4734M] mutation in diamide resistance, demonstrating this mutation conferred subtle differences on the binding affinities of four diamides. Moreover, our results firstly demonstrated that SfRyR[I4734M] mutation could affect muscle function, thereby reducing the flight ability of S. frugiperda.},
}
@article {pmid41236489,
year = {2025},
author = {Wang, Y and Kong, Y and Zhang, Y and He, Y and Wang, W and Lu, Y and Meng, J and Yuan, H},
title = {Long-Term Attenuation of Vascular Hyperpermeability in a Hereditary Angioedema Mouse Model by Adenine Base Editing.},
journal = {Allergy},
volume = {},
number = {},
pages = {},
doi = {10.1111/all.70152},
pmid = {41236489},
issn = {1398-9995},
support = {//the National Natural Science Foundation of China/ ; //West China Hospital, Sichuan University/ ; },
abstract = {BACKGROUND: Hereditary angioedema (HAE) is a rare and potentially life-threatening disorder caused by dysregulated kallikrein-kinin signaling and bradykinin-induced vascular hyperpermeability. Current therapies targeting this pathway are effective but require lifelong administration, underscoring the need for durable and potentially curative interventions. Adenine base editors (ABEs), engineered from CRISPR/Cas systems, enable precise single-nucleotide modifications with minimal genomic disruption, offering a promising strategy for long-term gene silencing.<br><br>METHODS: NG-ABE8e was delivered via AAV8 or lipid nanoparticles (LNP) to disrupt the exon 2 splice donor site of Klkb1 in Serping1[+/-] mice. Editing outcomes were quantified by high-throughput sequencing; serum kallikrein levels were measured by ELISA; and vascular permeability was evaluated using Evans blue dye extravasation, fluorescent tracer leakage, and VE-cadherin immunostaining. Safety evaluations included off-target analysis, histopathology, serum biochemistry, activated partial thromboplastin time (aPTT), and systemic hemodynamic stability.<br><br>RESULTS: AAV8-NG-ABE8e induced >&#x2009;60% A&#x2022;T-to-G&#x2022;C conversion at the target site, promoting exon 2 skipping and reducing Klkb1 mRNA and serum kallikrein levels by >&#x2009;85%, an effect sustained for at least 1&#x2009;year. AAV8-NG-ABE8e also reversed bradykinin-driven vascular hyperpermeability and attenuated inflammatory gene signatures. Prolongation of aPTT was observed only when circulating kallikrein levels declined below 2&#x2009;&#x3bc;g/mL. Similarly, LNP-mediated delivery of NG-ABE8e mRNA resulted in >&#x2009;90% reductions in Klkb1 expression and serum kallikrein levels. No evidence of significant long-term toxicity was detected.<br><br>CONCLUSION: These findings demonstrate that, in a murine model, NG-ABE8e-mediated disruption of Klkb1 enables durable suppression of serum kallikrein and vascular stabilization, suggesting its potential as a promising single-intervention strategy for the treatment of HAE.},
}
@article {pmid41236435,
year = {2025},
author = {Chen, Y and Wang, Y and Liu, L and Yu, X and Zhang, Y and Xi, M and Xu, J and Yang, H and Xie, C and Wang, D},
title = {Targeting BnNAC038 improves drought tolerance with low yield penalty in Brassica napus.},
journal = {The Plant journal : for cell and molecular biology},
volume = {124},
number = {3},
pages = {e70571},
doi = {10.1111/tpj.70571},
pmid = {41236435},
issn = {1365-313X},
support = {241111112400//Key Research and Development Project of Henan Province/ ; 2023-YBNY-023//Key Research and Development Project of Shaanxi Province/ ; 32070324//National Natural Science Foundation of China/ ; 32301811//National Natural Science Foundation of China/ ; 2024YFD1200400//the National Key Research and Development Project from the Ministry of Agriculture and Rural Affairs of China/ ; },
mesh = {*Brassica napus/genetics/physiology/growth &amp; development ; Droughts ; *Plant Proteins/genetics/metabolism/physiology ; Gene Expression Regulation, Plant ; Photosynthesis/genetics ; CRISPR-Cas Systems ; Drought Resistance ; },
abstract = {Drought stress severely limits crop productivity in Brassica napus, yet strategies to enhance drought tolerance without compromising yield remain elusive. Here, we identify BnNAC038 as a negative regulator of drought responses in Brassica napus. CRISPR/Cas9-generated bnnac038 mutants exhibited improved drought survival, reduced water loss, and enhanced stomatal closure under drought conditions compared to wild-type (WT) plants. RNA-sequencing (RNA-seq) and DNA affinity purification sequencing (DAP-seq) analyses revealed that BnNAC038 directly represses drought-responsive genes, including BnSnRK2.6 (a key ABA signaling kinase), and genes involved in photosynthesis (BnPPC2) and gluconeogenesis (BnPGK). Field trials demonstrated that bnnac038 plants exhibit enhanced photosynthesis, accumulate more sucrose and glucose under drought, and exhibit increased biomass and seed yield compared to WT. Genetic interaction studies further showed that BnSnRK2.6 acts downstream of BnNAC038 to mediate drought tolerance. Our results indicate that targeted editing of BnNAC038 enhances drought tolerance while minimizing yield loss, providing a new strategy for developing drought-resilient Brassica napus varieties with minimal yield penalty.},
}
@article {pmid41236144,
year = {2025},
author = {Apsley, EJ and Riepsaame, J and Cheng, YC and Cowley, SA and Becker, EBE},
title = {Cerebellar organoids model cell type-specific FOXP2 expression during human cerebellar development.},
journal = {Disease models &amp; mechanisms},
volume = {18},
number = {11},
pages = {},
doi = {10.1242/dmm.052290},
pmid = {41236144},
issn = {1754-8411},
support = {BB/M011224/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; //UK Research and Innovation/ ; },
mesh = {Humans ; *Forkhead Transcription Factors/metabolism/genetics ; *Organoids/metabolism ; *Cerebellum/metabolism/growth &amp; development/embryology/cytology ; Induced Pluripotent Stem Cells/metabolism/cytology ; Purkinje Cells/metabolism ; Gene Expression Regulation, Developmental ; Cell Differentiation/genetics ; *Models, Biological ; CRISPR-Cas Systems/genetics ; },
abstract = {Human cerebellar development is unique and cannot be fully replicated in animal models. Although human stem cell-derived cerebellar organoid models are increasingly being applied to model cerebellar diseases, their potential to provide insight into normal human cerebellar development remains underexplored. Here, we used CRISPR-based gene editing in cerebellar organoids as an approach for modelling specific features of early human cerebellar development. Forkhead box protein P2 (FOXP2) is a transcription factor associated with speech and language development that is highly expressed in the developing brain. However, little attention has been directed to the study of FOXP2 in the early developing cerebellum. We generated a fluorescent FOXP2 reporter line in human induced pluripotent stem cells to enable the characterisation of FOXP2-expressing cells during cerebellar organoid differentiation. Through transcriptomic profiling of FOXP2 reporter cerebellar organoids and cross-referencing with existing cerebellar datasets, we describe the expression and identify potential downstream targets of FOXP2 in the early developing human cerebellum. Our results highlight expression of FOXP2 in early human Purkinje cells and cerebellar nuclei neurons, and the vulnerability of these cell populations to neurodevelopmental disorders.},
}
@article {pmid41234307,
year = {2025},
author = {Tuncel, A and Kim, HU and Kim, MC},
title = {Editorial: Engineering future crops through genome editing.},
journal = {Frontiers in plant science},
volume = {16},
number = {},
pages = {1720325},
pmid = {41234307},
issn = {1664-462X},
}
@article {pmid41233858,
year = {2025},
author = {Mayer, J and Pack, M and Montenarh, M and Götz, C},
title = {Gene expression changes in pancreatic α-cell lines following knock-out Of either CK2α or CK2α'.},
journal = {Biological research},
volume = {58},
number = {1},
pages = {69},
pmid = {41233858},
issn = {0717-6287},
mesh = {*Casein Kinase II/genetics/metabolism ; *Glucagon-Secreting Cells/metabolism ; Animals ; Mice ; Cell Line ; Gene Knockout Techniques ; *Gene Expression/genetics ; },
abstract = {BACKGROUND: Protein kinase CK2 is known to exist as a tetramer of two catalytic α- or α'- subunits and two non-catalytic β-subunits, or as multimers of this tetramer. Moreover, CK2α (CSNK2A1) and CK2α' (CSNK2A2) are also active in the absence of CK2β (CSNK2B). Very little is known about specific functions of the individual subunits of protein kinase CK2.<br><br>RESULTS: In order to study the effects of CK2&#x3b1; and CK2&#x3b1;' on gene expression, we used the Mus musculus pancreatic &#x3b1;-cell line &#x3b1;TC1 and two derivatives with either CK2&#x3b1; (KO1 cells) or CK2&#x3b1;' (KO2 cells) expression knocked-out by CRISPR/Cas technology. We found numerous genes deregulated in both KO1 and KO2 cells compared to the parental cells. Applying stringent thresholds, 266 genes were found down-regulated and 153 genes up-regulated in KO1 cells, 233 genes were found down-regulated and 84 genes up-regulated in KO2 cells. Dozens of genes were found deregulated in a similar fashion in both KO1 and KO2 cells. We found altered expression of genes involved in the differentiation of pancreatic cells, including Hox genes, and in the regulation of glucagon synthesis or secretion. Moreover, many of the deregulated genes play an important role in developmental processes and in neuronal cell biology.<br><br>CONCLUSION: Our findings reveal individual and shared functions of the CK2&#x3b1; and CK2&#x3b1;' catalytic subunits, in particular regarding their involvement in regulating gene expression.},
}
@article {pmid41232012,
year = {2025},
author = {Maire, A and Bikard, D},
title = {Programmable DNA insertion in native gut bacteria.},
journal = {Science (New York, N.Y.)},
volume = {390},
number = {6774},
pages = {676-677},
doi = {10.1126/science.aec3823},
pmid = {41232012},
issn = {1095-9203},
mesh = {Animals ; *Gastrointestinal Microbiome/genetics ; Mice ; *Gene Editing/methods ; CRISPR-Cas Systems ; *Bacteria/genetics ; *Mutagenesis, Insertional ; DNA, Bacterial/genetics ; },
abstract = {A gene-editing approach enables modification of bacteria within the mouse gut.},
}
@article {pmid41231980,
year = {2025},
author = {Gelsinger, DR and Ronda, C and Ma, J and Kar, OB and Edwards, M and Huang, Y and Mavros, CF and Sun, Y and Perdue, T and Vo, PL and Ivanov, II and Sternberg, SH and Wang, HH},
title = {Metagenomic editing of commensal bacteria in vivo using CRISPR-associated transposases.},
journal = {Science (New York, N.Y.)},
volume = {390},
number = {6774},
pages = {eadx7604},
doi = {10.1126/science.adx7604},
pmid = {41231980},
issn = {1095-9203},
mesh = {Animals ; Mice ; *Gastrointestinal Microbiome/genetics ; *Gene Editing/methods ; *Bacteroides/genetics/growth &amp; development ; Humans ; Metagenomics/methods ; *CRISPR-Cas Systems ; Symbiosis ; Mice, Inbred C57BL ; Metagenome ; Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {Although metagenomic sequencing has revealed a rich microbial biodiversity in the mammalian gut, methods to genetically alter specific species in the microbiome are highly limited. Here, we introduce Metagenomic Editing (MetaEdit) as a platform technology for microbiome engineering that uses optimized CRISPR-associated transposases delivered by a broadly conjugative vector to directly modify diverse native commensal bacteria from mice and humans with new pathways at single-nucleotide genomic resolution. Using MetaEdit, we achieved in vivo genetic capture of native murine Bacteroides by integrating a metabolic payload that enables tunable growth control in the mammalian gut with dietary inulin. We further show in vivo editing of segmented filamentous bacteria, an immunomodulatory small-intestinal microbial species recalcitrant to cultivation. Collectively, this work provides a paradigm to precisely manipulate individual bacteria in native communities across gigabases of their metagenomic repertoire.},
}
@article {pmid41231855,
year = {2025},
author = {Liu, S and Bai, J and Zhan, B and Yao, J and Zhang, J and Yi, J and Dong, M and Li, Q and Shen, Y and Chen, Y and Zhao, Y},
title = {Development of an RNA aptamer-assisted CRISPR/Cas9 system for efficiently generating and isolating Cas9-free mutants in plant.},
journal = {PLoS genetics},
volume = {21},
number = {11},
pages = {e1011931},
pmid = {41231855},
issn = {1553-7404},
mesh = {*CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; *Aptamers, Nucleotide/genetics ; Plants, Genetically Modified/genetics ; Mutation ; *Arabidopsis/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; },
abstract = {The CRISPR/Cas9 gene-editing system is a powerful tool in plant genetic engineering; however, screening for Cas9-free edited plants remains complex and time-consuming. To address this limitation, we developed an RNA aptamer-assisted CRISPR/Cas9 system, termed 3WJ-4 × Bro/Cas9. In this system, the engineered RNA aptamer 3WJ-4 × Bro functions as a transcriptional reporter, serving as an alternative to traditional fluorescent proteins and thus avoiding their potential interference with Cas9 activity. Compared to the conventional GFP/Cas9 system, 3WJ-4 × Bro/Cas9 showed more efficient transformation and higher accuracy in fluorescence-based selection of positive T1 transformants, without significantly affecting plant growth. Furthermore, 3WJ-4 × Bro/Cas9 achieved a 78.6% increase in the T1 mutation rate compared to GFP/Cas9, with the homozygous mutation rate reaching 1.78%. In addition, 3WJ-4 × Bro/Cas9 enabled fluorescence-based visual screening in the T2 generation for rapid identification of Cas9-free mutants, improving sorting efficiency by 30.2% over the GFP-based method. Moreover, 3WJ-4 × Bro/Cas9 enabled more efficient generation of homozygous double-target mutants compared to GFP/Cas9. These results demonstrate that the 3WJ-4 × Bro/Cas9 system provides a non-transgenic, efficient, and broadly applicable strategy for plant genome editing and selection.},
}
@article {pmid40958664,
year = {2025},
author = {Yang, L and Zhao, Z and Lei, R and Zhang, Y and Wu, P},
title = {A one-pot RPA/CRISPR-bio-dCas9 lateral flow assay for rapid and on-site detection of Monilinia fructicola in stone and pome fruits.},
journal = {Pest management science},
volume = {81},
number = {12},
pages = {8564-8574},
doi = {10.1002/ps.70159},
pmid = {40958664},
issn = {1526-4998},
support = {//the National Key Research and Development Program of China (2021YFC2600400)/ ; //the Basic Scientific Research Foundation of the Chinese Academy of Quality and Inspection &amp; Testing (2024JK051)/ ; },
mesh = {*Plant Diseases/microbiology ; *Fruit/microbiology ; *Ascomycota/isolation &amp; purification/genetics ; *Nucleic Acid Amplification Techniques/methods ; CRISPR-Cas Systems ; },
abstract = {BACKGROUND: Brown rot, a devastating fungal disease affecting stone and pome fruits, leads to substantial economic losses worldwide, impacting production, post-harvest storage, and transportation. Monilinia fructicola, the primary causal agent of brown rot, is especially challenging to manage because of its ability to establish latent infections in fruit, making early detection and control extremely difficult, enabling the disease to spread unnoticed. Therefore, it is essential to develop fast and accurate detection technologies.<br><br>RESULTS: In this study, a one-pot detection method for M. fructicola, integrating CRISPR-bio-dCas9, recombinase polymerase amplification (RPA), and a lateral flow assay (LFA) was developed. This method could detect the M. fruticola genome in less than 30&#x2009;min from sample collection to result, with a detection limit of 4 copies/&#x3bc;L of M. fructicola. The RPA/CRISPR-bio-dCas9 LFA method simplified M. fructicola detection by eliminating the need for probes, additional reporters, or specialized equipment, thereby reducing costs and complexity. Furthermore, the streamlined single-tube workflow minimized cross-contamination risks, enabling non-expert workers to perform efficient pathogen screening.<br><br>CONCLUSIONS: The RPA/CRISPR-bio-dCas9 LFA enabled advanced brown rot management by addressing the limitations of traditional detection (slow speed, high cost, complexity). With high sensitivity and a short detection time using an equipment-free design, it enabled practical on-site detection of M. fructicola, allowing timely interventions to reduce pre- and post-harvest losses in fruit production. &#xa9; 2025 Society of Chemical Industry.},
}
@article {pmid40810834,
year = {2025},
author = {Xu, M and Chu, J and Li, M and Ren, X and Chen, X and Li, X and Cheng, H and Wang, C and Yang, F},
title = {A Streamlined One-Step Bioprocess for Isomaltulose Production in Bacillus subtilis Through Multicopy Genomic Integration of Sucrose Isomerase Gene.},
journal = {Applied biochemistry and biotechnology},
volume = {197},
number = {10},
pages = {6820-6833},
pmid = {40810834},
issn = {1559-0291},
support = {GXTZYKF202406//Opening Project of Guangxi Key Laboratory of Green Processing of Sugar Resources/ ; 2024-MSLH-037//Liaoning Provincial Science and Technology Program Joint Program/ ; 202410152013//Dalian Polytechnic University College Student Innovation and Entrepreneurship Training Program Project/ ; },
mesh = {*Bacillus subtilis/genetics/metabolism/enzymology ; *Isomaltose/analogs &amp; derivatives/biosynthesis ; *Glucosyltransferases/genetics/metabolism ; CRISPR-Cas Systems ; },
abstract = {Isomaltulose, a sucrose isomer with a low glycemic index and non-cariogenic properties, is extensively used in the food industry. The industrial production of this functional sugar relies on enzymatic biotransformation using sucrose isomerase (SIase). However, conventional bioprocesses involve expressing and isolating the SIase enzyme, followed by using the purified SIase to convert sucrose into isomaltulose, resulting in a multi-step and high-cost process that hindered the broader applications of isomaltulose. In this study, we reported a streamlined one-step bioprocess that integrates extracellular SIase secretion and direct isomaltulose biosynthesis in the culture medium of an engineered B. subtilis strain. Using CRISPR/Cas9 technology, we engineered B. subtilis to integrate multiple SIase expression cassettes into the genome while concurrently replacing genes within the sacP-sacA-ywdA and sacB-levB-yveA operons, which are crucial for sucrose hydrolysis in B. subtilis. This strategy synergistically increased the genomic copy number of SIase gene while limited sucrose consumption by native pathways, thereby maximizing substrate availability for SIase-mediated catalysis. The resulting engineered strain, containing four copies of the SIase expression cassettes, achieved an extracellular SIase activity of 8.2 U/mL in shake flasks. When cultured in a medium containing 200 g/L sucrose, this strain produced a maximum isomaltulose titer of 162.1 g/L with a yield of 0.81 g/g and a productivity of 13.5 g/L/h. These findings demonstrate an integrated bioprocess that eliminates costly enzyme isolation procedure and reduces fermentation complexity, presenting a commercially feasible strategy for sustainable isomaltulose production.},
}
@article {pmid40759871,
year = {2025},
author = {Luo, X and Su, B and Lai, P and Li, M and Deng, MR and Zhu, H},
title = {Development of an NADPH Regeneration System for L-threonine Production in Escherichia coli.},
journal = {Applied biochemistry and biotechnology},
volume = {197},
number = {10},
pages = {6575-6591},
pmid = {40759871},
issn = {1559-0291},
support = {2021YFC2100900//National Key Research and Development Program of China/ ; 2021JC06N628//Guangdong Special Support Program/ ; 2022GDASZH-2022010101//GDAS' Project of Science and Technology Development/ ; },
mesh = {*NADP/metabolism ; *Escherichia coli/metabolism/genetics ; *Threonine/biosynthesis ; Escherichia coli Proteins/genetics/metabolism ; CRISPR-Cas Systems ; },
abstract = {NADPH is essential for the biosynthesis of L-threonine, and a deficiency in its supply significantly constrains L-threonine production. To address the challenge of inadequate NADPH availability that adversely affects L-threonine synthesis, we developed an NADPH regeneration system aimed at enhancing the NADPH supply and subsequently improving L-threonine production. Through overexpression of the zwf and gnd genes, which are involved in NADPH generation within the pentose phosphate pathway (PPP), the NADPH/NADP[+] ratio in the strain was elevated 4.1-fold compared with the control strain, resulting in a 2.0-fold increase in L-threonine production. Subsequently, integration of the asd and thrA1034 genes, which are linked to NADPH consumption, enhanced L-threonine production by 3.6-fold. Moreover, the application of promoter engineering facilitated a 7.1-fold increase in L-threonine production compared with the control strain. Finally, we employed the CRISPR-Cas12f1 system to delete the pgi gene to further examine its impact on L-threonine production. The results indicated an increase in the NADPH/NADP[+] ratio and a subsequent enhancement in L-threonine production following deletion of the pgi gene. Consequently, the NADPH regeneration system developed in this study demonstrates potential to effectively improve L-threonine production and may serve as a novel strategy for L-threonine synthesis.},
}
@article {pmid40751408,
year = {2025},
author = {Bao, Z and Chang, X and Cheng, L and Lin, W and Xu, W and Shi, J},
title = {A highly specific and ultrasensitive approach to detect Hylurgus ligniperda based on RPA-CRISPR-LbaCas12a-LFD system.},
journal = {Pest management science},
volume = {81},
number = {12},
pages = {7874-7884},
doi = {10.1002/ps.70099},
pmid = {40751408},
issn = {1526-4998},
support = {2023YFE0116200//National Key Research and Development Program of China/ ; 2023YFC2605200//National Key Research and Development Program of China/ ; },
mesh = {Animals ; *CRISPR-Cas Systems ; *Weevils/genetics/classification ; *Nucleic Acid Amplification Techniques/methods ; Sensitivity and Specificity ; China ; Recombinases ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {BACKGROUND: Hylurgus ligniperda is an invasive bark beetle that poses a serious threat to global coniferous forests and the timber trade. Its broad host range, high reproductive potential, and strong environmental adaptability enable it to establish and spread rapidly in newly invaded regions. In October 2020, H. ligniperda was first reported in Shandong Province, China. Developing a rapid, sensitive, and accurate field detection method is critical for early interception and effective management.<br><br>RESULTS: We developed a detection method for H. ligniperda based on recombinase polymerase amplification (RPA) coupled with CRISPR/Cas12a, with results monitored via fluorescence signals and lateral flow dipstick (LFD). The mitochondrial COI gene was selected as the target sequence, and key parameters-including incubation time, temperature, and concentrations of Cas12a protein and CRISPR RNA (crRNA)-were optimized. The RPA-CRISPR-LbaCas12a-LFD assay exhibited high specificity and sensitivity, successfully distinguishing H. ligniperda from five closely related species, and detecting target DNA at concentrations as low as 1 copy per &#x3bc;L. Finally, The field applicability of the detection system was validated using samples from global geographic populations.<br><br>CONCLUSION: This study establishes a portable, rapid, and sensitive visual detection system for H. ligniperda based on RPA-CRISPR-LbaCas12a-LFD, suitable for both laboratory and field applications. The method enables field detection without the need for specialized equipment, offering a robust tool for invasive pest surveillance, port quarantine, and early warning. &#xa9; 2025 Society of Chemical Industry.},
}
@article {pmid41231321,
year = {2025},
author = {Kumari, S and Keshari, AK and Singh, SK and Pandey, S and Singh, A},
title = {CRISPR-Based genome editing in pulses: current approaches, challenges, and future prospects.},
journal = {Plant molecular biology},
volume = {115},
number = {6},
pages = {126},
pmid = {41231321},
issn = {1573-5028},
mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; *Genome, Plant/genetics ; *Crops, Agricultural/genetics ; *Fabaceae/genetics ; Plant Breeding/methods ; Plants, Genetically Modified/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Genetic Engineering/methods ; },
abstract = {Legumes are the second most important food crop after cereals for the world population. It is a significant protein source for developing countries and integral to global food security. However, various agroecological constraints and biotic and abiotic factors often compromise the production of pulses. Legumes are long-term neglected crops worldwide and follow traditional breeding, leading to a time-consuming, labor-intensive, less economically feasible program associated with linkage drag. Recent sequencing attempts in the twenty-first century, with the development of an enormous repertoire of genetic and genomic resources, allowed scientists to accelerate the improvement of legumes with modern genome editing tools. One such promising tool is CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats), which has revolutionized and transformed the landscape of genetic engineering. The emergence of CRISPR/Cas systems has redefined precision breeding, offering unprecedented control over genome manipulation in legume crops. It has tremendous potential for crop improvement and can precisely make changes at genomic locations with incredible accuracy. Therefore, identifying the desired genes and their precise manipulation has enormous implications for legume crop improvement. This review will give an overview of the genome editing tools available for crop improvement and the efficiency of different transformation methods in legume crops. It will also discuss the current status of genome editing in legume crops, including challenges and future perspectives.},
}
@article {pmid41230228,
year = {2025},
author = {Park, S and Mani, V and Ha, K and Kim, JA and Lee, S},
title = {Plant synthetic biology: from knowledge to biomolecules.},
journal = {Frontiers in plant science},
volume = {16},
number = {},
pages = {1562216},
pmid = {41230228},
issn = {1664-462X},
abstract = {Plant synthetic biology is rapidly emerging as an innovative approach to solving complex problems in human health and agriculture. Although conventional metabolic engineering primarily focuses on microbial systems for large-scale biomolecules production, these platforms often face limitations in expressing plant-derived enzymes and synthesizing structurally complex molecules. In contrast, recent advances in plant synthetic biology have integrated multidisciplinary tools, from molecular biology and biochemistry to synthetic circuit design and computational modeling, to engineer plant systems with enhanced traits. These include improved yield, nutritional quality, environmental resilience, and synthesis of pharmaceutically relevant functional biomolecules. This review focuses on the fundamental technologies that have enabled such advances, which include DNA synthesis, programmable gene circuits, and CRISPR/Cas-based genome editing. We discussed recent applications of reprogramming plant metabolic pathways and existing obstacles, such as transformation efficiency, regulatory bottlenecks, and pathway instability. This review provides key case studies and a forward-looking perspective on the evolution of plant synthetic biology as a robust foundation for sustainable biomanufacturing and production of functional biomolecules.},
}
@article {pmid41229344,
year = {2025},
author = {Sures, K and Esser, SP and Bornemann, TLV and Moore, CJ and Soares, AR and Plewka, J and Figueroa-Gonzalez, PA and Ruff, SE and Moraru, C and Probst, AJ},
title = {Acquisition of Spacers from Foreign Prokaryotic Genomes by CRISPR-Cas Systems in Natural Environments.},
journal = {Genome biology and evolution},
volume = {17},
number = {11},
pages = {},
pmid = {41229344},
issn = {1759-6653},
mesh = {*CRISPR-Cas Systems ; *Genome, Archaeal ; *Archaea/genetics ; Genome, Bacterial ; *Clustered Regularly Interspaced Short Palindromic Repeats ; *Bacteria/genetics ; Metagenome ; Evolution, Molecular ; *DNA, Intergenic/genetics ; },
abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) systems of bacteria and archaea provide immunities against mobile genetic elements, like viruses. In addition, protospacer analyses revealed a very specific acquisition of CRISPR spacers derived from genomes of related species or from closely interacting episymbiont genomes as recently shown for subsurface archaea. However, the origin of most of the spacers that can be found in CRISPR-Cas systems from natural environments has not been deciphered. Here, by analyzing CRISPR-Cas systems of metagenome-assembled genomes (MAGs) from two subsurface environments spanning more than 1 Tb of sequencing data, we show that a substantial proportion of CRISPR spacers are acquired from DNA of other prokaryotes inhabiting the same environment. As such, we found that the number of respective spacers can be up to three times higher than the number of self-targeting spacers. Statistical analyses demonstrated that the acquisition of CRISPR spacers from other prokaryotic genomes is partly explained by the relative abundance of the MAG containing the protospacer, as well as by other factors, such as the total number of CRISPR arrays present in a MAG with the respective spacers. Further, we found that spacer acquisition from foreign prokaryotic DNA occurs in almost all types of CRISPR-Cas systems, but shows preferences for subtypes of CRISPR-Cas systems that differ across the investigated ecosystems. Taken together, our results shed new light on the diversity of CRISPR spacers in natural microbial communities and provide an explanation for some of the many unmatched spacers in public databases.},
}
@article {pmid41229123,
year = {2025},
author = {Ma, Y and Wei, W and Yang, Z and Zhou, Y and Dong, T and Wang, T and Xia, X and Ma, Y and Zhou, M and Gao, Y and Yu, B and Wang, C and Ruan, L and Hong, K and Gu, C},
title = {Exosomes as Nonviral Carrier for Targeted Delivery of CRISPR/Cas12a for Therapeutic HIV-1Proviral DNA Editing.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.ymthe.2025.11.012},
pmid = {41229123},
issn = {1525-0024},
abstract = {Current strategies to treat HIV infection including traditional cART and immunotherapy can effectively suppress viral replication but unable to eliminate the latent viral reservoir, particularly within circulating immune cells. Although genome editing by CRISPR-Cas provides a promising cure for HIV-1, gene delivery efficiency in vivo remains an obstacle to overcome. Here, we developed an exosome-mediated targeted CRISPR-Cas12a delivery system (EMT-Cas12a), an engineered exosome system enabling targeted delivery of mRNA of Cas12a and crRNAs to CD4+ T cells. The EMT-Cas12a system uniquely optimizes cell-specific targeting, CRISPR-Cas12a expression, crRNAs maturation, nuclear entry efficiency, accuracy cleavage with major Delins and achieving dramatically HIV suppression in both cellular and humanized mouse models. Compared to single-crRNA approaches, the multiple crRNA arrays strategy demonstrates enhanced antiviral efficacy in HIV -infected mouse model, ex vivo expanded PBMCs from HIV+ subjects and especially in vitro cell line without detectable HIV DNA. Critically, the system exhibits no detectable off-target effects and restores CD4+ T cell counts in vivo and ex vivo PBMCs, indicating its dual therapeutic potential for viral clearance and immune reconstitution. Altogether, in vitro and in vivo excision of HIV-1 proviral DNA can be achieved via EMT-Cas12a delivery, which could advance efforts toward human clinical trials.},
}
@article {pmid41139833,
year = {2025},
author = {Neo, DM and Ben-Zion, I and Bagnall, J and Solomon, MY and Bond, AN and Gath, E and Zhang, S and Shoresh, N and Gomez, J and Hung, DT},
title = {A Multiplexed, Target-Based Phenotypic Screening Platform Using CRISPR Interference in Mycobacterium abscessus.},
journal = {ACS infectious diseases},
volume = {11},
number = {11},
pages = {3263-3275},
doi = {10.1021/acsinfecdis.5c00623},
pmid = {41139833},
issn = {2373-8227},
mesh = {*Mycobacterium abscessus/genetics/drug effects ; Microbial Sensitivity Tests ; Mycobacterium Infections, Nontuberculous/microbiology/drug therapy ; *Anti-Bacterial Agents/pharmacology ; Drug Discovery/methods ; Bacterial Proteins/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats ; *CRISPR-Cas Systems ; Phenotype ; Humans ; },
abstract = {The rise of difficult-to-treat Mycobacterium abscessus infections presents a growing clinical challenge due to the immense arsenal of intrinsic, inducible and acquired antibiotic resistance mechanisms that render many existing antibiotics ineffective against this pathogen. Moreover, the limited success in discovery of novel compounds that inhibit novel pathways underscores the need for innovative drug discovery strategies. Here, we report a strategic advancement in PROSPECT (PRimary screening Of Strains to Prioritize Expanded Chemistry and Targets), which is an antimicrobial discovery strategy that measures chemical-genetic interactions between small molecules and a pool of bacterial mutants, each depleted of a different essential protein target, to identify whole-cell active compounds with high sensitivity. Applying this modified strategy to M. abscessus, in contrast to previously described versions of PROSPECT which utilized protein degradation or promoter replacement strategies for generating engineered hypomorphic strains, here we leveraged CRISPR interference (CRISPRi) to more efficiently generate mutants each depleted of a different essential gene involved in cell wall synthesis or located at the bacterial surface. We applied this platform to perform a pooled PROSPECT pilot screen of a library of 782 compounds using CRISPRi guides as mutant barcodes. We identified a range of active hits, including compounds targeting InhA, a well-known mycobacterial target but under-explored in the M. abscessus space. The unexpected susceptibility to isoniazid, traditionally considered to be ineffective in M. abscessus, suggested a complex interplay of several intrinsic resistance mechanisms. While further complementary efforts will be needed to change the landscape of therapeutic options for M. abscessus, we propose that PROSPECT with CRISPRi engineering provides an increasingly accessible, high-throughput target-based phenotypic screening platform and thus represents an important step toward accelerating early stage drug discovery.},
}
@article {pmid41086891,
year = {2025},
author = {Sun, J and Huang, Y and Bai, J and Wang, J and Langford, PR and Zhang, Y and Li, G},
title = {A CRISPR/Cas12a-based DNAzyme chemiluminescence platform for rapid detection of all Streptococcus suis and individually S. suis serotypes 7 and 9.},
journal = {International journal of biological macromolecules},
volume = {330},
number = {Pt 4},
pages = {148280},
doi = {10.1016/j.ijbiomac.2025.148280},
pmid = {41086891},
issn = {1879-0003},
mesh = {*Streptococcus suis/genetics/isolation &amp; purification/classification ; *DNA, Catalytic/metabolism/genetics ; Serogroup ; *CRISPR-Cas Systems/genetics ; *Luminescent Measurements/methods ; Swine ; Animals ; Biosensing Techniques/methods ; Luminescence ; *Endodeoxyribonucleases/genetics ; Bacterial Proteins ; CRISPR-Associated Proteins ; },
abstract = {Streptococcus suis is a zoonotic pathogen that can infect pigs and humans with causing meningitis, sepsis, endocarditis, and arthritis. S. suis serotypes 7 and 9 cause substantial economic losses to the swine industry and pose a major threat to public health, thus, accurate and rapid detection is important for the prevention and control of epidemic disease. In this study, we developed a platform, combining recombinase polymerase amplification (RPA) with a CRISPR/Cas12a detection system to rapidly detect all S. suis serotypes and individually differentiates serotypes 7 and 9. This was achieved by targeting recN of S. suis and serotype-specific cpsH genes of serotypes 7 and 9, respectively. Both fluorescence and G4 DNAzyme chemiluminescence visualization biosensing methods had high specificity and sensitivity, no cross-reaction was found with common pig pathogens, closely related Streptococcus spp., or other S. suis serotypes. Compared to traditional identification techniques, these two methods are rapid and convenient. Notably, the G4 DNAzyme chemiluminescence method provides a clear, direct visual interpretation of results without the need for specialized equipment, which is particularly advantageous for point-of-care testing. Thus, this platform has the potential to significantly enhance diagnostic capabilities and ultimately benefit both animal and public health.},
}
@article {pmid40865049,
year = {2025},
author = {Shang, Y and Li, S and Xu, T and Sun, Y and Li, F},
title = {An Optimized Platform Overcomes Excessive Tumor Immune Rejection Induced by the CRISPR/Cas9 Lentiviral System.},
journal = {Cancer research},
volume = {85},
number = {22},
pages = {4315-4328},
doi = {10.1158/0008-5472.CAN-25-0809},
pmid = {40865049},
issn = {1538-7445},
support = {2022YFA1103900//National Natural Science Foundation of China (NSFC)/ ; 82372794//National Natural Science Foundation of China (NSFC)/ ; 82172744//National Natural Science Foundation of China (NSFC)/ ; GZB20240544//China Postdoctoral Science Foundation (China Postdoctoral Foundation Project)/ ; 2024M752432//China Postdoctoral Science Foundation (China Postdoctoral Foundation Project)/ ; 24ZR1469100//Natural Science Foundation of Shanghai Municipality ()/ ; //Shanghai Municipal Education Commission ()/ ; },
mesh = {*CRISPR-Cas Systems ; Animals ; *Lentivirus/genetics ; Mice ; Humans ; Gene Knockout Techniques/methods ; Integrases/genetics ; *Neoplasms/immunology/genetics/therapy ; Genetic Vectors/genetics ; Adenoviridae/genetics ; Cell Line, Tumor ; Female ; },
abstract = {UNLABELLED: The clustered regularly interspaced short palindromic repeats/Cas9 lentiviral system has emerged as a powerful tool for gene knockout in cancer immunology research. However, the persistent expression of exogenous elements, such as Cas9 and resistance markers, often triggers excessive tumor immune rejection. This can lead to prolonged experimental timelines, increased data variability, biased outcomes, and even experimental failures. To address this challenge, several strategies have been investigated that offer partial solutions, but they have failed so far to comprehensively resolve the issue. In response, we developed the v2-Blast-lox2272 (VL)-adenovirus expressing Cre recombinase (AdCre) system, a strategy that enables efficient excision of exogenous expression elements following gene knockout. The VL-AdCre system effectively reduced tumor immune rejection in allograft models, streamlined experimental workflows, and improved the reliability of research outcomes. Overall, this optimized clustered regularly interspaced short palindromic repeats/Cas9 lentiviral gene knockout system offers a robust and practical solution for studying gene functions in vivo and advancing immunotherapeutic strategies.<br><br>SIGNIFICANCE: The VL-AdCre system enables effective removal of exogenous components following gene knockout, thus avoiding tumor immune rejection and enhancing the accuracy and reproducibility of in vivo cancer immunology research.},
}
@article {pmid41226739,
year = {2025},
author = {Arana, &#xc1;J and Veiga-Rua, S and Cora, D and Gónzalez-Gómez, MA and Seijas, A and Carballeda, M and Polo, D and Cuesta, A and Piñeiro, Y and Rivas, J and Novo, M and Al-Soufi, W and Martínez, P and Sánchez, L and Robledo, D},
title = {Comparative Analysis of CRISPR/Cas9 Delivery Methods in Marine Teleost Cell Lines.},
journal = {International journal of molecular sciences},
volume = {26},
number = {21},
pages = {},
doi = {10.3390/ijms262110703},
pmid = {41226739},
issn = {1422-0067},
support = {CRISPR-NANOXEN//Universidade de Santiago de Compostela/ ; Optimizing CRISPR/Cas9 genome editing to improve disease resistance in aquaculture//Fundaci&#xf3;n Caixa Rural Galega Tom&#xe1;s Notario Vacas/ ; investment line n&#xba; 1 and component number 17, which includes the Complementary RTDI Plan for Marine Science//Spain's Recovery and Resilience Plan/ ; No 101076432&#x202f;(FishTRIM)//European Union ERC Starting Grant programme 2022/ ; (BBS/E/20002172, BBS/E/D/30002275, BBS/E/D/10002070 and BBS/E/RL/230002A)//Oportunius programme of the Axencia Galega the Innovaci&#xf3;n (GAIN, Xunta de Galicia)/ ; RYC2023-044793-I//Ministerio de Ciencia, Innovaci&#xf3;n y Universidades/ ; 10.13039/501100011033//Agencia Estatal de Investigaci&#xf3;n/ ; },
mesh = {Animals ; *CRISPR-Cas Systems ; *Gene Editing/methods ; Cell Line ; Electroporation/methods ; *Fishes/genetics ; Transfection/methods ; *Gene Transfer Techniques ; },
abstract = {Gene editing technologies such as CRISPR/Cas9 have revolutionized functional genomics, yet their application in marine fish cell lines remains limited by inefficient delivery. This study compares three delivery strategies-electroporation, lipid nanoparticles (LNPs), and magnetofection using gelatin-coated superparamagnetic iron oxide nanoparticles (SPIONs)-for CRISPR/Cas9-mediated editing of the ifi27l2a gene in DLB-1 and SaB-1 cell lines. We evaluated transfection and editing efficiency, intracellular Cas9 localization, and genomic stability of the target locus. Electroporation achieved up to 95% editing in SaB-1 under optimized conditions, but only 30% in DLB-1, which exhibited locus-specific genomic rearrangements. Diversa LNPs enabled intracellular delivery and moderate editing (~25%) in DLB-1 but yielded only minimal editing in SaB-1, while SPION-based magnetofection resulted in efficient uptake but no detectable editing, highlighting post-entry barriers. Confocal imaging and fluorescence correlation spectroscopy suggested that nuclear localization and Cas9 aggregation may influence editing success, highlighting the importance of intracellular trafficking in CRISPR/Cas9 delivery. Our findings demonstrate that CRISPR/Cas9 delivery efficiency is cell line-dependent and governed by intracellular trafficking and genomic integrity. These insights provide a practical framework for optimizing gene editing in marine teleosts, advancing both basic research and selective breeding in aquaculture.},
}
@article {pmid41226719,
year = {2025},
author = {Amiri, S and Adibzadeh, S and Khazaei Monfared, Y and Kaboli, S and Arashkia, A and Barkhordari, F and Mahmoudian, M and Kheirandish, MH and Trotta, F and Davami, F},
title = {Efficient Delivery of CRISPR-Cas9 RNP Complexes with Cyclodextrin-Based Nanosponges for Enhanced Genome Editing: TILD-CRISPR Integration.},
journal = {International journal of molecular sciences},
volume = {26},
number = {21},
pages = {},
doi = {10.3390/ijms262110682},
pmid = {41226719},
issn = {1422-0067},
support = {BD-66002183//Pasteur Institute of Iran/ ; },
mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; Animals ; *Cyclodextrins/chemistry ; CHO Cells ; Cricetulus ; *Ribonucleoproteins/genetics ; Green Fluorescent Proteins/genetics ; *Nanostructures/chemistry ; },
abstract = {The CRISPR-Cas9 system has transformed biomedical research by enabling precise genetic modifications. However, efficient delivery of CRISPR components remains a major hurdle for therapeutic applications. To address this, we employed a new modified cationic hyper-branched cyclodextrin-based polymer (Ppoly) system to deliver an integrating GFP gene using the TILD-CRISPR method, which couples donor DNA linearization with RNP complexes. The physicochemical properties, loading efficiency, and cellular uptake of RNP with Ppoly were studied. After transfection, antibiotic selection and single-cell cloning were performed. Junction PCR was then performed on the isolated clones, and we compared the knock-in efficiency of Ppoly with that of the commercial CRISPRMAX™ reagent (Thermo Fisher, Invitrogen™, Waltham, MA, USA). The results demonstrate the encapsulation efficiency of over 90% for RNP and Ppoly, and cell viability remaining above 80%, reflecting the minimal toxicity of this approach. These attributes facilitated successful GFP gene integration using the TILD-CRISPR with RNP delivered via cyclodextrin-based nanosponges. The present method achieved a remarkable 50% integration efficiency in CHO-K1 cells, significantly outperforming the 14% observed with CRISPRMAX™ while maintaining lower cytotoxicity. This study highlights a promising platform for precise and efficient genome editing, with strong potential for therapeutic and regenerative medicine applications.},
}
@article {pmid41226716,
year = {2025},
author = {Zhao, R and Zhu, J and Wang, J and Wang, D and Liu, X and Han, L and Li, S},
title = {Functional Characterization of Fp2Cas9, a Cold-Adapted Type II-C CRISPR Nuclease from Flavobacterium psychrophilum.},
journal = {International journal of molecular sciences},
volume = {26},
number = {21},
pages = {},
doi = {10.3390/ijms262110681},
pmid = {41226716},
issn = {1422-0067},
support = {2023YFD2400300//National Key Research and Development Program of China/ ; 2024XT01, 2023TD45//the Central Public Interest Scientific Institution Basal Research Fund, CAFS/ ; Identification, Evaluation, and Breeding of Characteristic Aquatic Animal Germplasm Resources in Heilongjiang Province.//Heilongjiang Province Seed Industry Innovation and Development Fund Project/ ; },
mesh = {*Flavobacterium/enzymology/genetics ; Zebrafish/genetics ; Gene Editing/methods ; *CRISPR-Cas Systems ; Animals ; Cold Temperature ; *CRISPR-Associated Protein 9/metabolism/genetics ; *Bacterial Proteins/genetics/metabolism ; },
abstract = {Cas9 with specialized temperature adaptations are essential for broadening the application of CRISPR-based genome editing across diverse biological contexts. Although Cas9 orthologs from thermophilic and mesophilic organisms have been characterized for high- and moderate-temperature applications, cold-active variants remain largely unexplored, limiting genome engineering in low-temperature systems such as aquaculture species. Here, we report the functional characterization of Fp2Cas9, a cold-adapted Type II-C nuclease from Flavobacterium psychrophilum. In vitro assays showed that Fp2Cas9 efficiently cleaves double-stranded DNA with a refined PAM requirement of 5'-SNAAAG-3', and that its engineered sgRNA scaffold (sgRNA-V2) supports programmable DNA targeting. Notably, Fp2Cas9 retains 75% cleavage efficiency at 5 °C, approximately 2.5-fold higher than SpCas9 under the same conditions, but shows a marked reduction in activity at 35 °C. In vivo, a nuclear-localized variant (2NLS-Fp2Cas9) mediated efficient mutagenesis of the zebrafish slc45a2 gene, yielding ~60% indel frequencies and pigmentation-deficient phenotypes in ~43% of injected embryos. Collectively, these findings establish Fp2Cas9 as a cold-adapted Cas9 with reliable activity at low temperatures. This work adds a valuable tool to the CRISPR-Cas9 toolkit and may facilitate genome editing in cold-water organisms and other low-temperature systems.},
}
@article {pmid41226505,
year = {2025},
author = {Qian, Y and Liu, X and Wang, B and Li, D and Wu, Z and Tong, J},
title = {Genome-Wide Identification of the LsaPHR1 Gene Family and Preliminary Functional Validation of LsaPHR1.1 in Phosphorus Tolerance in Lactuca sativa.},
journal = {International journal of molecular sciences},
volume = {26},
number = {21},
pages = {},
doi = {10.3390/ijms262110466},
pmid = {41226505},
issn = {1422-0067},
support = {KJCX20240508//Special Innovation Ability Construction Fund of Beijing Academy of Agricultural and Forestry Sciences/ ; QNJJ202248//Beijing Academy of Agriculture and Forestry Science Youth Research Fund/ ; KYCX202506//Innovation and Development Program of Beijing Vegetable Research Center/ ; CARS-24-B-02//China Agriculture Research System of the MOF and MARA/ ; KJCX20200205//Project for Scientific and Technological Innovation Capability Building of the Beijing Academy of Agriculture and Forestry Sciences/ ; },
mesh = {*Lactuca/genetics/metabolism ; *Phosphorus/metabolism ; *Plant Proteins/genetics/metabolism ; Gene Expression Regulation, Plant ; Stress, Physiological/genetics ; *Multigene Family ; CRISPR-Cas Systems ; Chlorophyll/metabolism ; Gene Editing ; },
abstract = {Phosphorus (P) is a limiting nutrient for plant growth and productivity. Improving P use efficiency is important for crop production. In Lactuca sativa (lettuce), five phosphate starvation response 1 (PHR1) genes were identified and characterized through a bioinformatics approach. The expression patterns of LsaPHR1s were examined using qRT-PCR under various treatments, including devoid phosphorus (DP), low phosphorus (LP), high phosphorus (HP), darkness, ABA, IAA, and MeJA. The results indicate that LsaPHR1s in lettuce responded to phosphorus stress, hormones, and darkness. Furthermore, we engineered LsaPHR1.1 knock-out mutants via CRISPR/Cas9-mediated genome editing. Then, the mutants were subjected to phosphorus stress (DP, LP, and HP). In contrast to WT, the mutants improved nitrate and ammonium contents, increased antioxidant enzyme activity, and elevated antioxidant and chlorophyll contents. Our results offer a potential strategy for improving phosphorus stress tolerance in lettuce, which holds great significance for maintaining yield and quality.},
}
@article {pmid41225502,
year = {2025},
author = {Huang, S and Zhou, F and Jiang, Z and Jiang, S and Yang, Q and Yang, L and Huang, J and Shi, J and Ding, Y and Li, E and Li, Y},
title = {Development and validation of PmMAD7 for efficient gene editing in Penaeus monodon.},
journal = {BMC biotechnology},
volume = {25},
number = {1},
pages = {125},
pmid = {41225502},
issn = {1472-6750},
support = {2022YFD2401900//National Key R &amp; D Program of China/ ; },
mesh = {Animals ; *Gene Editing/methods ; *Penaeidae/genetics ; *CRISPR-Cas Systems/genetics ; },
abstract = {BACKGROUND: Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-based gene editing has become a promising approach for enhancing traits in aquaculture species. Nevertheless, traditional CRISPR-Cas systems encounter challenges, including significant off-target effects and strict protospacer adjacent motif (PAM) requirements, which constrain their use in crustaceans such as Penaeus monodon. To address these limitations, this research has developed PmMAD7, a codon-optimized CRISPR system specifically designed for P. monodon, which incorporates nuclear localization signals to improve editing efficiency and precision.<br><br>RESULTS: This research successfully synthesized and delivered PmMAD7 mRNA and crRNAs targeting the ECH1 and AQP4 genes into the hemocytes of P. monodon. Quantitative PCR analysis demonstrated that PmMAD7 achieved significant gene silencing, reducing the expression levels of ECH1 and AQP4 by 81.5% and 78.33%, respectively. Next-generation sequencing confirmed targeted insertions and deletions at the gene loci, with knockout efficiencies of 14.81% for ECH1 and 20.57% for AQP4, which were significantly higher than those obtained with LbCas12a (7.14% and 12.43%, respectively). Furthermore, functional analysis indicated that ECH1 knockout resulted in increased cell volume and mortality, while AQP4 knockout led to decreased cell volume and reduced viability. These specific results highlight the first successful demonstration of MAD7-based genome editing in shrimp. The broader PAM compatibility and enhanced editing efficiency of PmMAD7 provide a versatile platform for gene editing in shrimp.<br><br>CONCLUSION: PmMAD7 constitutes an enhanced CRISPR editing tool specifically designed for P. monodon, exhibiting superior precision, expanded PAM compatibility, and enhanced editing efficacy relative to conventional Cas12a systems. These results lay the groundwork for the advancement of gene editing applications in crustaceans and contribute to sustainable genetic improvements in aquaculture.},
}
@article {pmid41223249,
year = {2025},
author = {Pan, X and Huang, P and Ali, SS and Renslo, B and Greenberg, Z and Erwin, N and Li, Y and Ding, Z and Hutchinson, TE and Warnecke, A and Fernandez, NE and Staecker, H and He, M},
title = {Extracellular vesicle-mediated gene editing for the treatment of nonsyndromic progressive hearing loss in adult mice.},
journal = {Science translational medicine},
volume = {17},
number = {824},
pages = {eadn3993},
doi = {10.1126/scitranslmed.adn3993},
pmid = {41223249},
issn = {1946-6242},
mesh = {Animals ; *Extracellular Vesicles/metabolism ; *Gene Editing/methods ; *Hearing Loss/therapy/genetics ; Mice ; CRISPR-Cas Systems/genetics ; Disease Models, Animal ; Genetic Therapy/methods ; Ribonucleoproteins/metabolism ; Hair Cells, Auditory/metabolism ; Electroporation ; Humans ; RNA, Guide, CRISPR-Cas Systems/metabolism ; },
abstract = {The clinical translation of gene therapy has been challenging in part because of the limitations of current delivery approaches. Herein, we report an efficient nonviral genome editor delivery approach using extracellular vesicles (EVs) carrying single-guide RNA (sgRNA): CRISPR-Cas9 ribonucleoprotein (RNP) complexes for in vivo gene therapy. By leveraging a high-throughput microfluidic droplet-based electroporation system (μDES), we achieved a 10-fold enhancement in loading efficiency and more than 1000-fold increase in processing throughput for loading RNP complexes into EVs compared with conventional high-voltage pulsed electroporation. μDES generated uniform microdroplets containing EVs and RNPs by applying direct current-controlled low voltage (up to 60 V) to transiently permeabilize membranes and enable efficient cargo encapsulation while maintaining EV integrity at both the protein and morphological levels. In the Myo7a[WT/Sh1] mouse model of autosomal dominant progressive hearing loss, which may model MYO7A-associated DFNA11 hearing loss in humans, we demonstrated the effective delivery of RNPs by EVs into cochlear hair cells by cross-sectional and whole-mount confocal imaging. The injection of RNP-EVs via the posterior semicircular canal in 4-week-old Myo7a[WT/Sh1] mice resulted in a reduction in Myo7a[Sh1] messenger RNA expression and evidence of hearing preservation, as measured by auditory brainstem responses, compared with untreated ears and EV only-injected mice. This study highlights the potential of μDES-produced RNP-EVs for gene editing as a treatment for progressive nonsyndromic hearing loss in patients.},
}
@article {pmid41223195,
year = {2025},
author = {Kimata, K and Satou, K},
title = {Improved CRISPR/Cas9 off-target prediction with DNABERT and epigenetic features.},
journal = {PloS one},
volume = {20},
number = {11},
pages = {e0335863},
pmid = {41223195},
issn = {1932-6203},
mesh = {*CRISPR-Cas Systems/genetics ; Humans ; *Epigenesis, Genetic ; *Gene Editing/methods ; Genome, Human ; Deep Learning ; },
abstract = {CRISPR/Cas9 is a powerful genome editing tool, but its clinical application is hindered by off-target effects. Accurate computational prediction of these unintended edits is crucial for ensuring the safety and efficacy of therapeutic applications. While various deep learning models have been developed, most are trained only on task-specific data, failing to leverage the vast knowledge embedded in entire genomes. To address this limitation, we introduce a novel approach that integrates DNABERT, a deep learning model pre-trained on the human genome, with epigenetic features (H3K4me3, H3K27ac, and ATAC-seq). We conducted a comprehensive benchmark of our model, DNABERT-Epi, against five state-of-the-art methods across seven distinct off-target datasets. Our results demonstrate that the pre-trained DNABERT-based models achieve competitive or even superior performance. Rigorous ablation studies quantitatively confirmed that both genomic pre-training and the integration of epigenetic features are critical factors that significantly enhance predictive accuracy. Furthermore, by applying advanced interpretability techniques (SHAP and Integrated Gradients), we identified the specific epigenetic marks and sequence-level patterns that influence the model's predictions, offering insights into its decision-making process. This study is the first to establish the significant potential of a pre-trained DNA foundation model for CRISPR/Cas9 off-target prediction. Our findings underscore that leveraging both large-scale genomic knowledge and multi-modal data is a key strategy for advancing the development of safer genome editing tools.},
}
@article {pmid41222688,
year = {2025},
author = {Yuan, A and Sun, T and Deng, Z and Ye, L and Shang, Y and Xie, W and Peng, H},
title = {A multifunctional approach: merging CRISPR/Cas technology with DNA nanomachines for advanced biosensing.},
journal = {Analytical and bioanalytical chemistry},
volume = {},
number = {},
pages = {},
pmid = {41222688},
issn = {1618-2650},
support = {2023YFA0915102//National Key Research and Development Program of China/ ; 22276199//National Natural Science Foundation of China/ ; 22306195//National Natural Science Foundation of China/ ; XDB0750100//Strategic Priority Research Program of the Chinese Academy of Sciences/ ; },
abstract = {CRISPR/Cas systems have revolutionized nucleic acid recognition, offering unparalleled programmability for biosensing. Simultaneously, DNA nanomachines have emerged as powerful bioanalytical tools due to their structural precision, biocompatibility, and multifunctional capabilities. The strategic integration of these platforms synergistically enhances sensitivity, specificity, and multiplexing potential, creating next-generation bioanalytical systems. While promising, this convergence presents unique engineering challenges and necessitates critical evaluation. This review systematically analyzes the target recognition mechanisms of CRISPR/Cas systems and critically evaluates prevalent signal readout modalities. A core focus is the critical assessment of innovative biosensing strategies that leverage DNA nanomachines-particularly dynamic walker systems-coupled with CRISPR/Cas activation. We highlight representative integrated platforms, detailing their operational principles and objectively examining their demonstrated advantages against inherent limitations such as signal leakage, complex assembly, and in vivo applicability constraints. By providing a critical analysis of the synergistic potential and current constraints of CRISPR/Cas-DNA nanomachine integration, this review aims to guide rational design towards robust, clinically translatable precision diagnostics and therapeutics.},
}
@article {pmid41221123,
year = {2025},
author = {van Karnebeek, CDM and Gailus-Durner, V and Engelke, UF and Seisenberger, C and Marschall, S and Dragano, NRV and da Silva-Buttkus, P and Leuchtenberger, S and Fuchs, H and Hrabě de Angelis, M and Wevers, RA and Coughlin, CR and Lefeber, DJ},
title = {New treatment for pyridoxine-dependent epilepsy due to ALDH7A1 deficiency: first proof-of-principle of upstream enzyme inhibition in the mouse.},
journal = {Brain communications},
volume = {7},
number = {6},
pages = {fcaf397},
pmid = {41221123},
issn = {2632-1297},
abstract = {Pyridoxine-dependent epilepsy (PDE) due to recessive ALDH7A1 mutations is characterized by intractable epilepsy that is often unresponsive to antiseizure medications. Irrespective of pyridoxine (vitamin B6) supplementation and lysine reduction therapy, patients present severe residual neurocognitive deficits. We evaluated upstream inhibition of 2-aminoadipic semialdehyde synthase (AASS) as a novel therapeutic strategy to reduce the accumulating metabolites (α-aminoadipic semialdehyde, Δ[1]-piperideine-6-carboxylate, pipecolic acid, 6-oxo-pipecolic acid and 2S,6S-/2s,6R-oxopropylpiperidine-2-carboxylic acid) considered neurotoxic. We utilized an existing mouse knockout model of hyperlysinaemia (Aass-knockout) and generated a PDE model, a Aldh7a1 single knockout model via CRISPR/Cas (clustered regularly interspaced short palindromic repeats and CRISPR-associated protein) and generated the double-knockout Aass/Aldh7a1 mice. Next-generation metabolomics screening was performed to measure all known biomarkers in brain, liver and plasma of wild-type and mutant mice. Metabolomics confirmed the known metabolite markers for Aldh7a1-knockout and Aass knockout mice in all samples. The potentially neurotoxic metabolites (Δ[1]-piperideine-6-carboxylate, pipecolic acid, 6-oxo-pipecolic acid and 2S,6S-/2s,6R-oxopropylpiperidine-2-carboxylic acid) significantly decreased in double-knockout Aass/Aldh7a1 mice brain and liver tissues compared to Aldh7a1-knockout mice. Plasma analysis revealed a significant reduction of known biomarkers, suggesting a reliable monitoring option in human patients. We demonstrate the first mammalian evidence that AASS inhibition is a viable strategy to rescue abnormal brain metabolism associated with PDE. This may target the intellectual disability and neurologic deficits caused by persistent lysine catabolic-related neurotoxicity despite adequate vitamin B6 supplementation.},
}
@article {pmid41220980,
year = {2025},
author = {Yuan, X and Yu, X and Zhao, W and Sun, J},
title = {A Practical CRISPR-Based Method for Rapid Genome Editing in Caulobacter crescentus.},
journal = {Bio-protocol},
volume = {15},
number = {21},
pages = {e5490},
pmid = {41220980},
issn = {2331-8325},
abstract = {The RNA-guided Cas enzyme specifically cuts chromosomes and introduces a targeted double-strand break, facilitating multiple kinds of genome editing, including gene deletion, insertion, and replacement. Caulobacter crescentus and its relatives, such as Agrobacterium fabrum and Sinorhizobium meliloti, have been widely studied for industrial, agricultural, and biomedical applications; however, their genetic manipulations are usually characterized as time-consuming and labor-intensive. C. crescentus and its relatives are known to be CRISPR/Cas-recalcitrant organisms due to intrinsic limitations of SpCas9 expression and possible CRISPR escapes. By fusing a reporting gene to the C terminus of SpCas9M and precisely manipulating the expression of SpCas9M, we developed a CRISPR/SpCas9M-reporting system and achieved efficient genome editing in C. crescentus and relatives. Here, we describe a protocol for rapid, marker-less, and convenient gene deletion by using the CRISPR/SpCas9M-reporting system in C. crescentus, as an example. Key features • CRISPR-SpCas9M-reporting system overcomes the limitation of CRISPR escape and achieves a high apparent editing efficiency. • The method enables multiple kinds of genome editing, generating in-frame and marker-less chromosomal modifications. • The method completes a cycle of genome editing within one week. • The method could be readily applied for genome editing in C. crescentus, A. fabrum, and S. meliloti.},
}
@article {pmid41219964,
year = {2025},
author = {George, NA and Zhou, Z and Anantharaman, K and Hug, LA},
title = {Discarded diversity: novel megaphages, auxiliary metabolic genes, and virally encoded CRISPR-Cas systems in landfills.},
journal = {Virology journal},
volume = {22},
number = {1},
pages = {370},
pmid = {41219964},
issn = {1743-422X},
support = {R35GM143024/GM/NIGMS NIH HHS/United States ; 2016-03686//Natural Sciences and Engineering Research Council of Canada/ ; Tier II chair//Canada Research Chairs/ ; },
mesh = {*Bacteriophages/genetics/classification/isolation &amp; purification ; *CRISPR-Cas Systems ; *Waste Disposal Facilities ; Metagenomics ; Archaea/virology/genetics ; *Bacteria/virology/genetics ; Genome, Viral ; Host Microbial Interactions ; Computational Biology ; North America ; },
abstract = {BACKGROUND: Viruses are the most abundant microbial entities on the planet, impacting microbial community structure and ecosystem services. Despite outnumbering bacteria and archaea by an order of magnitude, viruses have been comparatively underrepresented in reference databases. Metagenomic examinations have illustrated that viruses of bacteria and archaea have been specifically understudied in engineered environments. Here we employed metagenomic and computational biology methods to examine the diversity, host interactions, and genetic systems of viruses predicted from 27 samples taken from three municipal landfills across North America.<br><br>RESULTS: We identified numerous viruses that are not represented in reference databases, including the third largest bacteriophage genome identified to date (~&#x2009;678 kbp), and noted a large diversity of viruses in landfills that has limited overlap across landfills and is distinct from viromes in other systems. Host-virus interactions were examined via host CRISPR spacer to viral protospacer mapping which captured hyper-targeted viral populations and six viral populations predicted to infect hosts across multiple phyla. Auxiliary metabolic genes (AMGs) were identified with the potential to augment hosts' methane, sulfur, and contaminant degradation metabolisms, including AMGs not previously reported in the literature. CRISPR arrays and CRISPR-Cas systems were identified from predicted viral genomes, including the two largest bacteriophage genomes to contain these genetic features. Some virally encoded Cas effector-like proteins appear distinct relative to previously reported Cas effectors and are interesting targets for potential genome editing tools.<br><br>CONCLUSIONS: Our observations indicate landfills, as heterogeneous contaminated sites with unique selective pressures, are key locations for diverse viruses and atypical virus-host dynamics.},
}
@article {pmid41219611,
year = {2025},
author = {Javaid, MM and Ahmed, J and Ahmed, M and Awan, MJA and Waqas, MAB and Ali, Z and Saeed, NA},
title = {CRISPR-Cas9 based editing of the susceptibility allele TaLr34 enhances leaf rust resistance in bread wheat without yield penalty.},
journal = {Functional &amp; integrative genomics},
volume = {25},
number = {1},
pages = {236},
pmid = {41219611},
issn = {1438-7948},
mesh = {*Triticum/genetics/microbiology/growth &amp; development ; *CRISPR-Cas Systems ; *Disease Resistance/genetics ; *Plant Diseases/genetics/microbiology ; *Gene Editing ; Alleles ; *Plant Proteins/genetics/metabolism ; Basidiomycota/pathogenicity ; Plant Leaves/microbiology/genetics ; Plant Breeding ; },
abstract = {Wheat (Triticum aestivum L.) is widely grown and consumed cereal crop around the world, but most wheat-producing regions suffer from rust diseases, especially stripe and leaf rust, which has caused a devastating global pandemic and severely reduced grain yields. The most effective way to control rust problem in wheat is to sow and breed durable, rust resistant wheat varieties. Conventional breeding for disease-resistant crops primarily relies on resistance (R) genes; however, the effectiveness of R gene-mediated resistance is often compromised by mutations in the pathogen. In this study, we employed CRISPR-Cas9-based genome editing as an advanced breeding tool to enhance rust resistance in the bread wheat cultivar Galaxy-13 by knocking out the homologs of the susceptibility allele TaLr34, specifically targeting the conserved regions within exon 11. Out of 21 transformed plants, five carried successful editing and exhibited resistance to moderate resistance against leaf rust. The TaLr34 mutants were evaluated for leaf rust resistance under both glasshouse and field conditions over three consecutive growing seasons at multiple geographical locations. Our results demonstrate that CRISPR-Cas9-mediated knockout of TaLr34 provides a robust strategy for achieving durable leaf rust resistance in the high-yielding elite wheat cultivar Galaxy-13 without compromising grain yield and agronomic performance.},
}
@article {pmid41218116,
year = {2025},
author = {Wang, X and Cramer, Z and Leu, NA and Monaghan, K and Durning, K and Adams-Tzivelekidis, S and Rhoades, JH and Heintz, J and Tian, Y and Rico, J and Mendez, D and Petroni, R and King, AC and Kim, MS and Matsuda, R and Hanselman, O and Shin, AE and Carrera Rodríguez, MF and Brodsky, IE and Rustgi, A and Li, N and Lengner, CJ and Andrés Blanco, M},
title = {Forward genetic screening in engineered colorectal cancer organoids identifies regulators of metastasis.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {122},
number = {46},
pages = {e2510910122},
doi = {10.1073/pnas.2510910122},
pmid = {41218116},
issn = {1091-6490},
support = {P30-DK050306/GF/NIH HHS/United States ; F31CA250267-02/GF/NIH HHS/United States ; F31AI160741-01/GF/NIH HHS/United States ; R01 CA279317-01/GF/NIH HHS/United States ; },
mesh = {*Colorectal Neoplasms/genetics/pathology ; Animals ; *Organoids/pathology/metabolism ; Mice ; Humans ; *Neoplasm Metastasis/genetics ; CRISPR-Cas Systems ; *Genetic Testing/methods ; Cell Movement/genetics ; Cell Line, Tumor ; Neoplasm Invasiveness ; *Adenocarcinoma/genetics/pathology ; },
abstract = {Metastatic outgrowth requires that cancer cells delaminate from the primary tumor, intravasate, survive in circulation, extravasate, migrate to, and proliferate at a distal site. Recurrent genetic drivers of metastasis remain elusive, suggesting that unlike the early steps of oncogenesis, metastasis drivers may be variable. We develop a framework for identifying metastasis regulators using CRISPR/Cas9-based screening in a genetically defined organoid model of colorectal adenocarcinoma. We conduct in vitro screens for invasion and migration alongside orthotopic, in vivo screens for gain of metastasis in a syngeneic mouse model. We identify CTNNA1 and BCL2L13 as bona fide metastasis-specific suppressors which do not confer any selective advantage in primary tumors. CTNNA1 loss promotes cell invasion and migration, and BCL2L13 loss promotes anchorage-independent survival and non-cell-autonomous changes to macrophage polarization. This study demonstrates proof of principle that large-scale genetic screening can be performed in tumor-organoid models in vivo and identifies novel regulators of metastasis.},
}
@article {pmid41215577,
year = {2025},
author = {Uombe, NPI and Velikkakam, T and Silveira, ACA and Rodrigues, CC and Borges, BC and Teixeira, TL and Pereira, CL and Servato, JPS and Melo, NS and Mortara, RA and Silveira, JFD and Silva, CVD},
title = {Trypanosoma cruzi P21 Is a Pleiotropic Protein That Is Involved in Parasite Host Cell Invasion and Intracellular Parasitism.},
journal = {MicrobiologyOpen},
volume = {14},
number = {6},
pages = {e70154},
pmid = {41215577},
issn = {2045-8827},
support = {//This study was supported by grants and fellowships from FAPESP, FAPEMIG, CAPES, and CNPq. FAPESP Grant number: 2016/15000-4 and 2019/05049-4 (Postdoctoral fellowship); FAPEMIG - RED-00198-23./ ; },
mesh = {*Trypanosoma cruzi/genetics/pathogenicity/growth &amp; development/physiology/metabolism ; Animals ; *Chagas Disease/parasitology ; Vero Cells ; Chlorocebus aethiops ; Mice ; *Host-Parasite Interactions ; *Protozoan Proteins/genetics/metabolism ; Gene Knockout Techniques ; Virulence ; Disease Models, Animal ; CRISPR-Cas Systems ; },
abstract = {We characterized the secreted Trypanosoma cruzi P21 protein and hypothesized its role in parasite invasion and multiplication. To investigate the role of T. cruzi P21 protein in host-parasite interactions, specifically focusing on the low-virulence G strain. P21 knockout parasites were generated using CRISPR/Cas9. Cell invasion, multiplication, egress, and tissue parasitism were assessed in vitro and in vivo, comparing knockout and control parasites. P21 knockout significantly reduced parasite invasion and multiplication in Vero cells. In vivo, knockout parasites also showed reduced heart tissue parasitism in infected mice, despite no observable systemic parasitemia. Accordingly, P21 knockout trypomastigote egress was reduced in Vero cells. P21 plays a pleiotropic role in T. cruzi infection, differentially impacting parasite biology in the low-virulent G strain. In the G strain, P21 promotes invasion and persistence, potentially through mechanisms distinct from its role in the Y strain previously described. This highlights its potential as a therapeutic target for Chagas disease, warranting further investigation into strain-specific functions.},
}
@article {pmid41185939,
year = {2025},
author = {Kong, H and Liu, X and Xia, K and Guan, Y and Zhang, J and Zhang, Y and Liu, B and Ge, Z and Li, Q and Zuo, X and Wang, L and Li, J and Zhu, Y and Fan, C},
title = {Magnetically Driven High-Speed Rolling Nanoclusters for Enhanced CRISPR/Cas9 Genome Editing.},
journal = {ACS applied materials &amp; interfaces},
volume = {17},
number = {45},
pages = {61707-61717},
doi = {10.1021/acsami.5c15498},
pmid = {41185939},
issn = {1944-8252},
mesh = {Humans ; *Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; *Magnetite Nanoparticles/chemistry ; Plasmids/genetics/chemistry/metabolism ; Jurkat Cells ; Protein Serine-Threonine Kinases/genetics ; Cell Cycle Proteins/genetics ; Viscosity ; },
abstract = {Despite the tremendous potential of the CRISPR/Cas9 gene-editing technology in precision therapeutics, intracellular delivery remains a major challenge. High cytoplasmic viscosity and lysosomal entrapment significantly impair the cytosolic transport and gene-editing efficiency. In this study, we demonstrate that both the size and magnetic responsiveness of Fe3O4 nanoclusters can be finely tuned by modulating ionic strength, enabling their rapid propulsion under external magnetic fields. Leveraging this property, we develop magnetic nanoparticle cluster nanorobots (MagCbots) of approximately 200 nm in size by electrostatically assembling Fe3O4 nanoclusters with CRISPR-Cas9 plasmids. Under magnetic actuation, MagCbots exhibit rapid rotation in highly viscous intracellular environments, achieving a linear velocity of ∼0.41 μm/s. MagCbots reduce intracellular viscosity by approximately 50% and enhance lysosomal escape efficiency by 3-fold compared to nonactuated counterparts. Their porous architecture not only offers high payload capacity but also protects plasmid DNA from enzymatic degradation. Notably, MagCbots enable efficient genome editing of both PD1 and PLK1 genes across various cell lines including hard-to-transfect Jurkat T cells. This magnetically driven nanorobot platform presents a promising strategy for active intracellular delivery and holds significant potential for advancing gene therapy and related biomedical applications.},
}
@article {pmid41183022,
year = {2025},
author = {Rajamani, S and Vilchez, L and Cracovia, N and Dule, D and Vata, A and Landaverde, S and Iyengar, A and Dubrovsky, EB},
title = {Mitochondrial-specific perturbation of Drosophila RNase Z in neurons leads to motor impairments, disrupted learning and neurodegeneration.},
journal = {PLoS genetics},
volume = {21},
number = {11},
pages = {e1011938},
doi = {10.1371/journal.pgen.1011938},
pmid = {41183022},
issn = {1553-7404},
mesh = {Animals ; *Mitochondria/genetics/metabolism/pathology ; *Neurons/metabolism/pathology ; *Drosophila Proteins/genetics/metabolism ; Drosophila melanogaster/genetics ; *Endoribonucleases/genetics/metabolism ; Mutation ; Learning/physiology ; CRISPR-Cas Systems ; *Neurodegenerative Diseases/genetics/pathology ; Animals, Genetically Modified ; Reactive Oxygen Species/metabolism ; },
abstract = {Clinical studies have linked a rare form of neurological disorder to the highly conserved RNase Z gene, which encodes an endoribonuclease responsible for the processing of nuclear and mitochondrial primary tRNA transcripts. Patients harboring mutant variants of this gene exhibit a spectrum of neurological dysfunction; however, no studies to date have established the causality of RNase Z-linked neuropathology. We employed CRISPR/Cas9 technology to create flies with a neuron-specific knockout of the RNase Z gene, which is rescued with transgenes encoding a wild-type or a mutant copy of RNase Z. Neuronal activity of RNase Z is vital, as mutants display striking morphological abnormalities in central and peripheral neurons, along with attenuated motor circuit function and associative learning performance. Neuron-specific mutations of RNase Z also led to mitochondrial fragmentation and elevated ROS production. By employing the rescue transgene encoding RNase Z devoid of a mitochondrial targeting signal (MTS), we segregated the mitochondrial activity of RNase Z from that in other compartments, allowing us to assess organelle-specific contributions to disease etiology and progression. We found that mutating mitochondrial RNase Z was sufficient to induce the neuropathology in flies, as they recapitulate the salient phenotypes observed in the pan-neuronal mutants. Collectively, our study validates the pathogenicity of mutant RNase Z and establishes mitochondrial-specific contributions to neuropathology.},
}
@article {pmid41170612,
year = {2025},
author = {Pu, J and Zhou, S and Ma, W and Liu, C and Guo, L and Yan, B and Huo, Z},
title = {Light-controlled CRISPR-Cas12a one-pot platform for ultrasensitive cell-free DNA detection in systemic lupus erythematosus diagnosis.},
journal = {Analytical methods : advancing methods and applications},
volume = {17},
number = {44},
pages = {8916-8922},
doi = {10.1039/d5ay01286d},
pmid = {41170612},
issn = {1759-9679},
mesh = {*Lupus Erythematosus, Systemic/diagnosis/blood/genetics ; Humans ; *Cell-Free Nucleic Acids/blood/genetics/analysis ; *CRISPR-Cas Systems/genetics ; *Biosensing Techniques/methods ; Limit of Detection ; },
abstract = {Systemic lupus erythematosus (SLE), as a complex autoimmune disease with heterogeneous clinical manifestations, presents significant challenges for early diagnosis. Circulating cell-free DNA (cfDNA) has emerged as a promising disease monitoring biomarker due to its correlation with SLE pathological progression in terms of concentration and fragmentation patterns. However, existing detection methods lack sufficient sensitivity and practicality for clinical application. To address this, we developed a spatiotemporally resolved light-controlled biosensor by integrating a photoactivatable CRISPR-Cas12a system with TdT-mediated poly-A tail extension, achieving three major innovations: (1) implementation of NPOM-dt modified crRNA for precise regulation; (2) optimization of 365 nm UV activation protocol to eliminate interference in one-pot reactions; and (3) establishment of a three-phase "Extension-Activation-Detection" workflow. The platform demonstrates outstanding performance with a detection limit of 0.42 pM, excellent linearity (R[2] = 0.9956) in the 0-0.1 nM range, and the novel DNA Integrity Index (DII) as a diagnostic indicator - showing significantly higher values in SLE patients (9.82 × 10[3] nmol g[-1]) versus healthy controls (4.2 × 10[3] nmol g[-1], P < 0.0001) with an AUC of 0.8947. This study provides an innovative detection platform combining high sensitivity with clinical utility for early SLE diagnosis.},
}
@article {pmid41165493,
year = {2025},
author = {Zhang, J and Li, X and Wang, M and Ren, S and Li, M and Liu, Y and Lu, F and Li, Q and Li, Y},
title = {Rational Construction of a Robust Bacillus amyloliquefaciens Cell Factory for Acid-Stable α Amylase Production.},
journal = {Journal of agricultural and food chemistry},
volume = {73},
number = {45},
pages = {28883-28895},
doi = {10.1021/acs.jafc.5c04227},
pmid = {41165493},
issn = {1520-5118},
mesh = {*Bacillus amyloliquefaciens/genetics/metabolism/enzymology ; *Bacterial Proteins/genetics/metabolism/chemistry ; *alpha-Amylases/genetics/chemistry/metabolism/biosynthesis ; Fermentation ; Gene Editing ; Prophages/genetics/metabolism ; Enzyme Stability ; CRISPR-Cas Systems ; Multigene Family ; },
abstract = {With the development of synthetic biology and biotechnology, chassis engineering has become the main means of industrial protein production, but it has been limited by the lack of efficient gene editing methods and effective engineering strategies. Bacillus amyloliquefaciens shows potential for expressing heterologous proteins, but its cells undergo early autolysis, hindering further application. In this study, an autolysis-related prophage gene cluster was rationally deleted by establishing an efficient CRISPR-nCas9 editing process, and the prophage mutant strain was constructed, which prevented cell lysis. Based on the prophage mutant strain, we screened secondary metabolite biosynthetic gene clusters that hindered the expression of heterologous proteins, and we made reasonable deletions to further improve their efficient expression. Finally, an optimized yield of acid-stable α amylase (2,46,089.21 U/mL) was obtained in a 5-L fed-batch fermentation. Therefore, we successfully constructed an ideal candidate strain for the expression of heterologous proteins, which provides an important research basis for the development of more chassis strains.},
}
@article {pmid41144643,
year = {2025},
author = {Huang, C and Guo, LJ and Li, JY and Chen, YW and Wu, B and Liu, YW and Zhang, CZ and Lin, WW and Yang, Y and Chen, JY and Liu, ZJ},
title = {A CRISPR-Cas12a Fluorescent Aptasensor for Point-of-Care Drug Concentration Detection: A Synergistic Regulation by DNA Aptamer and RNA Blocker.},
journal = {Analytical chemistry},
volume = {97},
number = {44},
pages = {24415-24424},
doi = {10.1021/acs.analchem.5c03744},
pmid = {41144643},
issn = {1520-6882},
mesh = {*Aptamers, Nucleotide/chemistry ; *CRISPR-Cas Systems ; *Biosensing Techniques/methods ; *Point-of-Care Systems ; *Vancomycin/analysis/blood ; Limit of Detection ; *RNA/chemistry/antagonists &amp; inhibitors ; Humans ; *Fluorescent Dyes/chemistry ; *Anti-Bacterial Agents/analysis ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {The combination of CRISPR-Cas12a with aptamers can potentially improve the sensitivity, specificity, and speed of non-nucleic acid target detection. Nevertheless, current CRISPR-Cas12a aptasensors, solely dependent on aptamer affinity and overlooking the enzymatic regulation of CRISPR-Cas12a, may produce false-positive signals. We proposed a CRISPR-Cas12a aptasensor synergistically regulated by an aptamer and RNA, which incorporated aptamer-mediated drug recognition and CRISPR-powered signal amplification in a one-pot format. Herein, a regulatory RNA probe synergized with the conformational switching of a drug-induced aptamer, enabling precise regulation of Cas12a trans-cleavage activity via toehold-mediated strand displacement (TMSD). This dual regulatory mechanism transformed the aptamer-activated CRISPR-Cas12a sensing process into a TMSD-driven conditional reaction, avoiding false-positive signals and thus achieving better detection performance with a lower detection limit. With the vancomycin (VCM) aptamer as a model, the aptasensor can detect VCM within 30 min from 2% serum, 1% synovial fluid, and 1% cerebrospinal fluid, with a detection limit of 13.62, 7.56, and 6.75 nM, respectively. The proposed aptasensor was incorporated into a custom 3D-printed portable fluorometer and underwent clinical validation in 22 VCM serum samples, reporting no significant difference when compared with the enzyme-multiplied immunoassay technique, confirming the reliability for point-of-care quantification. It further received cross-validation with a quinine aptamer, suggesting universality [a linear range of 10-250 nM (R[2] = 0.985) and a detection limit of 0.42 nM]. By integration of aptamer selectivity with CRISPR programmability, this work presents a novel robust biosensing paradigm for point-of-care drug concentration detection.},
}
@article {pmid41122778,
year = {2025},
author = {Hasselbeck, S and Wang, J and Bai, Z and Hüfner, T and Hummer, G and Grote, P and Cheng, X},
title = {A Chemically Induced CRISPR/dCas13[FCPF] Platform for Precise and Programmable RNA Regulation.},
journal = {Journal of medicinal chemistry},
volume = {68},
number = {21},
pages = {22633-22649},
doi = {10.1021/acs.jmedchem.5c01609},
pmid = {41122778},
issn = {1520-4804},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; Gene Editing/methods ; *RNA/genetics/metabolism ; Alternative Splicing/drug effects ; Survival of Motor Neuron 2 Protein/genetics ; HEK293 Cells ; Exons ; },
abstract = {Alternative splicing enhances proteomic diversity, yet its dysregulation drives cancer, neurodegeneration, and inherited disease. Small-molecule splicing modulators, while clinically validated, like risdiplam, often lack locus specificity, producing off-target effects. CRISPR/Cas13 enables programmable transcript-level targeting, but dCas13 fusion effectors are bulky and can hamper delivery and RNA homeostasis. Building on our previous Chem-CRISPR/dCas9[FCPF] system for epigenome editing, we now introduce Chem-CRISPR/dCas13[FCPF], a modular platform that covalently tethers a perfluorobiphenyl-tagged small molecule to dCas13 via a four-residue FCPF π-clamp tag. Guided by crRNAs, dRfxCas13d[FCPF] recruits a risdiplam-derived conjugate to the SMN2 exon 7 splice region, inducing exon inclusion at ligand doses ∼500-fold lower than those of free risdiplam and with no detectable effects at known risdiplam-sensitive transcripts in our assays. The approach generalizes to additional transcripts by crRNA redesign. By coupling CRISPR addressability with dose-sparing chemical action, Chem-CRISPR/dCas13[FCPF] establishes a proximity-induced, chemically controllable route to precise RNA modulation suitable for therapeutic exploration.},
}
@article {pmid41108054,
year = {2025},
author = {Mariki, A and Kohlmeier, KA and Mousavi, SM and Shabani, M},
title = {A systematic review of CRISPR applications in demyelinating peripheral nervous system disorders.},
journal = {Regenerative medicine},
volume = {20},
number = {11},
pages = {653-662},
pmid = {41108054},
issn = {1746-076X},
mesh = {Humans ; Animals ; *Demyelinating Diseases/therapy/genetics ; *CRISPR-Cas Systems/genetics ; Genetic Therapy/methods ; *Peripheral Nervous System Diseases/therapy/genetics ; Charcot-Marie-Tooth Disease/therapy/genetics ; Schwann Cells/metabolism/pathology ; Gene Editing ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; },
abstract = {AIM: This review evaluates CRISPR-based strategies for myelin regeneration in peripheral demyelinating disorders, with a focus on Guillain - Barré syndrome (GBS) and Charcot - Marie - Tooth disease type 1A (CMT1A). It aims to identify current therapeutic approaches, delivery systems, and gaps in the literature.<br><br>MATERIALS &amp; METHODS: A systematic literature search was conducted across PubMed, Scopus, Web of Science, and Google Scholar for studies published from 2010 onward, following PRISMA guidelines. Study quality was assessed using OHAT and SYRCLE tools, and 14 articles met the inclusion criteria.<br><br>RESULTS: In GBS, CRISPR interventions primarily targeted antiviral immune regulation (AXL, IFI6, IFNL2), inhibition of viral entry mechanisms (Integrin &#x3b1;v&#x3b2;5, SPCS1), and Schwann cell repair. In CMT1A, therapeutic approaches focused on correcting PMP22 overexpression. Lentiviral transduction was the most frequently used delivery method, with no major adverse effects reported. However, most studies were in vitro, and only two were in vivo, highlighting the need for further validation in animal models.<br><br>CONCLUSIONS: CRISPR technology shows considerable potential for addressing peripheral nerve demyelination through precise genetic modifications that may enhance Schwann cell function and support myelin repair. Nevertheless, the field remains at an early discovery stage, with no near-term clinical applicability demonstrated.},
}
@article {pmid41061783,
year = {2025},
author = {Ma, Y and Zhao, Q and Yuan, J and Wang, D and Chen, X and Yu, Y and Li, J and Yu, M and Yuan, J and Lou, J and Du, S and Wen, Y and Wang, Y and Wu, R and Yan, QG and Huang, X and Zheng, Y and Zhao, F and Cao, SJ},
title = {RIPK4-p53 interaction drives aflatoxin B1-induced renal mitochondrial apoptosis via Ser15 phosphorylation: A CRISPR-Cas9 mechanistic study.},
journal = {International journal of biological macromolecules},
volume = {330},
number = {Pt 3},
pages = {148130},
doi = {10.1016/j.ijbiomac.2025.148130},
pmid = {41061783},
issn = {1879-0003},
mesh = {*Apoptosis/drug effects ; Animals ; *CRISPR-Cas Systems ; *Aflatoxin B1/toxicity ; Phosphorylation/drug effects ; *Mitochondria/metabolism/drug effects ; *Tumor Suppressor Protein p53/metabolism ; *Kidney/metabolism/drug effects/pathology ; Swine ; Cell Line ; *Protein Serine-Threonine Kinases/metabolism/genetics ; Protein Binding ; },
abstract = {Aflatoxin B1 (AFB1), recognized as a highly toxic and carcinogenic mycotoxin, contaminates more than 25 % of the global grain supply, thereby presenting a substantial public health threat and posing significant risks to renal health. However, the host factors that mediate the associated damage remain inadequately understood. This study aimed to identify key host factors in AFB1-induced cytotoxicity using a genome-wide CRISPR/Cas9 screen and elucidate the underlying molecular mechanisms. We developed a porcine kidney epithelial (PK15) cell model, followed by knockout validation, CCK-8 assays, qRT-PCR, western blotting, AO-EB staining, flow cytometry, and co-immunoprecipitation to dissect mechanistic pathways. Receptor-Interacting Protein Kinase 4 (RIPK4) was identified as a critical pro-apoptotic factor. RIPK4 knockout increased PK15 cell viability by ~50 % (P < 0.001) and reduced apoptosis by ~44 %(P < 0.001), accompanied by downregulation of APAF1, Cyt-c, cleaved-Caspase-9/-3, and p53 Ser15 phosphorylation, and upregulation of Bcl-2. Mechanistically, RIPK4 directly interacted with p53 via its N-terminal 1-490 aa region, enhancing its phosphorylation and pro-apoptotic activity. In conclusion, RIPK4 promotes AFB1 nephrotoxicity by activating p53-mediated mitochondrial apoptosis, identifying it as a novel therapeutic target. Future studies should validate these findings in vivo models and explore the potential of RIPK4-specific inhibitors for mitigating nephrotoxicity.},
}
@article {pmid41025776,
year = {2025},
author = {Messina, G and Goerner, A and Bennett, C and Brennan, E and Carruthers, VB and Martorelli Di Genova, B},
title = {Impact of equilibrative nucleoside transporters on Toxoplasma gondii infection and differentiation.},
journal = {mBio},
volume = {16},
number = {11},
pages = {e0220725},
pmid = {41025776},
issn = {2150-7511},
support = {P20 GM125498/GM/NIGMS NIH HHS/United States ; },
mesh = {*Toxoplasma/genetics/growth &amp; development/metabolism ; Animals ; Mice ; *Toxoplasmosis/parasitology ; *Protozoan Proteins/genetics/metabolism ; *Nucleoside Transport Proteins/genetics/metabolism ; *Equilibrative Nucleoside Transport Proteins/genetics/metabolism ; CRISPR-Cas Systems ; },
abstract = {UNLABELLED: Toxoplasma gondii cannot synthesize purines de novo and must import them; however, the functional interplay among its four equilibrative nucleoside transporters (ENTs) homologs remains unclear. We systematically deconstructed this network by combining CRISPR-Cas9 knockouts with an auxin-inducible degron. Across all phenotypic assays, tachyzoite replication, nucleoside-analog sensitivity, alkaline-stress-induced differentiation, and murine cyst formation, the ΔTgENT2 strain was indistinguishable from the parental line, indicating that TgENT2 is dispensable under the conditions tested. In contrast, the double mutant ΔTgAT1ΔTgENT3 exhibited delayed bradyzoite differentiation in vitro and produced smaller brain cysts in vivo. This double deletion triggered approximately threefold transcriptional upregulation of TgENT1, whose product we partially localized to the plant-like vacuolar compartment (PLVAC). Conditional depletion of TgENT1 caused complete intracellular growth arrest, PLVAC swelling, and a purine-starvation-like transcriptomic program enriched for nucleoside phosphatases and cyclic-nucleotide phosphodiesterases. These findings reveal a compensatory salvage pathway in which the parasite reroutes purine acquisition through a vacuolar route when plasma-membrane import is compromised. Although this response sustains tachyzoite proliferation, it fails during the energetically demanding transition to bradyzoites, creating a metabolic bottleneck that impairs chronic infection. Our work reveals an adaptable yet ultimately limited purine-import network and identifies TgENT1, along with the vacuolar salvage axis it mediates.<br><br>IMPORTANCE: In this manuscript, we demonstrate that Toxoplasma gondii employs a flexible transporter network that redirects to a vacuolar salvage route when primary transporters are compromised. Disrupting this backup pathway disrupts parasite growth, exposing an Achilles' heel in purine homeostasis. Because nucleoside transporters are druggable, these findings suggest that the purine import machinery and TgENT1 may be potential targets for therapies against T. gondii infections.},
}
@article {pmid40985725,
year = {2025},
author = {Sayson, SG and Ashbaugh, A and Bauer, LC and Smulian, AG},
title = {Extracellular vesicle-mediated delivery of genetic material for transformation and CRISPR/Cas9-based gene editing in Pneumocystis murina.},
journal = {mBio},
volume = {16},
number = {11},
pages = {e0182525},
pmid = {40985725},
issn = {2150-7511},
support = {R61 AI187097/AI/NIAID NIH HHS/United States ; 1R61AI187097//National Institute of Allergy and Infectious Diseases/ ; },
mesh = {*Extracellular Vesicles/metabolism/genetics ; *Gene Editing/methods ; *CRISPR-Cas Systems ; *Pneumocystis/genetics ; Animals ; Mice ; *Transformation, Genetic ; *Gene Transfer Techniques ; Lung/microbiology ; Plasmids/genetics ; },
abstract = {Pneumocystis species are obligate fungal pathogens that cause severe pneumonia, particularly in immunocompromised individuals. The absence of robust genetic manipulation tools has impeded our mechanistic understanding of Pneumocystis biology and the development of novel therapeutic strategies. Herein, we describe a novel method for the stable transformation and CRISPR/Cas9-mediated genetic editing of Pneumocystis murina utilizing extracellular vesicles (EVs) as a delivery vehicle. Building upon our prior investigations demonstrating EV-mediated delivery of exogenous material to Pneumocystis, we engineered mouse lung EVs to deliver plasmid DNA encoding reporter genes and CRISPR/Cas9 components. Our initial findings demonstrated successful in vitro transformation and subsequent expression of mNeonGreen and Dhps[ARS] in P. murina organisms. Subsequently, we established stable in vivo expression of mNeonGreen in mice infected with transformed P. murina for a duration of up to 5 weeks. Furthermore, we designed and validated a CRISPR/Cas9 system targeting the P. murina Dhps gene, confirming DNA cleavage efficiency in vitro. Ultimately, we achieved successful in vivo CRISPR/Cas9-mediated homologous recombination, precisely introducing a Dhps[ARS] mutation into the P. murina genome, which was confirmed by Sanger sequencing across all tested animals. Here, we establish a foundational methodology for genetic manipulation in Pneumocystis, thereby opening avenues for functional genomics, drug target validation, and the generation of genetically modified strains for advanced research and potential therapeutic applications.IMPORTANCEPneumocystis species are obligate fungal pathogens and major causes of pneumonia in immunocompromised individuals. However, their strict dependence on the mammalian lung environment has precluded the development of genetic manipulation systems, limiting our ability to interrogate gene function, study antifungal resistance mechanisms, or validate therapeutic targets. Here, we report the first successful approach for stable transformation and CRISPR/Cas9-based genome editing of Pneumocystis murina, achieved through in vivo delivery of engineered extracellular vesicles containing plasmid DNA and encoding CRISPR/Cas9 components. We demonstrate sustained transgene expression and precise modification of the dhps locus via homology-directed repair. This modular, scalable platform overcomes a long-standing barrier in the field and establishes a foundation for functional genomics in Pneumocystis and other obligate, host-adapted microbes.},
}
@article {pmid40980888,
year = {2025},
author = {Danskog, K and Petersen, F and Frängsmyr, L and Gonzalez, G and Becker, M and Lenman, A and Arnberg, N},
title = {CD46 is a cellular receptor for species D human adenovirus.},
journal = {mBio},
volume = {16},
number = {11},
pages = {e0158725},
pmid = {40980888},
issn = {2150-7511},
support = {101098647//HORIZON EUROPE European Innovation Council/ ; 2023-01831//Vetenskapsr&#xe5;det/ ; 2019-01472//Vetenskapsr&#xe5;det/ ; 22 2005 Pj//Cancerfonden/ ; CAN 2018/771//Cancerfonden/ ; },
mesh = {Humans ; *Membrane Cofactor Protein/metabolism/genetics ; *Adenoviruses, Human/physiology/genetics/classification ; *Receptors, Virus/metabolism/genetics ; A549 Cells ; Viral Tropism ; CRISPR-Cas Systems ; Desmoglein 2/genetics/metabolism ; Virus Attachment ; Coxsackie and Adenovirus Receptor-Like Membrane Protein/genetics/metabolism ; },
abstract = {UNLABELLED: Human adenovirus species D (HAdV-D) contains two-thirds of all known HAdV types (116 in total) and is important as a vector in clinical applications. However, the broad tropism exhibited by several HAdV-D types poses challenges for their use as targeted gene delivery vectors. Since adenoviral tropism is largely governed by receptor usage, we aimed to determine the relative importance of known adenovirus receptors in mediating infection by different HAdV-D types. Here, we generated A549 single-cell CRISPR/Cas9 knockout clones of desmoglein 2 (DSG2), CD46, the coxsackievirus and adenovirus receptor (CAR), and cytidine monophosphate N-acetylneuraminic acid synthetase (CMAS; needed for biosynthesis of sialic acid-containing glycans), and assessed their relative importance for infection by 18 different HAdV-D types. We show that CD46 is the most important receptor for a majority of species D HAdVs. Minor changes in infection levels were noted with A549-ΔCAR and A549-ΔDSG2 cells, whereas A549-ΔCMAS cells displayed an increased susceptibility to infection. We proceed to show that HAdV-D types require CD46 for efficient attachment to A549 cells, and surface plasmon resonance analysis demonstrates that their hexon proteins bind CD46 in an avidity-dependent manner. Strategies to retarget HAdV-D vectors should thus consider hexon-CD46 interactions as a critical determinant of tropism, as CD46 is broadly expressed in vivo. These results increase our understanding of adenovirus-host interactions and will guide the development and targeting of vectors based on HAdV-D types.<br><br>IMPORTANCE: Several human adenovirus species D (HAdV-D) types are currently used, or under development, as viral vectors for vaccines and gene delivery. However, the unusually broad tropism observed in many HAdV-D types limits their specificity and effectiveness as targeted vectors. Since tropism is largely governed by receptor usage, and previous studies have reported conflicting findings on receptor preferences within this species, clarifying receptor usage is essential. In this study, we systematically investigated receptor usage in 18 different HAdV-D types and identified CD46 as the primary receptor. Since CD46 is widely expressed across human tissues, our findings explain the broad cellular tropism of these viruses and provide valuable insight for the rational design and refinement of HAdV-D-based vectors.},
}
@article {pmid40973826,
year = {2025},
author = {Petri, K and D'Ippolito, E and Künkele, A and Köhl, U and Busch, DH and Einsele, H and Hudecek, M},
title = {Next-generation T cell immunotherapies engineered with CRISPR base and prime editing: challenges and opportunities.},
journal = {Nature reviews. Clinical oncology},
volume = {22},
number = {12},
pages = {902-923},
pmid = {40973826},
issn = {1759-4782},
mesh = {Humans ; *Gene Editing/methods ; *T-Lymphocytes/immunology/transplantation ; *CRISPR-Cas Systems/genetics ; *Immunotherapy, Adoptive/methods ; *Neoplasms/therapy/immunology/genetics ; Receptors, Antigen, T-Cell/genetics ; },
abstract = {T cells can be reprogrammed with transgenic antigen recognition receptors, including chimeric antigen receptors and T cell receptors, to selectively recognize and kill cancer cells. Such adoptive T cell therapies are effective in patients with certain haematological cancers but challenges persist, including primary and secondary resistance, a lack of efficacy in patients with solid tumours, a narrow range of targetable antigens, and time-consuming and complex manufacturing processes. CRISPR-based genome editing is a potent strategy to enhance cellular immunotherapies. Conventional CRISPR-Cas9 systems are useful for gene editing, transgene knock-in or gene knockout but can result in undesired editing outcomes, including translocations and chromosomal truncations. Base editing and prime editing technologies constitute a new generation of CRISPR platforms and enable highly precise and programmable installation of defined nucleotide variants in primary T cells. Owing to their high precision and versatility, base editing and prime editing systems, hereafter collectively referred to as CRISPR 2.0, are advancing to become the new standard for precision-engineering of cellular immunotherapies. CRISPR 2.0 can be used to augment immune cell function, broaden the spectrum of targetable antigens and facilitate streamlined production of T cell therapies. Notably, CRISPR 2.0 is reaching clinical maturity, with multiple clinical trials of CRISPR 2.0-modified cellular therapies currently ongoing. In this Review, we discuss emerging CRISPR 2.0 technologies and their progress towards clinical translation, highlighting challenges and opportunities, and describe strategies for the use of CRISPR 2.0 to advance cellular immunotherapy for haematological malignancies and solid tumours in the future.},
}
@article {pmid40973646,
year = {2025},
author = {Dinneen, E and Dasgupta, P and Sharma, A and Nisaa, K and Silva-García, CG},
title = {A single-copy knock-in system: one plasmid to target all chromosomes in C. elegans.},
journal = {G3 (Bethesda, Md.)},
volume = {15},
number = {11},
pages = {},
pmid = {40973646},
issn = {2160-1836},
support = {//NIA/ ; R00AG065508/GF/NIH HHS/United States ; A24058//American Federation for Aging Research/ ; //Brown University Division of Research Seed Award/ ; },
mesh = {Animals ; *Caenorhabditis elegans/genetics ; *Plasmids/genetics ; CRISPR-Cas Systems ; *Chromosomes/genetics ; *Gene Knock-In Techniques/methods ; Transgenes ; },
abstract = {Successful transgenesis in model organisms has significantly helped us understand gene function, regulation, genetic networks, and potential applications. Here, we introduce a single-copy knock-in system that uses 1 plasmid to target all chromosomes in Caenorhabditis elegans (SKI PLACE), designed for inserting a transgene by CRISPR/Cas9. The SKI PLACE system uses the pSKI plasmid to insert a desired transgene at specific harbor loci on each chromosome. The pSKI plasmid contains multiple restriction sites for cloning and serves as a CRISPR/Cas9-based insertion repair template because it has 2 synthetic and long homology arms that recombine with the SKI PLACE cassettes. This system also uses a single crRNA guide, which acts as a Co-CRISPR enrichment marker. Overall, the SKI PLACE system is flexible; with the same SKI PLACE cassette on each chromosome, researchers can select the insertion site, work with 1 plasmid, and streamline tracking using standard primers.},
}
@article {pmid40972573,
year = {2025},
author = {Pfefferle, A and Malmberg, KJ},
title = {Rewiring natural killer cells for next-generation cancer therapies.},
journal = {Cancer cell},
volume = {43},
number = {11},
pages = {1980-1982},
doi = {10.1016/j.ccell.2025.08.007},
pmid = {40972573},
issn = {1878-3686},
mesh = {*Killer Cells, Natural/immunology ; Humans ; *Neoplasms/therapy/immunology/genetics ; *Immunotherapy/methods ; CRISPR-Cas Systems ; Animals ; },
abstract = {Harnessing the cytotoxic potential of natural killer (NK) cells for cancer immunotherapy has proven challenging. In this issue of Cancer Cell, Biederstädt et al. and Nikolic et al. utilize genome-wide CRISPR screening to uncover novel regulators of NK cell function, paving the way for developing next-generation NK cell therapies.},
}
@article {pmid40902837,
year = {2025},
author = {Huang, M and Ewadi, A and Servatian, N and Noormohamadi, H and Aminov, Z and Muzammil, K and Esfahani, MA and Abak, N and Soleimani Samarkhazan, H},
title = {Epigenetic mechanisms and next-gen editing platforms in hematology: From molecular basis to therapeutic frontiers.},
journal = {Critical reviews in oncology/hematology},
volume = {215},
number = {},
pages = {104916},
doi = {10.1016/j.critrevonc.2025.104916},
pmid = {40902837},
issn = {1879-0461},
mesh = {Humans ; *Gene Editing/methods ; *Epigenesis, Genetic ; *Hematologic Diseases/therapy/genetics ; CRISPR-Cas Systems ; Animals ; *Hematology/methods ; Genetic Therapy/methods ; },
abstract = {Epigenetic regulation is fundamental to hematopoiesis, influencing stem cell fate, lineage commitment, and the development of hematologic diseases. Recent technological innovations have transitioned from traditional genetic editing towards programmable, reversible epigenetic modulation without altering the DNA sequence. This review explores the evolution of epigenetic editing platforms, from zinc finger proteins and TALEs to the transformative CRISPR-dCas9 system, and introduces next-generation technologies leveraging dCas12, dCas13, and modular RNA-guided systems. By fusing catalytically inactive CRISPR variants with chromatin or RNA-modifying enzymes, these tools enable precise control of gene expression and epitranscriptomic landscapes. In hematology, these advances offer novel strategies to modulate oncogenes, reactivate silenced tumor suppressors, and correct epigenetic dysregulation in malignancies such as leukemia, lymphoma, and myelodysplastic syndromes, as well as in inherited disorders like β-thalassemia and sickle cell disease. The integration of epigenetic editing into immune engineering, particularly in enhancing CAR-T and NK cell therapies, underscores its growing clinical impact. Together, these next-generation approaches herald a paradigm shift, enabling safer, more dynamic, and tunable interventions for blood disorders. This review highlights the current landscape and future directions of epigenetic editing, positioning it as a cornerstone of precision hematologic therapy.},
}
@article {pmid40845844,
year = {2025},
author = {Biederstädt, A and Basar, R and Park, JM and Uprety, N and Shrestha, R and Reyes Silva, F and Dede, M and Watts, J and Acharya, S and Xiong, D and Liu, B and Daher, M and Rafei, H and Banerjee, P and Li, P and Islam, S and Fan, H and Shanley, M and Jin, J and Kumar, B and Woods, V and Lin, P and Tiberti, S and Nunez Cortes, AK and Jiang, XR and Biederstädt, I and Zhang, P and Li, Y and Rawal, S and Liu, E and Muniz-Feliciano, L and Deyter, GM and Shpall, EJ and Fowlkes, NW and Chen, K and Rezvani, K},
title = {Genome-wide CRISPR screens identify critical targets to enhance CAR-NK cell antitumor potency.},
journal = {Cancer cell},
volume = {43},
number = {11},
pages = {2069-2088.e11},
pmid = {40845844},
issn = {1878-3686},
support = {P30 CA016672/CA/NCI NIH HHS/United States ; R01 CA280827/CA/NCI NIH HHS/United States ; P50 CA281701/CA/NCI NIH HHS/United States ; R01 CA211044/CA/NCI NIH HHS/United States ; P01 CA148600/CA/NCI NIH HHS/United States ; P50 CA127001/CA/NCI NIH HHS/United States ; S10 OD024977/OD/NIH HHS/United States ; R01 CA288617/CA/NCI NIH HHS/United States ; },
mesh = {Humans ; *Killer Cells, Natural/immunology/transplantation/metabolism ; *Immunotherapy, Adoptive/methods ; Animals ; Gene Editing/methods ; Mice ; *CRISPR-Cas Systems ; *Receptors, Chimeric Antigen/genetics/immunology ; *Neoplasms/therapy/immunology/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Xenograft Model Antitumor Assays ; Cell Line, Tumor ; Tumor Microenvironment/immunology ; },
abstract = {Adoptive cell therapy using engineered natural killer (NK) cells is a promising approach for cancer treatment, with targeted gene editing offering the potential to further enhance their therapeutic efficacy. However, the spectrum of actionable genetic targets to overcome tumor and microenvironment-mediated immunosuppression remains largely unexplored. We performed multiple genome-wide CRISPR screens in primary human NK cells and identified critical checkpoints regulating resistance to immunosuppressive pressures. Ablation of MED12, ARIH2, and CCNC significantly improved NK cell antitumor activity against multiple treatment-refractory human cancers in vitro and in vivo. CRISPR editing augmented both innate and CAR-mediated NK cell function, associated with enhanced metabolic fitness, increased secretion of proinflammatory cytokines, and expansion of cytotoxic NK cell subsets. Through high-content genome-wide CRISPR screening in NK cells, this study reveals critical regulators of NK cell function and provides a valuable resource for engineering next-generation NK cell therapies with improved efficacy against cancer.},
}
@article {pmid40845150,
year = {2025},
author = {Carballar-Lejarazú, R and Pham, TB and Tushar, T and James, AA},
title = {Mutant allele formation and inheritance during Cas9/guide RNA-mediated gene drive in a population modification mosquito strain for human malaria control.},
journal = {Genetics},
volume = {231},
number = {3},
pages = {},
pmid = {40845150},
issn = {1943-2631},
support = {//University of California Irvine Malaria Initiative/ ; INV-043645/GATES/Gates Foundation/United States ; AI170692/NH/NIH HHS/United States ; //AAJ/ ; },
mesh = {Animals ; *Anopheles/genetics/parasitology ; *Gene Drive Technology/methods ; Alleles ; *Malaria/prevention &amp; control/parasitology ; *RNA, Guide, CRISPR-Cas Systems/genetics ; Humans ; Mutation ; Mosquito Vectors/genetics ; *CRISPR-Cas Systems ; Female ; Male ; },
abstract = {Gene-drive systems are under development for the population modification of anopheline vectors of human malaria parasites. The key to their success is the fixation of genes in target mosquito populations that encode molecules preventing parasite transmission. High-efficiency Cas9/guide RNA (gRNA)-based gene-drive systems can facilitate this objective. A potential challenge to these systems is the presence of naturally-occurring or drive system-induced sequence polymorphisms in the genomic target site that could impede Cas9/gRNA-mediated cleavage and negatively impact gene-drive dynamics and fixation. Careful choice of the target site can mitigate the impact of natural variation, and here we analyze drive system-mediated, target-site mutagenesis in the outcross and testcross progeny of an Anopheles gambiae strain homo- and hemizygous for the TP13-based gene-drive system. The resulting data allow for estimation of the rates at which drive-system activity generates mutant target-site alleles in the germline and the impact of inherited paternal- and maternal-effect mutations. Functional and nonfunctional mutant alleles are recovered from the germlines at average rates per target gene/generation of 0.08% for paternal and 0.33% for maternal testcross lineages, with an overall average rate of 0.21%. Clustering effects amplify the potential inheritance frequencies of the mutant alleles. Mutations originating in the germlines represent 47% of the total inherited in testcross progeny, with the balance coming from mutant alleles generated by paternal and maternal effects inherited through the respective parental lineages. This approach allows the estimation of potential cleavage-resistant allele formation and inheritance for this drive system in this species and provides empirically derived values to inform more realistic data-driven gene-drive modeling.},
}
@article {pmid40827355,
year = {2025},
author = {Spilsbury, K and Wu, J and Reidy, M and Kropp, PA},
title = {The mitochondrial trans-2-enoyl-CoA reductase is necessary for mitochondrial homeostasis in C. elegans.},
journal = {Genetics},
volume = {231},
number = {3},
pages = {},
doi = {10.1093/genetics/iyaf166},
pmid = {40827355},
issn = {1943-2631},
support = {//Intramural Research Program/ ; //Digestive and Kidney Diseases/ ; //National Heart, Blood, and Lung Institute/ ; //Kenyon College/ ; },
mesh = {Animals ; *Caenorhabditis elegans/genetics/metabolism ; *Mitochondria/metabolism/genetics/enzymology ; *Homeostasis ; *Caenorhabditis elegans Proteins/genetics/metabolism ; Fatty Acids/metabolism/biosynthesis ; CRISPR-Cas Systems ; *Acyl-CoA Dehydrogenases/genetics/metabolism ; Oxidoreductases Acting on CH-CH Group Donors ; },
abstract = {Fatty acids function not only as signaling molecules and for energy storage but also as essential cofactors for mitochondrial enzymes. These fatty acid cofactors are produced by the mitochondrial fatty acid synthesis (mtFAS) pathway, the terminal enzyme of which is mitochondrial trans-2-enoyl-CoA reductase (MECR). Dysfunction of MECR prevents the synthesis of fatty acids and is the monogenic cause of Mitochondrial Enoyl-CoA Protein Associated Neurodegeneration (MEPAN) syndrome, a rare mitochondrial disease characterized by dystonia, basal ganglia degeneration, and optic nerve atrophy. Given the necessity of mtFAS products for mitochondrial function, MECR should be essential. Yet, evidence from MEPAN individuals and model organisms with MECR loss of function indicate that mitochondrial function is not as severely impaired as would be expected. However, many of these studies have been limited to single cells or cell types. To better understand the role of MECR and its products in a multicellular system, we used CRISPR/Cas9 to knock out its 2 orthologs in Caenorhabditis elegans, MECR-1 and MECR-2. We found that only MECR-1 is necessary for normal mitochondrial function, germline development, and neuromuscular function. We thus establish a model in which further studies of MECR/MECR-1 can clarify its biochemical, developmental, and physiological roles.},
}
@article {pmid40784261,
year = {2026},
author = {Wang, Y and Fu, L and Li, S and Tao, D and Gong, P and Yang, Y and Ruan, J and Xie, S and Wang, C and He, D},
title = {A rapid and highly sensitive CRISPR assay utilizing Cas12a orthologs for the detection of Novel Duck Reovirus.},
journal = {Talanta},
volume = {297},
number = {Pt B},
pages = {128680},
doi = {10.1016/j.talanta.2025.128680},
pmid = {40784261},
issn = {1873-3573},
mesh = {Nucleic Acid Amplification Techniques/methods ; Animals ; *Orthoreovirus, Avian/genetics/isolation &amp; purification ; Ducks/virology ; *CRISPR-Associated Proteins/genetics/metabolism ; *Endodeoxyribonucleases/genetics/metabolism ; *CRISPR-Cas Systems ; *Molecular Diagnostic Techniques/methods ; Limit of Detection ; *Reoviridae Infections/diagnosis/veterinary/virology ; Poultry Diseases/virology/diagnosis ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Bacterial Proteins ; },
abstract = {The Novel Duck Reovirus (NDRV) seriously threatens the global poultry industry due to the lack of effective therapies. Preventive measures for NDRV heavily depend on early disease detection, highlighting the need for rapid and sensitive diagnostic methods. This study used Cas12a orthologs Gs12-18 to develop a visual CRISPR-based detection assay targeting the NDRV S3 gene. Comparative analysis of candidate Cas12a proteins, Gs12-16 and Gs12-18, showed that Gs12-18 has significantly better trans-cleavage activity, making it especially suitable for highly sensitive nucleic acid detection. We integrated Gs12-18 with loop-mediated isothermal amplification (LAMP) technology to create a LAMP-CRISPR/Gs12-18 detection platform. This method enables visual detection of the NDRV S3 gene with high specificity and sensitivity, with a detection limit of 38 copies per reaction. It does not require complex equipment and is suitable for point-of-care testing. This research provides a reliable diagnostic tool for the early prevention and control of NDRV.},
}
@article {pmid40591237,
year = {2025},
author = {Sharma, J and Biswas, R and Khare, P},
title = {Design principle of successful genome editing applications using CRISPR-based toolkits.},
journal = {Journal of applied genetics},
volume = {66},
number = {4},
pages = {1065-1077},
pmid = {40591237},
issn = {2190-3883},
mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Streptococcus pyogenes/genetics/enzymology ; Plasmids/genetics ; Genetic Engineering/methods ; RNA, Guide, CRISPR-Cas Systems/genetics ; Synthetic Biology/methods ; },
abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) proteins are the most promising toolkit of synthetic biology for genetic engineering applications across species. Essentially, the Type II CRISPR system, featuring Cas9 nuclease from Streptococcus pyogenes complexed with sgRNA, introduces targeted DNA cleavage, enabling modifications with exceptional precision. This technology can be utilized for not only editing but also modulating gene expressions, thereby finding widespread utility in various biotechnological applications. Here we discuss strategies to construct a consolidated platform aiming at developing a CRISPR-based gene editing system in microbial hosts such as yeast. Employing the well-known gene editing enzymes, i.e., Cpf1 and dCas9, two independent strategies to develop a one-pot plasmid system have been proposed. Furthermore, approaches to reduce off-target cleavages introduced by non-specific targeting of CRISPR complex have been discussed. Finally, an overarching discussion on advanced strategies to design robust CRISPR components is provided for streamlining future genome editing applications.},
}
@article {pmid40579953,
year = {2025},
author = {Ye, Z and Fan, G and Wei, Y and Li, L and Liu, F},
title = {CRISPR/Cas9-mediated germline mutagenesis in the subsocial parasitoid wasp, Sclerodermus guani.},
journal = {Insect molecular biology},
volume = {34},
number = {6},
pages = {939-947},
doi = {10.1111/imb.13014},
pmid = {40579953},
issn = {1365-2583},
support = {Qian Ke He Ji Chu ZK [2024] General 423//Guizhou Provincial Science and Technology Department/ ; 32401587//National Natural Science Foundation of China/ ; QSXM [2022] B09//Guizhou Normal University/ ; Qian Jiao Ji [2024] 54//Department of Education of Guizhou Province/ ; },
mesh = {Animals ; *Wasps/genetics/growth &amp; development ; *CRISPR-Cas Systems ; Female ; Male ; *Mutagenesis ; *Kynurenine 3-Monooxygenase/genetics/metabolism ; Insect Proteins/genetics/metabolism ; Phylogeny ; Gene Editing ; },
abstract = {The ectoparasitoid wasp Sclerodermus guani (Hymenoptera: Bethylidae), as a subsocial insect, is widely applied in biological control against beetle vectors of pine wood nematodes. Despite significant advances in behavioural research, functional genetics in S. guani remains underdeveloped due to the absence of efficient gene manipulation tools. In this study, we employed CRISPR-mediated mutagenesis to achieve germline gene knockout targeting the eye pigment-associated gene kynurenine 3-monooxygenase (KMO). Phylogenetic analysis revealed that S. guani KMO shares a close relationship with its homologue in Prorops nasuta (Hymenoptera: Bethylidae). Two single-guide RNAs (sgRNAs), coupled with Cas9 protein with and without nuclear localisation signal (NLS) were tested. Both sgRNAs induced specific in vitro DNA cleavage and in vivo heritable indels at the target genomic loci. Homozygous null mutant females and males exhibit a white-eye phenotype, which was identified during pupal stage. Optimal editing efficiency in vivo was achieved using the Cas9-NLS variant. Given the complication of germline gene editing in eusocial Hymenopterans, the application of CRISPR in the subsocial parasitoid wasp S. guani provides an accessible research platform for the molecular evolution of insect sociality.},
}
@article {pmid39681701,
year = {2025},
author = {Uijttewaal, ECH and Lee, J and Sell, AC and Botay, N and Vainorius, G and Novatchkova, M and Baar, J and Yang, J and Potzler, T and van der Leij, S and Lowden, C and Sinner, J and Elewaut, A and Gavrilovic, M and Obenauf, A and Schramek, D and Elling, U},
title = {CRISPR-StAR enables high-resolution genetic screening in complex in vivo models.},
journal = {Nature biotechnology},
volume = {43},
number = {11},
pages = {1848-1860},
pmid = {39681701},
issn = {1546-1696},
support = {TFRI Project #1107//Terry Fox Research Institute (Institut de Recherche Terry Fox)/ ; },
mesh = {Animals ; Mice ; *CRISPR-Cas Systems/genetics ; *Genetic Testing/methods ; RNA, Guide, CRISPR-Cas Systems/genetics ; Humans ; Gene Editing/methods ; },
abstract = {Pooled genetic screening with CRISPR-Cas9 has enabled genome-wide, high-resolution mapping of genes to phenotypes, but assessing the effect of a given genetic perturbation requires evaluation of each single guide RNA (sgRNA) in hundreds of cells to counter stochastic genetic drift and obtain robust results. However, resolution is limited in complex, heterogeneous models, such as organoids or tumors transplanted into mice, because achieving sufficient representation requires impractical scaling. This is due to bottleneck effects and biological heterogeneity of cell populations. Here we introduce CRISPR-StAR, a screening method that uses internal controls generated by activating sgRNAs in only half the progeny of each cell subsequent to re-expansion of the cell clone. Our method overcomes both intrinsic and extrinsic heterogeneity as well as genetic drift in bottlenecks by generating clonal, single-cell-derived intrinsic controls. We use CRISPR-StAR to identify in-vivo-specific genetic dependencies in a genome-wide screen in mouse melanoma. Benchmarking against conventional screening demonstrates the improved data quality provided by this technology.},
}
@article {pmid39604565,
year = {2025},
author = {Cullot, G and Aird, EJ and Schlapansky, MF and Yeh, CD and van de Venn, L and Vykhlyantseva, I and Kreutzer, S and Mailänder, D and Lewków, B and Klermund, J and Montellese, C and Biserni, M and Aeschimann, F and Vonarburg, C and Gehart, H and Cathomen, T and Corn, JE},
title = {Genome editing with the HDR-enhancing DNA-PKcs inhibitor AZD7648 causes large-scale genomic alterations.},
journal = {Nature biotechnology},
volume = {43},
number = {11},
pages = {1778-1782},
pmid = {39604565},
issn = {1546-1696},
support = {855741-DDREAMM-ERC-2019-SyG//EC | Horizon 2020 Framework Programme (EU Framework Programme for Research and Innovation H2020)/ ; DFG CA 311/4-1//Deutsche Forschungsgemeinschaft (German Research Foundation)/ ; },
mesh = {*Gene Editing/methods ; Humans ; *DNA-Activated Protein Kinase/antagonists &amp; inhibitors ; *Pyrazoles/pharmacology ; CRISPR-Cas Systems/genetics ; *Recombinational DNA Repair/drug effects/genetics ; Protein Kinase Inhibitors/pharmacology ; Pyrimidines ; },
abstract = {The DNA-PKcs inhibitor AZD7648 enhances CRISPR-Cas9-directed homology-directed repair efficiencies, with potential for clinical utility, but its possible on-target consequences are unknown. We found that genome editing with AZD7648 causes frequent kilobase-scale and megabase-scale deletions, chromosome arm loss and translocations. These large-scale chromosomal alterations evade detection through typical genome editing assays, prompting caution in deploying AZD7648 and reinforcing the need to investigate multiple types of potential editing outcomes.},
}
@article {pmid41219913,
year = {2025},
author = {Muttappagol, M and Sharma, M and Adhikari, DN and Suma, R and Borgohain, T and Saikia, B and Keithellakpam, A and Dutt, A and Laskar, A and Hiremath, S and Chikkaputtaiah, C},
title = {Towards Climate-Resilient Crops: Integrative Strategies for Reproductive Stage Abiotic Stress Tolerance.},
journal = {Physiologia plantarum},
volume = {177},
number = {6},
pages = {e70635},
doi = {10.1111/ppl.70635},
pmid = {41219913},
issn = {1399-3054},
support = {MMP025301//Council of Scientific and Industrial Research, India/ ; CRG/2022/007073 TSA//Anusandhan National Research Foundation (ANRF), Government of India/ ; BT/PR38410/GET/119/310/2020//Department of Biotechnology, Government of India (DBT)/ ; },
abstract = {Abiotic stresses such as drought, heat, cold, and salinity, threaten global crop productivity, particularly during the highly sensitive reproductive phase. These stresses disrupt female gametophyte formation, ovule development, fertilization, and seed maturation, resulting in reduced fertility and yield losses. While the mechanisms of vegetative stress tolerance are relatively well studied, reproductive-stage resilience, especially the resilience of the female gametophyte, remains insufficiently understood, despite its decisive role in determining yield stability. Recent advances provide an opportunity to close this gap through an integrative perspective. Physiological assessments and high-throughput phenotyping offer reliable tools to evaluate gametophyte and embryo performance under stress. Multi-omics approaches, including genomics, transcriptomics, proteomics, and metabolomics, are covering regulatory pathways underlying reproductive success. Emerging technologies such as CRISPR/Cas genome editing, biomarker discovery and machine learning further accelerate trait identification and their application in breeding pipelines. The novelty of this review lies in its focus on the female gametophyte as an overlooked determinant of stress resilience and in presenting a systems-level framework that integrates physiology, omics, breeding, and AI. This integration does more than catalogue stress effects; it connects mechanistic insights with breeding strategies and scales them with computational tools. The ultimate outcome is climate-resilient varieties that sustain productivity under stress, which support farmer livelihoods and contribute to global food security.},
}
@article {pmid41219189,
year = {2025},
author = {Uchigashima, M and Iguchi, R and Fujii, K and Shiku, K and Kumar, P and Liu, X and Isogai, M and Hoshino, C and Abe, M and Nozumi, M and Okamura, Y and Igarashi, M and Sakimura, K and Bise, R and Lavis, LD and Mikuni, T},
title = {Single-cell synaptome mapping of endogenous protein subpopulations in mammalian brain.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {9705},
pmid = {41219189},
issn = {2041-1723},
support = {JP19dm0207080, JP21wm0525014, and JP24wm0625117//Japan Agency for Medical Research and Development (AMED)/ ; JPMJPR16F9//MEXT | JST | Precursory Research for Embryonic Science and Technology (PRESTO)/ ; CDA00043/2019-C//Human Frontier Science Program (HFSP)/ ; N/A//Uehara Memorial Foundation/ ; JP20K21461, JP20H03349, JP20H05918, JP23K18160, and JP24K02130//MEXT | Japan Society for the Promotion of Science (JSPS)/ ; JPMJFR231M//MEXT | Japan Science and Technology Agency (JST)/ ; N/A//Kowa Life Science Foundation/ ; },
mesh = {Animals ; *Synapses/metabolism ; *Brain/metabolism/cytology ; *Single-Cell Analysis/methods ; Mice ; *Neurons/metabolism ; CRISPR-Cas Systems ; Mice, Inbred C57BL ; },
abstract = {Different spatial or temporal protein populations, such as cell-surface/intracellular or pre-existing/nascent subpopulations, determine the basal and activity-induced functions of individual synapses within a neuron in vivo. Here, we developed a simple and generalizable platform to image different spatial and temporal subpopulations of endogenous proteins at thousands of synapses in single neurons in the mammalian brain. The platform is based on the development, improvement and integration of CRISPR-Cas9-mediated protein labeling methods, chemical tag labeling techniques, and a semi-automatic analytical pipeline. The combined platform enables whole-cell mapping of total, cell-surface, intracellular, pre-existing, nascent or nascent-and-surface populations of endogenous proteins, such as receptor, scaffold and signaling proteins, at thousands of synapses in individual neurons in living or fixed mouse brain. Our single-cell "synaptome" mapping of endogenous protein subpopulations comprehensively visualizes the spatial representation of synapse diversity in protein localization, trafficking and turnover, providing valuable insights into single-cell organization and computations in the brain.},
}
@article {pmid41218598,
year = {2025},
author = {Nazarudeen, A and Aswathy, VA and Rauf, AA and Aswathy, UV and Mahesh Chandran, VR and Abhirami, N and Sudhina, S and Chandran, A and Ayyappan, JP},
title = {Targeting Circular RNAs (circRNAs) in Atherosclerosis Using CRISPR Technology.},
journal = {The journal of gene medicine},
volume = {27},
number = {11},
pages = {e70048},
doi = {10.1002/jgm.70048},
pmid = {41218598},
issn = {1521-2254},
support = {//University of Kerala/ ; //Start-up Research Grant from the Science and Engineering Research Board (SERB), India/ ; //Start-up grant for new faculty from the University Grants Commission (UGC), India/ ; //University of Kerala research support in the form of Plan and Nonplan funds/ ; },
mesh = {*RNA, Circular/genetics ; Humans ; *Atherosclerosis/genetics/therapy/metabolism ; *CRISPR-Cas Systems/genetics ; Animals ; Gene Editing/methods ; Genetic Therapy/methods ; Lipid Metabolism/genetics ; },
abstract = {Atherosclerosis is a chronic inflammatory condition that remains a major global cause of cardiovascular morbidity and death. Circular RNAs (circRNAs), emerging as key regulators of biological processes, have been linked to atherosclerosis because of their functions in inflammation, lipid metabolism, and plaque stability. This review explores the biogenesis and cellular functions of circRNAs, highlighting specific circRNAs, such as circANRIL, circHIPK, and circRSF1, which influence atherosclerosis progressions and development. CRISPR-Cas technology, specifically Cas9 and Cas13, has transformed the way atherosclerosis is studied and potentially treated. Targeting PCSK9, LDLR, and APOB to modify lipid metabolism, including lowering LDL cholesterol and repairing mutations in familial hypercholesterolemia, has been made possible using CRISPR-Cas9 in atherosclerosis models. In parallel, CRISPR-Cas13 offers a novel approach for RNA-level intervention by selectively editing circRNAs, providing a dynamic approach to regulate atherosclerosis-related pathways. In order to convert these findings into therapeutic treatments, future research should focus on elucidating the mechanics of circRNA, which in turn determines CRISPR-Cas13, and designing specific delivery systems. This review paper demonstrates the revolutionary promise of circRNA research and CRISPR innovation in the treatment of atherosclerosis and underscores the need for extensive preclinical validation to bridge the gap towards clinical use.},
}
@article {pmid41217051,
year = {2025},
author = {Alam, SS and Mehdi, A and Zafar, A and Ali, S and Rehman, AU and Liaqat, I and Peng, L and Kanwal, F and Afzal, S and Haq, IU and Aftab, MN},
title = {Advances in microbial biofuel production by metabolic and enzyme engineering, synthetic biology, metagenomics, and genome editing applications.},
journal = {Emerging topics in life sciences},
volume = {},
number = {},
pages = {},
doi = {10.1042/ETLS20240002},
pmid = {41217051},
issn = {2397-8554},
abstract = {Microorganisms are the primary source of genetic diversity on earth due to their unparalleled metabolic and functional variability. With the depletion of fossil fuels, a sustainable alternative approach is the use of biofuels, where plant biomass as feedstock is essentially degraded to sugars with the aid of microbe-derived enzymes, followed by the conversion of those sugars to biofuels. Several cellulolytic and non-cellulolytic enzymes are involved in biofuel synthesis. Molecular cloning, along with the advancements in genetic and metabolic engineering in microbial cells, plays a significant contribution to biofuel overproduction. Advanced molecular technologies such as metagenomics and synthetic biology approaches are also being used to construct effective microorganisms for biofuel manufacturing. Obtaining novel enzymes from undiscovered microbial consortia and functional gene analysis is possible through a metagenomics approach. While synthetic biology provides engineered biological systems to generate required biofuel productivity, the CRISPR-Cas genome editing tool is another revolutionary tool being utilized for efficient biofuel production. This article provides a brief overview of different methods of biofuel production using microorganisms.},
}
@article {pmid41216030,
year = {2025},
author = {Naki, D and Gophna, U},
title = {Evolutionary insights into provirus-encoded CRISPR-Cas systems in halophilic archaea.},
journal = {microLife},
volume = {6},
number = {},
pages = {uqaf033},
pmid = {41216030},
issn = {2633-6693},
abstract = {Prokaryotic microorganisms coexist with mobile genetic elements (MGEs), which can be both genetic threats and evolutionary catalysts. In Haloferax lucentense, a halophilic archaeon, we have recently identified an unusual genomic arrangement: a complete type I-B CRISPR-Cas system encoded on a megaplasmid and an incomplete type I-B system within an integrated provirus in the main chromosome. The provirus-encoded system lacks the adaptation genes (cas1, cas2, and cas4), suggesting its potential reliance on the megaplasmid-encoded CRISPR-Cas module for the acquisition of new spacers. This arrangement suggests a potential instance of "adaptive outsourcing," where a provirus might leverage a co-resident MGE for a key function. Through comparative genomics, we show that similar proviral CRISPR-Cas systems are found in distantly related haloarchaea (e.g. Natrinema and Halobacterium), indicating probable virus-mediated horizontal transfer and suggesting they may function as mobile defense modules. Phylogenetic analysis highlights distinct evolutionary origins of the two systems: the plasmid system clusters with other Haloferax CRISPR-Cas systems, while the proviral system clusters with those from other genera, consistent with horizontal acquisition. Interestingly, spacer analysis reveals that the proviral systems predominantly target viral sequences, while the plasmid system appears to target both plasmids and viral sequences, a distribution mirroring broader trends observed in other plasmid- and chromosome-encoded CRISPR systems. This observed targeting preference suggests a potential for complementarity that could support a model of cooperative immunity, where each system may protect its genetic "owner" from competition and, indirectly, the host.},
}
@article {pmid41214117,
year = {2025},
author = {Bakthavachalam, V and Sanborn, MA and Mathayan, M and Salunkhe, SS and Wood, JM and Maienschein-Cline, M and Setty, S and Kabeer, BSA and Rehman, J and Prabhakar, BS},
title = {CRISPR/Cas9-mediated deletion of MADD induces cell cycle arrest and apoptosis in anaplastic thyroid cancer cells.},
journal = {Scientific reports},
volume = {15},
number = {1},
pages = {39264},
pmid = {41214117},
issn = {2045-2322},
support = {VA Merit Review, Award # 5 I01 BX004697//United States (U.S.) Department of Veteran's Affairs Biomedical Laboratory Research and Development Services (BLRD)/ ; },
mesh = {*Apoptosis/genetics ; *CRISPR-Cas Systems/genetics ; Humans ; *Thyroid Carcinoma, Anaplastic/genetics/pathology/metabolism ; Animals ; Cell Line, Tumor ; *Cell Cycle Checkpoints/genetics ; Mice ; *Thyroid Neoplasms/genetics/pathology ; Cell Proliferation/genetics ; *Death Domain Receptor Signaling Adaptor Proteins/genetics ; Gene Expression Regulation, Neoplastic ; Cell Movement/genetics ; Gene Deletion ; Guanine Nucleotide Exchange Factors ; },
abstract = {Anaplastic thyroid cancer (ATC) is an aggressive malignancy with a poor prognosis and limited treatment options. Previous studies have shown that selective downregulation of the MADD (MAP-kinase-activating death domain-containing protein) gene isoform increases ATC cell susceptibility to TRAIL-induced apoptosis. However, the existence of multiple MADD gene isoforms raised the possibility of functional compensation. This study aimed to definitively evaluate the role of MADD in ATC by employing CRISPR-Cas9 to Cas9 to target exon 3, a conserved exon expressed in all known MADD isoforms, resulting in functional knockout of MADD expression. CRISPR-Cas9-mediated MADD knockout, performed in three ATC cell lines (8505 C, C643, and HTH7) with distinct mutational backgrounds, significantly impaired ATC cell function in vitro, as indicated by reduced viability, increased apoptosis, decreased migration, and G0/G1 cell cycle arrest. RNA-seq analysis revealed alterations in genes related to cell survival, proliferation, and metastasis. In the orthotopic ATC mouse model, MADD deletion dramatically suppressed tumor growth, reduced lung metastases, and prolonged survival. Our findings demonstrate that MADD plays a crucial role in ATC cell survival, proliferation, and metastasis. The consistent effects observed across multiple cell lines and in vivo models suggest that MADD may represent a promising therapeutic target for this aggressive malignancy.},
}
@article {pmid41213972,
year = {2025},
author = {Chisholm, CG and Bartlett, R and Brown, ML and Proctor, EJ and Farrawell, NE and Gorman, J and Delerue, F and Ittner, LM and Vine-Perrow, KL and Ecroyd, H and Cashman, NR and Saunders, DN and McAlary, L and Lum, JS and Yerbury, JJ},
title = {Development of a targeted BioPROTAC degrader selective for misfolded SOD1.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {9713},
pmid = {41213972},
issn = {2041-1723},
mesh = {Animals ; *Superoxide Dismutase-1/metabolism/genetics/chemistry ; *Amyotrophic Lateral Sclerosis/genetics/metabolism/pathology/drug therapy ; Mice, Transgenic ; Mice ; Protein Folding ; Humans ; Disease Models, Animal ; Proteolysis ; Motor Neurons/metabolism/pathology ; Ubiquitin-Protein Ligases/metabolism/genetics ; CRISPR-Cas Systems ; Disease Progression ; HEK293 Cells ; },
abstract = {The accumulation of misfolded proteins underlies a broad range of neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS). Due to their dynamic nature, these misfolded proteins have proven challenging to target therapeutically. Here, we specifically target misfolded disease variants of the ALS-associated protein superoxide dismutase 1 (SOD1), using a biological proteolysis targeting chimera (BioPROTAC) composed of a SOD1-specific intrabody and an E3 ubiquitin ligase. Screening of intrabodies and E3 ligases for optimal BioPROTAC construction reveals a candidate capable of degrading multiple disease variants of SOD1, preventing their aggregation in cells. Using CRISPR/Cas9 technology to develop a BioPROTAC transgenic mouse line, we demonstrate that the presence of the BioPROTAC delays disease progression in the SOD1[G93A] mouse model of ALS. Delayed disease progression is associated with protection of motor neurons, a reduction of insoluble SOD1 accumulation and preservation of innervated neuromuscular junctions. These findings provide proof-of-concept evidence and a platform for developing BioPROTACs as a therapeutic strategy for the targeted degradation of neurotoxic misfolded species in the context of neurodegenerative diseases.},
}
@article {pmid41212595,
year = {2025},
author = {Wu, Y and Su, J and Sun, J and Li, Z and Yang, J and Wu, C and Liu, Z and Wang, J and Wang, Y and Liu, J and Duan, L and Wang, C and Zhu, G and Wen, X},
title = {Rapid detection of Seneca Valley virus based on reverse transcription recombinase polymerase amplification and CRISPR-Cas13a.},
journal = {Virulence},
volume = {16},
number = {1},
pages = {2585719},
pmid = {41212595},
issn = {2150-5608},
mesh = {Animals ; Swine ; *CRISPR-Cas Systems ; *Swine Diseases/diagnosis/virology ; Sensitivity and Specificity ; *Picornaviridae/isolation &amp; purification/genetics ; *Picornaviridae Infections/diagnosis/veterinary/virology ; Recombinases/genetics ; *Nucleic Acid Amplification Techniques/methods ; Reverse Transcription ; },
abstract = {The Seneca Valley virus (SVV) is an emerging novel etiological agent that triggers vesicular stomatitis in pigs. SVV leads to considerable financial detriment to the global swine industry. Availability of expeditious, simple, and precise detection techniques would aid in the diagnosis and control of Seneca Valley disease. In this study, we developed a novel SVV detection assay based on reverse transcription recombinase polymerase amplification (RT-RPA)/CRISPR-Cas13a. This technique can specifically identify SVV with no cross-reactivity with other viruses, such as the porcine reproductive and respiratory syndrome virus, pseudorabies virus, classical swine fever virus, and foot-and-mouth disease virus. It boasts a detection threshold as sensitive as 2.43 copies/μL, matching the proficiency of reverse transcriptase quantitative PCR in clinical diagnostics. The streamlined RT-RPA/CRISPR-Cas13a assay for SVV minimizes procedural complexity and the likelihood of contamination. Integrating this method with nucleic acid acquisition via nucleic acid releaser enables a turnaround time of only 40 min from sample acquisition to result determination, underscoring its suitability for on-site diagnostics and affirming its role as a robust instrument for the monitoring and detection of SVV.},
}
@article {pmid41208113,
year = {2025},
author = {Hermann, MD and Fernandez Perez, N and Yeroslaviz, A and Murray, PJ},
title = {Normal macrophage signaling and gene expression in Rosa26 Cas9-expressing mice.},
journal = {ImmunoHorizons},
volume = {9},
number = {11},
pages = {},
pmid = {41208113},
issn = {2573-7732},
support = {//Deutsche Forschungsgemeinschaft/ ; //Max-Planck-Gesellschaft/ ; },
mesh = {Animals ; Mice ; *Macrophages/metabolism/immunology ; *Signal Transduction/genetics ; CRISPR-Cas Systems/genetics ; *CRISPR-Associated Protein 9/genetics/metabolism ; *RNA, Untranslated/genetics ; Mice, Inbred C57BL ; Toll-Like Receptor 3/metabolism ; Mice, Transgenic ; Toll-Like Receptor 4/metabolism ; },
abstract = {Cas9-expression from the Rosa26 "safe harbor" locus are widely used for gene manipulation and Crispr-based screening. Recently, experimental evidence suggested that macrophages isolated from Rosa26-Cas9 mice may have signaling differences compared to control mice in terms of TRIF signaling downstream of TLR3 and TLR4. As we frequently use the Rosa26-Cas9 mice made by Feng Zhang (Cas9-FZ, Jackson Laboratory stock No. 026179), arguably the strain with the widest distribution and utilization, we were motivated to test macrophage signaling in these mice under our conventional conditions. We used different macrophage polarization and signaling conditions combined with RNA sequencing and measurement of TLR signaling by immunoblotting. Our results suggest that the Cas9-FZ mice bear no obvious defects in any commonly used macrophage signaling pathway. We document the differences in our macrophage culture techniques compared to Raychowdhury et al., which may aid in how individual laboratories use Cas9-expressing macrophages, especially for focused or genome-wide screening.},
}
@article {pmid41206041,
year = {2025},
author = {Liu, Q and Pan, TT and Wang, LJ and Zhang, CY},
title = {Mirror-synchronized asymmetric CRISPR nanoswitch for single-molecule profiling of multiple circRNAs in different stages of breast cancer.},
journal = {Nucleic acids research},
volume = {53},
number = {20},
pages = {},
pmid = {41206041},
issn = {1362-4962},
support = {22474019//National Natural Science Foundation of China/ ; },
mesh = {*RNA, Circular/genetics ; *Breast Neoplasms/genetics/pathology ; Humans ; *CRISPR-Cas Systems/genetics ; Female ; *RNA/genetics ; CRISPR-Associated Proteins/genetics/metabolism ; Single Molecule Imaging/methods ; Bacterial Proteins ; Endodeoxyribonucleases ; },
abstract = {Circular RNAs (circRNAs) represent a class of endogenous noncoding RNAs characterized by their covalently closed circular structures. They have been implicated in significant transcriptional and post-transcriptional regulation of gene expression. Here, we present a one-pot method for the detection of circRNAs based on engineered DNA hairpins and CRISPR-Cas12a signal amplification, which involves signal pre-amplification via coupled probe-mediated hairpin amplification of two palindromic hairpins and Cas12a signal generation via trans-cleavage. We demonstrate that this platform is sensitive (detection limit of 1.07 aM), specific (capable of single-mismatch discrimination), and fast (reaction time of 25 min) and can be used to detect different circRNAs from RNase R-treated RNA (both in vitro and in clinically relevant samples, including correct classification of disease progression). This method enables single-molecule profiling and can be extended to detect other types of nucleic acids.},
}
@article {pmid41205588,
year = {2025},
author = {Kumar, KRR},
title = {Plant genome editing goes viral: balancing innovation and biosafety.},
journal = {Trends in biotechnology},
volume = {43},
number = {11},
pages = {2684-2685},
doi = {10.1016/j.tibtech.2025.09.006},
pmid = {41205588},
issn = {1879-3096},
mesh = {*Gene Editing/methods ; *Genome, Plant/genetics ; *Plants, Genetically Modified/genetics ; Genetic Vectors/genetics ; CRISPR-Cas Systems ; Containment of Biohazards ; },
abstract = {Weiss and colleagues demonstrated a breakthrough in transgene-free heritable genome editing using viral vectors. While promising for controlled laboratory applications, the open-field use envisaged by Sajjad and colleagues raises ecological and biosafety concerns. Rigorous risk assessment is essential to harness innovation responsibly while safeguarding biodiversity and public trust.},
}
@article {pmid41190788,
year = {2025},
author = {Orzechowski, M and Hoikkala, V and Chi, H and McMahon, S and Gloster, T and White, MF},
title = {A viral SAVED protein with ring nuclease activity degrades the CRISPR second messenger cA4.},
journal = {The Biochemical journal},
volume = {482},
number = {22},
pages = {},
doi = {10.1042/BCJ20253271},
pmid = {41190788},
issn = {1470-8728},
mesh = {*Viral Proteins/metabolism/genetics/chemistry ; *Adenine Nucleotides/metabolism ; *CRISPR-Cas Systems ; *Second Messenger Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Oligoribonucleotides ; },
abstract = {Type III CRISPR systems typically generate cyclic oligoadenylate second messengers such as cyclic tetra-adenylate (cA4) on detection of foreign RNA. These activate ancillary effector proteins which elicit a diverse range of immune responses. The Calp (CRISPR associated Lon protease) system elicits a transcriptional response to infection when CalpL (Calp Lon protease) binds cA4 in its SAVED (SMODS associated and fused to various effectors domain) sensor domain, resulting in filament formation and activation of the Lon protease domain, which cleaves the anti-Sigma factor CalpT, releasing the CalpS (Calp Sigma factor) for transcriptional remodelling. Here, we show that thermophilic viruses have appropriated the SAVED domain of CalpL as an anti-CRISPR, AcrIII-2 (second anti-CRISPR of type III systems), which they use to degrade cA4. AcrIII-2 dimers sandwich cA4, degrading it in a shared active site to short linear products, using a mechanism highly reminiscent of CalpL. This results in inhibition of a range of cA4 activated effectors in vitro. This is the first example of a virally encoded SAVED domain with ring nuclease activity, highlighting the complex interplay between viruses and cellular defences.},
}
@article {pmid41187216,
year = {2025},
author = {Treaster, MJ and McCann, J and Solovei, KS and Palmieri, RJ and White, MA},
title = {A Y-linked duplication of anti-Mullerian hormone is the sex determination gene in threespine stickleback.},
journal = {PLoS genetics},
volume = {21},
number = {11},
pages = {e1011932},
pmid = {41187216},
issn = {1553-7404},
mesh = {Animals ; *Smegmamorpha/genetics ; Male ; *Anti-Mullerian Hormone/genetics ; *Sex Determination Processes/genetics ; Female ; *Y Chromosome/genetics ; *Gene Duplication ; CRISPR-Cas Systems ; Evolution, Molecular ; Sex Differentiation/genetics ; },
abstract = {Many taxa have independently evolved genetic sex determination where a single gene located on a sex chromosome controls gonadal differentiation. The gene anti-Mullerian hormone (amh) has convergently evolved as a sex determination gene in numerous vertebrate species, but how this gene has repeatedly evolved this novel function is not well understood. In the threespine stickleback (Gasterosteus aculeatus), amh was duplicated onto the Y chromosome (amhy) ~22 million years ago. To determine whether amhy is the primary sex determination gene, we used CRISPR/Cas9 and transgenesis to show that amhy is necessary and sufficient for male sex determination, consistent with the function of a primary sex determination gene. We find that amhy contributes to a higher total dosage of amh early in development and likely contributes to differential germ cell proliferation key to sex determination. The creation of sex-reversed lines also allowed us to investigate the genetic basis of secondary sex characteristics. Threespine stickleback have striking differences in behavior and morphology between sexes. Here we show one of the classic traits important for reproductive success, blue male nuptial coloration, is controlled by both sex-linked genetic factors as well as hormonal factors independent of sex chromosome genotype. This research establishes stickleback as a model to investigate how amh regulates gonadal development and how this gene repeatedly evolves novel function in sex determination. Analogous to the "Four Core Genotypes" model in house mice, sex-reversed threespine stickleback offer a new vertebrate model for investigating the separate contributions of gonadal sex and sex chromosomes to sexual dimorphism.},
}
@article {pmid41170863,
year = {2025},
author = {Liu, J and Feng, G and Guo, C and Liu, D and Li, Y},
title = {Opportunities and Challenges of CRISPR-Cas9 in the Sustainable Development of Animal Husbandry.},
journal = {Journal of agricultural and food chemistry},
volume = {73},
number = {45},
pages = {28575-28587},
doi = {10.1021/acs.jafc.5c09076},
pmid = {41170863},
issn = {1520-5118},
mesh = {*CRISPR-Cas Systems ; Animals ; Gene Editing ; Sustainable Development ; *Animal Husbandry/methods ; *Livestock/genetics/metabolism/growth &amp; development ; Animals, Genetically Modified/genetics/metabolism ; },
abstract = {Food security is of vital importance to the destiny of the entire globe. However, the realization of the United Nations' Sustainable Development Goals, which advocate for the eradication of hunger and the improvement of nutrition, appears somewhat bleak. The advent of gene editing has made precise modification of biological genomes a reality. Among these technologies, CRISPR-Cas9, due to its convenience and high efficiency, has been widely applied in fields such as agriculture. The application of CRISPR-Cas9 in genetically engineered animals can rapidly improve animal traits, reduce resource consumption, and has great potential in addressing food security issues. Therefore, this review focuses on the editing principles of CRISPR-Cas9, its developmental trajectory, and the opportunities and challenges it presents in animal husbandry, aiming to provide a theoretical reference for the further application of CRISPR-Cas9 to promote the sustainable development of animal husbandry.},
}
@article {pmid41162785,
year = {2025},
author = {Coda, DM and Watt, L and Glauser, L and Batiuk, MY and Burns, AM and Stahl, CL and Wong, LY and Gräff, J},
title = {Cell-type- and locus-specific epigenetic editing of memory expression.},
journal = {Nature genetics},
volume = {57},
number = {11},
pages = {2661-2668},
pmid = {41162785},
issn = {1546-1718},
support = {101043457//EC | EU Framework Programme for Research and Innovation H2020 | H2020 Priority Excellent Science | H2020 European Research Council (H2020 Excellent Science - European Research Council)/ ; 310030_219342//Schweizerischer Nationalfonds zur F&#xf6;rderung der Wissenschaftlichen Forschung (Swiss National Science Foundation)/ ; 310030_197752//Schweizerischer Nationalfonds zur F&#xf6;rderung der Wissenschaftlichen Forschung (Swiss National Science Foundation)/ ; },
mesh = {*Epigenesis, Genetic/genetics ; *Memory/physiology ; Animals ; *Gene Editing/methods ; Mice ; CRISPR-Cas Systems/genetics ; Promoter Regions, Genetic ; Nerve Tissue Proteins/genetics ; Neuronal Plasticity/genetics ; Male ; Cytoskeletal Proteins/genetics ; Neurons/metabolism/physiology ; Epigenome Editing ; },
abstract = {Epigenetic mechanisms have long been proposed to act as molecular mnemonics[1-3], but whether the epigenetic makeup of a single genomic site can guide learnt behaviors remains unknown. Here we combined CRISPR-based epigenetic editing tools[4,5] with c-Fos-driven engram technologies[6,7] to address this question in memory-bearing neuronal ensembles. Focusing on the promoter of Arc, which encodes a master regulator of synaptic plasticity[8], we found that its locus-specific and temporally controlled epigenetic editing is necessary and sufficient to regulate memory expression. Such effects occurred irrespective of the memory phase-during the initially labile period after learning and for fully consolidated memories-and were reversible within subject, testifying to their inherent plasticity. These findings provide a proof-of-principle that site-specific epigenetic dynamics are causally implicated in memory expression.},
}
@article {pmid41087678,
year = {2025},
author = {Kaplan, EG and Steger, RJ and Shah, ST and Drepanos, LM and Griffith, AL and Reint, G and Doench, JG},
title = {Activity-based selection for enhanced base editor mutational scanning.},
journal = {Nature genetics},
volume = {57},
number = {11},
pages = {2920-2929},
pmid = {41087678},
issn = {1546-1718},
mesh = {Humans ; *Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; *Mutation ; RNA, Guide, CRISPR-Cas Systems/genetics ; Tumor Suppressor Protein p53/genetics ; },
abstract = {Base editing is a CRISPR-based technology that enables high-throughput, nucleotide-level functional interrogation of the genome that is essential for understanding the genetic basis of human disease and informing therapeutic development. Base editing screens have emerged as a powerful experimental approach, yet significant cell-to-cell variability in editing efficiency introduces noise that may obscure meaningful results. Here we develop a co-selection method that enriches for cells with high base editing activity, substantially increasing editing efficiency at a target locus. We evaluate this activity-based selection method against a traditional screening approach by tiling guide RNAs across TP53, demonstrating its enhanced capacity to pinpoint specific mutations and protein regions of functional importance. We anticipate that this modular selection method will enhance the resolution of base editing screens across many applications.},
}
@article {pmid40996032,
year = {2025},
author = {Mofed, D and Gowripalan, A and Berrigan, J and Das, PK and Pujari, N and Ajasin, D and Haldar, S and McCullough, J and Zhang, Y and Kalpana, GV and Bresnick, A and Kielian, M and Wilson, DW and Zhang, J and Chandran, K and Prasad, VR},
title = {Influence of CCL2-mediated modulation of ALIX in the budding and replication of viruses from multiple families.},
journal = {mBio},
volume = {16},
number = {11},
pages = {e0224125},
doi = {10.1128/mbio.02241-25},
pmid = {40996032},
issn = {2150-7511},
support = {R01 AI125244/AI/NIAID NIH HHS/United States ; R01 AI153008/NH/NIH HHS/United States ; R01 AI125244/NH/NIH HHS/United States ; R01 AI075647/NH/NIH HHS/United States ; R01 AI185073/NH/NIH HHS/United States ; P30CA013330/NH/NIH HHS/United States ; S10OD026833-01/NH/NIH HHS/United States ; S10OD032169-01/NH/NIH HHS/United States ; },
mesh = {Humans ; HeLa Cells ; *Virus Replication ; *Endosomal Sorting Complexes Required for Transport/metabolism/genetics ; *Calcium-Binding Proteins/metabolism/genetics ; *Chemokine CCL2/genetics/metabolism ; *Virus Release ; HIV-1/physiology/genetics ; *Cell Cycle Proteins/metabolism/genetics ; Gene Knockout Techniques ; Signal Transduction ; CRISPR-Cas Systems ; Animals ; },
abstract = {UNLABELLED: Signaling by C-C motif ligand 2 (CCL2), a β-chemokine, modulates HIV-1 budding and release by mobilizing ALG-2-interacting protein X (ALIX) from the F-actin cytoskeleton to the cytosol. Immunodepleting CCL2 in the medium sequesters ALIX to F-actin. We developed a novel tool to study HIV budding and release without mutating viral late domains or silencing ESCRT genes, but by blocking CCL2 signaling using CRISPR-Cas9 knockout (KO) of the CCL2 or CCR2 genes. We knocked out CCL2 (CCL2KO) and CCR2 (CCR2KO) singly or together (double knockout) in HeLa cells and confirmed that knockout was associated with the absence of CCL2 or CCR2 expression. In KO cells, ALIX was associated with the F-actin cytoskeleton, while in control cells, it was associated with the cytosolic soluble fraction. In KO cells, HIV-1 production was profoundly reduced (10-fold). Strikingly, for CCL2KO cells, the addition of CCL2 mobilized ALIX to the soluble fraction, and virus production was stimulated to levels higher than those of untreated HeLa cells. We utilized these cells to test the involvement of ALIX in the budding and/or replication of several viruses, including Simian Immunodeficiency Virus (SIV), Equine Infectious Anemia Virus (EIAV), Herpes Simplex Virus type 1 (HSV-1), Dengue virus (DENV), and Hazara virus (HAZV). Budding and release of SIV and EIAV were both inhibited in CCL2KO cells and rescued by CCL2 addition. Replication of HSV-1 and DENV was unaffected in CCL2KO cells, confirming that ALIX is not involved in their replication. Finally, HAZV replication was affected by CCL2 signaling. Our studies indicate that CCL2 signaling and ALIX mobilization are important for several viral families.<br><br>IMPORTANCE: C-C motif ligand 2 (CCL2) plays a regulatory role in the budding and release of HIV-1 in macrophages and HeLa cells. CCL2 signaling mobilizes ALG-2-interacting protein X (ALIX) from the F-actin cytoskeleton to the soluble cytosol, where it is accessible for recruitment by the HIV-1 Gag polyprotein in the assembling virions at the plasma membrane. In previous studies, CCL2 immunodepletion, which blocks CCL2 signaling, resulted in ALIX sequestration to the F-actin cytoskeleton and inhibited virus production. Here, we developed a HeLa CCL2 gene knockout cell line and found that abrogation of CCL2 signaling can be restored by CCL2 addition, as evidenced by the restoration of ALIX to the cytosolic fraction and rescue of HIV-1 release. Employing such a system, we tested Simian Immunodeficiency Virus, Equine Infectious Anemia Virus, Herpes Simplex Virus type 1, Dengue, and Hazara virus for their dependence on ALIX for virus replication. The results indicate that CCL2 signaling and ALIX release from F-actin may play a role in the replication of several viruses.},
}
@article {pmid40980903,
year = {2025},
author = {Khurram, I and Choudhery, MS and Ghani, MU and Arif, T and Naeem, A and Mahmood, R and Niaz, A and Khan, MU},
title = {Gene Editing for Cystic Fibrosis: Advances and Prospects of CRISPR-Cas9 Therapy.},
journal = {Cell biology international},
volume = {49},
number = {12},
pages = {1564-1578},
doi = {10.1002/cbin.70082},
pmid = {40980903},
issn = {1095-8355},
mesh = {*Cystic Fibrosis/genetics/therapy/diagnosis ; Humans ; *Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; Cystic Fibrosis Transmembrane Conductance Regulator/genetics/metabolism ; *Genetic Therapy/methods ; Mutation/genetics ; Animals ; },
abstract = {Cystic fibrosis (CF) is an inherited, autosomal recessive disorder that is caused by mutations in the gene encoding cystic fibrosis transmembrane conductance regulator (CFTR). CFTR maintains the balance between water and salts by transporting chloride ions along various epithelial surfaces. CFTR impairment affects the function of several organs, including the lungs. Newborn screening, prenatal diagnosis, and pharmacological interventions have altered the prevalence and incidence of cystic fibrosis. Although CFTR modulators are a promising treatment option, their ability to target and correct only one mutation at a time restricts their therapeutic potential. The development of genome editing technologies such as Clustered Regularly Interspaced Short Palindromic Repeats-Cas(CRISPR-Cas9) has the potential to correct genetic mutations, including those associated with CF, thereby offering a permanent treatment by fixing the root cause of CF. This article summarizes cystic fibrosis development, prognosis, and diagnosis, as well as possibilities for correcting various types of CFTR gene mutations. The review focuses on the potential of gene editing technologies to repair CFTR mutations and their applications in the advancement of CF treatment.},
}
@article {pmid40975652,
year = {2025},
author = {Sajjad, MW and Naqvi, RZ and Amin, I},
title = {Viral genome editing: striking a balance between promises and precautions.},
journal = {Trends in biotechnology},
volume = {43},
number = {11},
pages = {2686-2687},
doi = {10.1016/j.tibtech.2025.09.010},
pmid = {40975652},
issn = {1879-3096},
mesh = {*Genome, Viral/genetics ; *Genetic Vectors/genetics ; *Genetic Engineering/methods ; Humans ; *Viruses/genetics ; *Gene Editing ; CRISPR-Cas Systems ; Agriculture ; },
abstract = {Viral vectors can provide transgene-free genome editing. The forward-looking frameworks for their use must be biosafety and freedom from ecological risk. The responsible innovations, showing a balance between promise and caution following regulatory frameworks, can guarantee that viral genome editing develops into a revolutionary but environmentally conscious strategy for sustainable agricultural improvement.},
}
@article {pmid40962831,
year = {2025},
author = {Su, X and Zhang, H and Hong, Y and Yang, Q and Wang, L and Le, T and Liu, J and Cheruvu, L and Labour, E and Zhang, S and Mendez-Maldonado, K and Kreimer, A and Song, H and Ming, GL and Duan, J and Pang, ZP},
title = {Mutations of schizophrenia risk gene SETD1A dysregulate synaptic function in human neurons.},
journal = {Molecular psychiatry},
volume = {30},
number = {12},
pages = {5680-5693},
pmid = {40962831},
issn = {1476-5578},
support = {ASF_23-004/ASF/Autism Science Foundation/United States ; R01MH125528//U.S. Department of Health &amp; Human Services | NIH | National Institute of Mental Health (NIMH)/ ; R01 MH106575/MH/NIMH NIH HHS/United States ; 74260//Robert Wood Johnson Foundation (RWJF)/ ; R01 AG081374/AG/NIA NIH HHS/United States ; R01 AG063175/AG/NIA NIH HHS/United States ; RM1 MH133065/MH/NIMH NIH HHS/United States ; ASF_23-004/ASF/Autism Science Foundation/United States ; },
mesh = {Humans ; *Schizophrenia/genetics/metabolism ; *Histone-Lysine N-Methyltransferase/genetics/metabolism ; Neurons/metabolism/physiology ; Induced Pluripotent Stem Cells/metabolism ; Mutation/genetics ; Synapses/genetics/metabolism ; Genetic Predisposition to Disease ; Haploinsufficiency/genetics ; CRISPR-Cas Systems/genetics ; },
abstract = {Schizophrenia (SCZ) is a complex neuropsychiatric disorder associated with both common risk variants of small effect sizes and rare risk variants of high penetrance. Rare premature open reading frame (ORF) termination variants in SETD1A (SET Domain Containing 1A) show a strong association with SCZ; however, it remains largely unclear how rare premature ORF termination variants in SETD1A contribute to the pathophysiology of SCZ. To understand the impact of SETD1A rare premature ORF termination variants in human neurons, we CRISPR/Cas9-engineered five isogenic pairs of human induced pluripotent stem cells (iPSCs), with a recurrent heterozygous patient-specific premature ORF termination mutation c.4582-2delAG in two donor lines and a heterozygous frameshift mutation c.4596_4597insG (p. Leu1533fs) in three donor lines. These two mutations are predicted to cause a premature stop codon in exon 16 of SETD1A, leading to SETD1A haploinsufficiency. We found that these presumably loss-of-function (LoF) mutations caused the SETD1A mRNAs to be degraded by nonsense-mediated decay (NMD), accompanied by a reduction of full-length SETD1A protein level in iPSCs. We then characterized the morphological, electrophysiological, and transcriptomic impacts of SETD1A[+/-] LoF mutations in iPSC-derived human excitatory neurons induced by NGN2. We found that the SETD1A[+/-] exon-16 LoF mutations altered dendrite complexity, dysregulated synaptic transmission, and synaptic plasticity, likely by dysregulating genes involved in synaptic function. These results provide mechanistic insights into how SETD1A[+/-] exon-16 patient-specific LoF mutations affect neuron phenotypes that may be relevant to the pathophysiology of SCZ.},
}
@article {pmid40817029,
year = {2025},
author = {Vidal, P and Giménez-Dejoz, J and Fernandez-Lopez, L and Romero, S and Nazemi, SA and Luengo, M and Gonzalez-Alfonso, JL and Martinez-Sugrañes, M and Robles-Martín, A and Almendral, D and Roda, S and Pérez-García, P and Kruse, L and Jaeger, KE and Streit, WR and Plou, FJ and Floor, M and Shahgaldian, P and Bargiela, R and Guallar, V and Ferrer, M},
title = {Computationally guided genome rewiring of Escherichia coli and its application for nanopolyethylene terephthalate (PET) biodegradation and upcycling.},
journal = {Trends in biotechnology},
volume = {43},
number = {11},
pages = {2874-2903},
doi = {10.1016/j.tibtech.2025.07.008},
pmid = {40817029},
issn = {1879-3096},
mesh = {*Polyethylene Terephthalates/metabolism/chemistry ; *Escherichia coli/genetics/metabolism ; Biodegradation, Environmental ; *Metabolic Engineering/methods ; *Genome, Bacterial ; *Nanoparticles/chemistry/metabolism ; CRISPR-Cas Systems ; Escherichia coli Proteins/genetics/metabolism ; Gene Editing/methods ; },
abstract = {Numerous strategies for the biodegradation and upcycling of polyethylene terephthalate (PET) are under investigation. Here, we present a proof-of-concept study for reprogramming the Escherichia coli BL21(DE3) strain to degrade PET nanoparticles (nPET) without introducing foreign DNA and compromising native cellular fitness. In brief, native proteins selected in silico from the genome were repurposed to acquire artificial PETase activity without compromising their function and were subsequently replaced via CRISPR/Cas9 editing. A variant of the transport protein LsrB, selected for its ability to bind PET, was engineered to degrade PET powder (at 37-60°C). Building on LsrB periplasmic localization, we engineered a strain that degrades nPET at 37°C. The strain was further engineered to grow on nPET degradation products and produce valuable compounds. Our method, which is applicable across diverse genomes and microbial chassis, expands the potential of metabolic engineering to address plastic biodegradation and upcycling while reducing reliance on foreign DNA.},
}
@article {pmid40473489,
year = {2025},
author = {Sen, MK and Sellamuthu, G and Mondal, SK and Varshney, RK and Roy, A},
title = {Epigenome editing for herbicide-resistant crops.},
journal = {Trends in plant science},
volume = {30},
number = {11},
pages = {1201-1210},
doi = {10.1016/j.tplants.2025.05.003},
pmid = {40473489},
issn = {1878-4372},
mesh = {*Gene Editing/methods ; *Crops, Agricultural/genetics/drug effects ; *Herbicide Resistance/genetics ; CRISPR-Cas Systems/genetics ; *Epigenome/genetics ; *Herbicides/pharmacology ; Plants, Genetically Modified/genetics ; Epigenome Editing ; },
abstract = {Herbicide resistance (HR) is fundamental for sustainable agriculture as global food security increasingly relies on efficient and eco-friendly weed management. Recent advances in CRISPR/dCas9-based epigenome editing offer a promising, non-genetic approach by precisely targeting regulatory regions of genes involved in herbicide sensitivity and detoxification. While CRISPR/Cas9 has successfully been used to develop HR crops, CRISPR/dCas9 remains underexplored in this field. We propose that CRISPR/dCas9-driven epigenome editing could enable time- and tissue-specific control of gene expression, allowing for the introduction of heritable HR traits without altering DNA sequences. This innovative approach could transform sustainable HR development, offering a powerful solution to enhance agricultural resilience and food security while aligning with eco-friendly weed management strategies.},
}
@article {pmid41215733,
year = {2025},
author = {Claisse, O and Mosterd, C and Marrec, CL and Samot, J},
title = {Defense Systems and Prophage Detection in Streptococcus mutans Strains.},
journal = {Molecular oral microbiology},
volume = {},
number = {},
pages = {e70014},
doi = {10.1111/omi.70014},
pmid = {41215733},
issn = {2041-1014},
support = {//Association France Parkinson/ ; },
abstract = {Although the species is extensively studied, limited data are available on antiphage defense systems (APDSs) in Streptococcus mutans. The present study aimed to explore the diversity and the occurrence of APDSs and to search for prophages in the genomes of clinical isolates of S. mutans using bioinformatics tools. Forty-four clinical isolates of S. mutans were obtained from saliva samples of people with Parkinson's disease. Genomic DNA was extracted, sequenced using Illumina MiSeq technology, and analyzed for the presence of defense systems using DefenseFinder and PADLOC. CRISPR-Cas systems were characterized using CRISPRCasFinder, and prophages were detected by the PhiSpy pipeline from RAST. AcrFinder and AcrHub were used to identify anti-CRISPR proteins. Each strain harbored between 6 and 12 APDS, with restriction-modification systems being the most prevalent, followed by the MazEF toxin-antitoxin system and CRISPR-Cas systems. Type II-C CRISPR-Cas systems were not identified here in S. mutans. Novel variations in type II-A signature protein Cas9 were identified, allowing their classification into four distinct groups. Variability in direct repeat sequences within the same CRISPR array was also observed, and 80% of the spacers were classified as targeting "dark matter". A unique prophage, phi_37bPJ2, was detected, showing high similarity with previously described phages. The AcrIIA5 protein encoded by phi_37bPJ2 was conserved and suggested to remain functionally active. This study reveals the diversity of APDSs in S. mutans and the limited presence of prophages. The findings provide a foundation for future research on the evolutionary dynamics of these systems and their role in S. mutans adaptation to phage pressure.},
}
@article {pmid41215480,
year = {2025},
author = {Ali, RM and Arshad, H and Zafar, AQ and Gull, S and Gul, A and Mansoor, F and Zahid, R},
title = {A Comprehensive Review on Engineering Lactic Acid Bacteria: Emerging Genetic Tools and Synthetic Biology Strategies.},
journal = {Biotechnology and applied biochemistry},
volume = {},
number = {},
pages = {},
doi = {10.1002/bab.70079},
pmid = {41215480},
issn = {1470-8744},
abstract = {Lactic acid bacteria (LAB) are pivotal in food, pharmaceutical, and environmental applications due to their metabolic versatility and probiotic potential. This review explores the advancements in genetic engineering and synthetic biology strategies to enhance LAB functionality. We examine the genomic architecture of key LAB species, such as Lactobacillus and Lactococcus, highlighting their natural genetic traits and metabolic constraints. Emerging genetic tools, including electroporation, conjugation, and CRISPR-Cas systems, have revolutionized LAB modification, enabling precise gene editing and expression control. Synthetic biology approaches, such as genetic circuits, riboswitches, and biosensor development, offer novel pathways for optimizing LAB for functional foods, mucosal therapeutics, and industrial biotechnology. We discuss applications in probiotic delivery, bioremediation, and agricultural enhancement, emphasizing LAB's role in producing bioactive metabolites and combating pathogens. Challenges, including plasmid instability, metabolic burden, and regulatory hurdles, are addressed alongside socio-ethical considerations for genetically modified LAB. The integration of genome-scale engineering and CRISPR-based technologies holds promise for overcoming these barriers, paving the way for next-generation LAB with enhanced stress tolerance and tailored functionalities. This review synthesizes current knowledge and future prospects, underscoring the transformative potential of engineered LAB in addressing global health, environmental, and industrial needs while navigating biosafety and public perception challenges.},
}
@article {pmid41213945,
year = {2025},
author = {Hoetzel, J and Wang, T and Suess, B},
title = {Beyond the niche - unlocking the full potential of synthetic riboswitches.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {9897},
pmid = {41213945},
issn = {2041-1723},
support = {SU402/12-1//Deutsche Forschungsgemeinschaft (German Research Foundation)/ ; WA 5722/1-1//Deutsche Forschungsgemeinschaft (German Research Foundation)/ ; },
abstract = {Synthetic riboswitches have undergone great development in the past decade, evolving into valuable regulatory tools. Operating entirely at the RNA level and independently of auxiliary proteins, they offer a promising alternative to protein-based systems such as TetON/OFF or CRISPR-Cas. As compact, modular RNA elements they unite sensing and regulatory functions within a single molecule, giving them the advantages of high modularity, portability and low metabolic burden. Here, we explore the unique features of synthetic riboswitches, highlight key applications, assess current bottlenecks and limitations and put them in context with emerging solutions, to emphasise the potential of synthetic riboswitches.},
}
@article {pmid41212235,
year = {2025},
author = {Yilmaz-Çolak, &#xc7;},
title = {The CRISPR-cas repertoire of Kluyvera ascorbata: insights from genomic data.},
journal = {Current genetics},
volume = {71},
number = {1},
pages = {28},
pmid = {41212235},
issn = {1432-0983},
abstract = {The genus Kluyvera has gained increasing attention due to their emerging role as opportunistic pathogens and their antibiotic resistance determinants. Various approaches have been employed to reveal genomic insights into the evolution and pathogenicity of Kluyvera species. However, detailed knowledge about Kluyvera-specific clustered regularly interspaced short palindromic repeats (CRISPR) is still missing. In this study, a genome-mining approach was employed for the characterization of CRISPR-Cas loci in a total of 13 complete Kluyvera genomes using CRISPRCasFinder and related tools. Out of 13 species, only K. ascorbata displayed multiple CRISPR-Cas arrays and a complete set of cas genes characteristics of a type I-E system. Spacer analysis revealed potential targets within phage and plasmid sequences, indicating historical exposure to mobile genetic elements. Furthermore, a phylogenetic tree constructed using the Cas3 protein sequence positioned K. ascorbata closely with other enteric bacteria, including Salmonella spp. and Citrobacter spp. This study provides the first detailed insight into the CRISPR-Cas architecture of K. ascorbata. Although there is no significant diversity of the CRISPR-Cas system identified in this species, it can emphasize a role as active immune defenses against invaders and offer a foundation for future functional and evolutionary investigations. Moreover, difficulties in identification of the genus Kluyvera can be overcome through the CRISPR-Cas system using next-generation diagnostics tools.},
}
@article {pmid41211267,
year = {2025},
author = {Ramesh, B and Fakoya, AO},
title = {Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas Unleashed: Transforming Gene Editing With Breakthroughs, Applications, and Ethical Dilemmas.},
journal = {Cureus},
volume = {17},
number = {11},
pages = {e95908},
pmid = {41211267},
issn = {2168-8184},
abstract = {The most significant breakthrough in gene editing is the advent of the clustered regularly interspaced short palindromic repeats (CRISPR)-Cas system. This innovative technology enables scientists to insert or delete genes using specific enzymes, facilitating modifications to genomes that can influence an organism's phenotype. The Cas9 enzyme is the most widely used within the CRISPR framework and has already received approval for treating sickle cell disease, with many other applications likely to follow. As this rapidly evolving field continues to advance, it holds great promise for addressing genetic disorders and diseases. This article will explore the various enzymes available in the CRISPR system, the range of diseases and conditions that could be treated using this technology, alternative gene therapy methods, and the ethical considerations surrounding its use.},
}
@article {pmid41211185,
year = {2025},
author = {Allmang, S and Witzel, HR and Hausen, A and Marquard, S and Eckert, C and Marnet, N and Hörner, N and Mayer, P and Heinrich, S and Dang, H and Roth, W and Gaida, MM},
title = {Aberrant Expression of A Disintegrin and Metalloproteinase With Thrombospondin Motifs 13 (ADAMTS13) in Pancreatic Cancer Leads to Dichotomic Functions.},
journal = {MedComm},
volume = {6},
number = {11},
pages = {e70462},
pmid = {41211185},
issn = {2688-2663},
abstract = {Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive cancers characterized by highly invasive growth into the surrounding peripancreatic fat tissue, where tumor cells can directly interact with adipocytes. Due to poor response to the currently available (radio)chemotherapies, there is an urgent need for advanced therapy concepts. The present study shows that ADAMTS13 (a disintegrin and metalloproteinase with thrombospondin motifs 13), a key factor in blood coagulation, is significantly overexpressed in human PDAC. Immunohistochemical analysis revealed that ADAMTS13 expression is associated with prolonged survival and negatively correlated with vascular density. In vitro and in vivo experiments demonstrate its partial induction by leptin. Mechanistically, CRISPR/Cas-mediated ADAMTS13 knockout in PDAC cells resulted in reduced migration and invasion. In an avian xenograft tumor model, ADAMTS13 loss led to increased vascularization, decreased vascular length, and diminished tumor growth, accompanied by reduced expression of multiple key angiogenic and angioplastic factors. Furthermore, loss of ADAMTS13 was associated with decreased expression of mesenchymal markers. In conclusion, we identified an aberrant expression and alternative function of ADAMTS13 in PDAC linked to tumor progression, plasticity, and angiogenesis, partly induced by the peripancreatic fat tissue, making this metalloproteinase an interesting target for personalized therapies.},
}
@article {pmid41210586,
year = {2025},
author = {Joo, JH and Lee, S and Kim, KP},
title = {Precision gene editing: The power of CRISPR-Cas in modern genetics.},
journal = {Molecular therapy. Nucleic acids},
volume = {36},
number = {4},
pages = {102733},
pmid = {41210586},
issn = {2162-2531},
abstract = {Gene editing has transformed molecular biology by enabling precise modifications to genomic DNA across a wide variety of organisms. Gene editing technologies make it possible to add, remove, or modify specific DNA sequences, with a range of applications including gene knockouts, therapeutic gene correction, and the design of targeted genetic traits. These techniques depend on two main DNA repair mechanisms: homology-directed repair (HDR), which facilitates precise changes to the genome, and non-homologous end joining (NHEJ), which often results in mutations such as deletions or frameshift errors. Among the diverse gene-editing platforms, the CRISPR-Cas system has emerged as the most extensively employed, owing to its simplicity, low cost, and efficiency. This review presents the evolution of gene-editing technologies, with a particular emphasis on the CRISPR-Cas system and its expanding applications in genetics, biotechnology, agriculture, and medicine. Furthermore, advanced gene editing approaches are discussed, offering an overview of emerging trends.},
}
@article {pmid41209346,
year = {2025},
author = {Wei, Y and Sun, J and Zhu, R},
title = {CRISPR-epigenetic crosstalk: From bidirectional regulation to therapeutic potential.},
journal = {Computational and structural biotechnology journal},
volume = {27},
number = {},
pages = {4496-4504},
pmid = {41209346},
issn = {2001-0370},
abstract = {Recent advances in epigenetics have elucidated the pivotal roles of epigenetic modifications in genomic regulation and disease pathogenesis. Concurrently, CRISPR-based technologies have transcended conventional gene-editing applications and have emerged as powerful tools for target gene screening, chromatin imaging, and epigenetic modulation. Notably, epigenetic landscapes substantially influence the CRISPR editing efficiency, whereas CRISPR itself can reshape epigenetic states, forming a dynamic CRISPR-Epigenetics Regulatory Circuit. This review systematically examines the bidirectional interplay between CRISPR systems and epigenetic modifications, emphasizing their collective impact on genome-editing precision, disease progression, and therapeutic development. Existing studies have predominantly focused on the application of CRISPR in epigenetic modifications or the impact of epigenetic landscapes on CRISPR, exhibiting unidirectional characteristics. However, accumulating evidence suggests a bidirectional interaction between the two. Here, a transformative "CRISPR-Epigenetics Regulatory Circuit" model is synthesized and presented, supported by three pivotal breakthroughs: demonstrating CRISPR as an active epigenetic programmer, synthesizing the epigenetic preconditioning therapeutic paradigm, and elucidating the first predictive mathematical model (EPIGuide). Further exploration of this circuit is expected to enhance CRISPR performance, optimize sgRNA design via epigenetic predictive models, and pioneer sequential epigenetic or gene editing therapies.},
}
@article {pmid41206864,
year = {2025},
author = {Docherty, JAD and Cook, R and Kiu, R and Dyball, X and Brown, TL and Kujawska, M and Smith, RL and Phillips, S and Watt, R and Telatin, A and Tiwari, SK and Hall, LJ and Adriaenssens, EM},
title = {Diverse defense systems and prophages in human-associated Bifidobacterium species reveal coevolutionary "arms race" dynamics.},
journal = {Cell reports},
volume = {44},
number = {11},
pages = {116542},
doi = {10.1016/j.celrep.2025.116542},
pmid = {41206864},
issn = {2211-1247},
abstract = {Bacteria of the genus Bifidobacterium are pivotal for human health, especially in early life, where they dominate the gut microbiome in healthy infants. Bacteriophages, as drivers of gut bacterial composition, can affect bifidobacterial abundance. Here, we use a bioinformatics approach to explore direct interactions between human-associated Bifidobacterium spp. and prophages, as evidenced by their genomes. Analysis of 1,086 bifidobacterial genomes reveals the presence of complex systems that prevent viral invasion, with 34 defense systems and 56 subtypes detected, including several different CRISPR-Cas systems. CRISPR spacers target almost three-quarters of bifidobacteria-derived prophages, indicating dynamic interactions. At least one prophage is present in ∼67% of strains, with phages exhibiting high genomic diversity and evidence of historical recombination. These prophages encode various defense and anti-defense systems, such as anti-CRISPR genes and restriction-modification mechanisms. Overall, this investigation reveals that coevolutionary "arms race" dynamics drive genomic diversity in both bifidobacteria and their phages.},
}
@article {pmid41206491,
year = {2025},
author = {Landesman, WJ and Hudson, TR and Bedore, SE and Suarez, MC and Hayden, MS},
title = {A CRISPR/LbCas12a system for Borrelia burgdorferi sensu stricto detection in blacklegged ticks.},
journal = {Journal of medical entomology},
volume = {},
number = {},
pages = {},
doi = {10.1093/jme/tjaf163},
pmid = {41206491},
issn = {1938-2928},
support = {P20GM103449//Institutional Development Award (IDeA) from the National Institute of General Medical Sciences of the National Institutes of Health/ ; U01CK000661/ACL/ACL HHS/United States ; /CC/CDC HHS/United States ; },
abstract = {CRISPR/Cas systems have the potential to revolutionize DNA detection of vector-borne pathogens with highly specific and user-friendly assays. One such system, named DNA Endonuclease Targeted CRISPR Trans Reporter (DETECTR), uses a guide RNA (gRNA) and Cas enzyme to bind to and cut DNA targets. Following cutting, Cas12a exhibits non-specific collateral cleavage of single-stranded DNA (ssDNA). A ssDNA reporter in the reaction allows the trans-cleavage activity to be harnessed as an amplified output signal upon recognition of the target by the Cas12a/gRNA complex. We developed a DETECTR assay to detect Borrelia burgdorferi sensu stricto, the primary Lyme disease pathogen in the United States, in blacklegged ticks (Ixodes scapularis) collected from forests in southern Vermont. We compared DETECTR to gel electrophoresis of PCR-amplified products and used quantitative real-time PCR (qPCR) of a different B. burgdorferi primer set for independent confirmation. We found that 123/125 of the samples had identical results for DETECTR and gel electrophoresis. Both assays identified the same 33 B. burgdorferi-positive samples and the same 90 B. burgdorferi-negative samples. On a subset of eight samples, we tested DETECTR using lateral flow test strips and obtained identical results to those obtained with the fluorescence-based DETECTR. The sensitivity of DETECTR was lower than qPCR, which detected nine additional B. burgdorferi-positive samples. When qPCR is not available, the DETECTR assay offers a robust alternative to gel electrophoresis that is more user-friendly and requires less time. Due to the highly specific nature of the assay, DETECTR provides additional confidence that a B. burgdorferi target is present.},
}
@article {pmid41206049,
year = {2025},
author = {Wu, JN and Li, C and Liu, Z and Li, X and Wang, J and Lou, X and Xia, F and Dai, J and Wu, T},
title = {Photocontrolled dissociation and toehold-mediated strand displacement-based synergistic regulation of CRISPR-Cas12a.},
journal = {Nucleic acids research},
volume = {53},
number = {20},
pages = {},
pmid = {41206049},
issn = {1362-4962},
support = {22&#x2009;474&#x2009;045//National Natural Science Foundation of China/ ; 82&#x2009;172&#x2009;372//National Natural Science Foundation of China/ ; HUST: 2024JYCXJ010//Central Universities/ ; S202410487577//Undergraduates of Hubei Province/ ; },
abstract = {The trans-cleavage activity of the CRISPR-Cas system holds broad potential across diverse fields, yet precise spatiotemporal regulation remains challenging due to the predominantly single-direction control strategies available. Here, we present a bidirectional, multi-round modulation strategy for CRISPR-Cas12a trans-cleavage activity, utilizing toehold-mediated strand displacement and photocontrolled dissociation. This approach enables dynamic transitions between on and off states: Cas12a activity is activated by an activator, inhibited by a photosensitive blocker, and reactivated via UV light. We further integrated this system with DNA cryptography, establishing a hierarchical temporal authorization system that enhanced cryptographic security. The method supported multi-round modulation, achieving restoration of 95.4% activity after multiple cycles in the on state while maintaining suppression to 12.4% in the off state. This precise control strategy provides a versatile tool for spatiotemporal regulation in CRISPR-based applications, with significant implications for advanced gene editing, diagnostics, and bioengineering.},
}
@article {pmid41206045,
year = {2025},
author = {Nakamura, J and Shiraishi, M and Yamamoto, J and Suzuki, K},
title = {Development of programmable RNA imaging with RNA-guided GFP via click chemistry.},
journal = {Nucleic acids research},
volume = {53},
number = {20},
pages = {},
pmid = {41206045},
issn = {1362-4962},
support = {25K21779//The Uehara Memorial Foundation and the Japan Society/ ; JPMJSP2138//JST/ ; //Japan Society for the Promotion of Science/ ; },
abstract = {The CRISPR-Cas system revolutionized molecular biology by guiding Cas proteins to target nucleic acid sequences using customizable guide RNAs, offering unparalleled precision and versatility. Inspired by this innovation, we developed RNA-guided green fluorescent protein (RGG), a simple and programmable platform for targeting nucleic acid. Using a streamlined click chemistry approach, known for its high efficiency and specificity, we conjugated dibenzocyclooctyne (DBCO)-modified guide nucleic acids, designed to complement target sequences, with azide-exposed proteins to construct RGG. Systematic optimization identified 30-nt RNA with 3'-DBCO modifications as the most effective configuration for RGG, enabling precise visualization of nuclear-localized RNAs, including NEAT1 and Satellite III RNA, in living cells. This establishes RGG as a customizable and efficient system for RNA imaging and molecular analysis, underscoring the potential of direct conjugation between guide nucleic acids and proteins to enable precise nucleic acid recognition and dynamic molecular modification in living cells.},
}
@article {pmid41205654,
year = {2025},
author = {Yu, L and Niu, M and Dong, Z and Dong, X and Han, Y and An, J and Jiang, T and Chen, Y and Feng, Y and Sun, Y and Li, H},
title = {High-efficiency inhibition of human adenovirus type 55 replication by CRISPR-Cas12a.},
journal = {The Journal of infection},
volume = {},
number = {},
pages = {106650},
doi = {10.1016/j.jinf.2025.106650},
pmid = {41205654},
issn = {1532-2742},
abstract = {OBJECTIVES: Human adenovirus 55 (HAdV-55), a highly pathogenic double-stranded DNA virus, presents a serious global public health challenge due to its rapid transmission and complex pathogenesis. Current antiviral treatment options for HAdV-55 are limited, with no specific antiviral drugs available. The CRISPR-Cas system, capable of precisely targeting viral genomes, has emerged as a novel approach for antiviral therapy. This study aimed to leverage targeted DNA cleavage activity of the CRISPR-Cas12a system to develop a therapeutic strategy for effectively inhibiting HAdV-55 replication.<br><br>METHODS: We developed a rapid and efficient screening platform for identifying antiviral targets by integrating CRISPR-Cas12a fluorescence detection technology with bioinformatics analysis. Using this platform, we systematically screened 194 candidate targets against HAdV-55.<br><br>RESULTS: The E1B-crRNA6-Cas12a system was identified, demonstrating a highly potent antiviral activity with 99.17% inhibitory efficiency and a selectivity index (SI) of 2482.80. This target significantly outperformed the clinical broad-spectrum anti-adenovirus drug cidofovir in both inhibitory efficacy and duration.<br><br>CONCLUSIONS: This study not only holds promise for providing safe and highly effective antiviral candidate targets for HAdV-55 therapy but also, through the construction of an interdisciplinary technical platform, is expected to enhance the translational potential of CRISPR antiviral technology for preclinical applications.},
}
@article {pmid41203870,
year = {2025},
author = {Chen, X and Xiao, L and Wang, Q and Zhou, L and Xu, Y and Zhou, C and Dai, M and Wang, F and Xu, H and Luo, D and Sirois, P and Li, K and Liao, D and Zhang, J},
title = {Coloring target and off-target effects of genetically modified nucleases by blue &amp; white colony assays.},
journal = {Biotechnology letters},
volume = {47},
number = {6},
pages = {128},
pmid = {41203870},
issn = {1573-6776},
support = {SKY2021047//the Suzhou Science and Technology Planning Project/ ; ND2022B04//Program of Clinical Research Center of Neurological Disease/ ; NS2021016//Funda&#xe7;&#xe3;o de Apoio ao Desenvolvimento do Ensino, Ci&#xea;ncia e Tecnologia do Estado de Mato Grosso do Sul/ ; },
mesh = {*Gene Editing/methods ; Escherichia coli/genetics ; *CRISPR-Cas Systems ; *Endonucleases/genetics/metabolism ; Humans ; beta-Galactosidase/genetics/metabolism ; },
abstract = {More sensitive evaluation of the off-target effects of gene editing nucleases is crucial for human gene therapy. Here we report chromogenic assays designed for sensitive evaluation of gene editing activities using CRISPR/Cas9 test system. Based on beta-galactosidase alpha complementation, qualitative and quantitative evaluations of the target and off-target effects of CRISPR/Cas9 were well established through the color alteration of the E.coli colonies. In addition to target effect analysis, these new assays provide extremely sensitive and efficient tool to profile the off-target effects with one or more bases mismatched between the targets and the gRNAs. Moreover, these assays allow the identification of gene editing effects for off-targets with one base mismatched PAM sites.},
}
@article {pmid41203686,
year = {2025},
author = {Sun, Y and Qu, K and Corsi, GI and Anthon, C and Pan, X and Xiang, X and Jensen, LJ and Lin, L and Luo, Y and Gorodkin, J},
title = {Deep learning models simultaneously trained on multiple datasets improve base-editing activity prediction.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {9821},
pmid = {41203686},
issn = {2041-1723},
support = {NNF21OC0068988//Novo Nordisk Fonden (Novo Nordisk Foundation)/ ; },
mesh = {*Deep Learning ; *Gene Editing/methods ; RNA, Guide, CRISPR-Cas Systems/genetics ; *CRISPR-Cas Systems/genetics ; Neural Networks, Computer ; Humans ; Software ; },
abstract = {CRISPR-derived base editors (BE) enable precise single nucleotide substitution without introducing double-stranded DNA breaks. Apart from the base editing enzymes, efficient base editing strongly depends on both the CRISPR guide RNA (gRNA) efficiency and the edited position. Here, we show that the accuracy of BE gRNA design can be significantly improved by generating more data and by introducing deep neural networks trained on multiple different datasets simultaneously. Generating ~20,000 gRNAs for A•T to G•C and C•G to T•A conversions, we present such deep learning models, which also allow users to do dataset-aware predictions. The methods are available online and as stand-alone software.},
}
@article {pmid41203361,
year = {2025},
author = {Xia, N and Gupta, N},
title = {Function and therapeutic potential of transketolase enzymes in parasitic protists.},
journal = {Methods in enzymology},
volume = {722},
number = {},
pages = {97-115},
doi = {10.1016/bs.mie.2025.07.010},
pmid = {41203361},
issn = {1557-7988},
mesh = {*Toxoplasma/enzymology/genetics/pathogenicity ; *Transketolase/genetics/metabolism ; Animals ; Mice ; *Protozoan Proteins/genetics/metabolism ; *Toxoplasmosis/parasitology/therapy ; CRISPR-Cas Systems ; Recombinant Proteins/genetics/metabolism ; Gene Editing ; Humans ; },
abstract = {Transketolase (TKT), the rate-limiting enzyme in the non-oxidative branch of the pentose phosphate pathway (PPP), catalyzes the reversible transfer of glycolaldehyde moieties between sugar phosphates. Here, we present wide-ranging methods to study TKT function in a clinically-relevant and globally-prevalent model intracellular pathogen, Toxoplasma gondii. The experimental framework utilizes heterologous expression of recombinant TgTKT proteins in a bacterial system, anti-TgTKT antibody production, CRISPR/Cas9-assisted genome editing to generate TgTKT-null mutants, and in-depth phenotyping, including [13]C-based metabolomic analysis to determine the carbon flux in the acutely-infectious tachyzoite stage of T. gondii. The phenotypic studies comprise the parasite proliferation in vitro and virulence assessment in a murine model of acute toxoplasmosis. The data suggest TgTKT as a metabolic rheostat regulating nucleotide biosynthesis and enabling adaptation of this highly promiscuous parasite in different nutritional environments. Last but not least, the work presents the therapeutic potential of the TgTKT-null mutant as a metabolically-attenuated vaccine, highlights the evolutionary divergence of TgTKT proteins, and provides a robust foundation to investigate TgTKT orthologs in other pathogenic protozoan organisms.},
}
@article {pmid41203292,
year = {2025},
author = {Yu, D and Duan, C},
title = {[Epigenetics and precise crop breeding for resistance].},
journal = {Sheng wu gong cheng xue bao = Chinese journal of biotechnology},
volume = {41},
number = {10},
pages = {3918-3938},
doi = {10.13345/j.cjb.250513},
pmid = {41203292},
issn = {1872-2075},
mesh = {*Epigenesis, Genetic/genetics ; *Crops, Agricultural/genetics ; *Plant Breeding/methods ; DNA Methylation/genetics ; Gene Editing ; *Disease Resistance/genetics ; CRISPR-Cas Systems ; },
abstract = {Epigenetics refers to a heritable phenomenon that dynamically modulates gene expression without altering the DNA sequence, through molecular mechanisms such as DNA methylation, histone modification, non-coding RNA, chromatin remodeling, and RNA modifications. In plants, these modifications are extensively involved in key biological processes, including flowering time, gametogenesis, stress responses, and immune defenses. Over the past few decades, the research on epigenetics has gradually shifted from fundamental studies primarily conducted in Arabidopsis thaliana to investigations in various crop species such as rice and tomato. This transition has revealed the multifaceted roles of epigenetic regulation in shaping agronomic traits. This review integrates current knowledge of epigenetic regulatory mechanisms and their functions in plant responses to both biotic and abiotic stresses. Epigenetic editing tools such as CRISPR-dCas9 enable targeted DNA methylation or histone acetylation. Emerging transformation technologies, including magnetic nanoparticles and virus-based delivery systems, have the potential to overcome the bottlenecks of plant regeneration, offering new possibilities for precise epigenetic editing. In future agriculture, it is essential to further elucidate multi-layered epigenetic regulatory mechanisms at the single-cell level, develop efficient delivery systems, and leverage artificial intelligence to advance the application of epigenetic breeding for sustainable agricultural development.},
}
@article {pmid41203276,
year = {2025},
author = {Ding, Y and Guo, W},
title = {[Advances in the application of genome editing technologies in plant pathogenic fungi].},
journal = {Sheng wu gong cheng xue bao = Chinese journal of biotechnology},
volume = {41},
number = {10},
pages = {3683-3700},
doi = {10.13345/j.cjb.250539},
pmid = {41203276},
issn = {1872-2075},
mesh = {*Gene Editing/methods ; *Fungi/genetics/pathogenicity ; *CRISPR-Cas Systems/genetics ; *Plant Diseases/microbiology ; *Plants/microbiology ; *Genome, Fungal/genetics ; },
abstract = {Filamentous fungi represent an important group of eukaryotic microorganisms with diverse ecological functions and ubiquitous distribution in various ecosystems. Among them, many species are closely associated with agriculture, functioning as major plant pathogens that cause yield losses and produce mycotoxins to compromise both the quality and safety of agricultural products. In recent years, the CRISPR/Cas system has emerged as a powerful and programmable genome editing tool, and it has been extensively applied to the genetic study of plant pathogenic fungi. This technology has greatly facilitated the investigation of pathogenic mechanisms, mycotoxin biosynthetic pathways and key gene functions, antifungal resistance, and rapid pathogen detection. This review summarizes the development of CRISPR/Cas systems and the key strategies for their application in plant pathogenic fungi and makes an outlook on the practical deployment. With the continuous advancement of gene editing technologies, emerging fungal-adapted editing systems hold great promise for advancing functional genomics and enabling innovations in disease-resistant breeding and sustainable crop protection.},
}
@article {pmid40178656,
year = {2025},
author = {Gao, C and Cai, Y and Wu, X and Song, J and Zheng, Q and Wang, M and Luo, Y and Luo, Y and Fei, X and Zhang, Y and Yang, Y and Kuai, L and Ru, Y and Hong, S and Tian, N and Li, B and Zhang, Z},
title = {CRISPR/Cas9-Mediated Knockout and Overexpression Studies Unveil the Role of PD-L1 in Immune Modulation in a Psoriasis-like Mouse Model.},
journal = {Inflammation},
volume = {48},
number = {5},
pages = {3519-3528},
pmid = {40178656},
issn = {1573-2576},
support = {20224Y0373, 20234Y0269, 20234Y0075//Shanghai Municipal Health Commission Health Industry Clinical Research Special Project/ ; No. 82174383, 82204954, 82304819, 82305232, 82374458, W2433194//Shanghai Municipal Health Commission Health Industry Clinical Research Special Project/ ; 22YF1441300//Shanghai Science and Technology Development Funds (Sailing Program)/ ; lczh2021-05, lcfy2022-04, lczh2023-01//Clinical Transformation Incubation Program in Hospital/ ; zyyzdxk-2023065//Evidence-based dermatology base sponsored by State Administration of Traditional Chinese Medicine, High-level Chinese Medicine Key Discipline Construction Project (Integrative Chinese and Western Medicine Clinic) of National Administration of TCM/ ; 2023ZZ02017//Shanghai Dermatology Research Center/ ; SHDC2023CRW009//Shanghai Dermatology Hospital Demonstration Research Ward Project/ ; shzyyzdxk-2024104//Shanghai Key Discipline Construction Project of Traditional Chinese Medicine/ ; 2024-QNRC2-B04//Youth Medical Talents-Specialist Program of Shanghai "Rising Stars of Medical Talents" Youth Development Program, and Youth Talent Promotion Project of China Association of Traditional Chinese Medicine (2024-2026) Category B/ ; },
mesh = {Animals ; *B7-H1 Antigen/genetics/immunology ; *Psoriasis/immunology/chemically induced/genetics/pathology/metabolism ; Mice ; *CRISPR-Cas Systems ; Disease Models, Animal ; Mice, Knockout ; Imiquimod ; },
abstract = {The role of programmed death-ligand 1 (PD-L1), an essential immune checkpoint protein, has garnered considerable interest in recent years due to its influence on immune responses, particularly inhibiting immature Th cells into Th17 cells. This study aims to examine the effect of PD-L1 on psoriasis progress, which is the condition characterized by an immune response dominated by Th17 cells. We constructed the PD-L1 knockout (PD-L1[KO]) and overexpression (PD-L1[OE]) mice through CRISPR/Cas9 technology to assess the impact of PD-L1 in an imiquimod (IMQ)-induced psoriasis-like mouse model. In comparison to IMQ, the ear thickness exhibited a reduction, the PASI score decreased, and HE sections revealed a thinning of the epidermal spines in PD-L1[OE] mice. PD-L1[KO] mice, however, showed opposite results. Moreover, immunohistochemical assessments of the skin lesion tissues demonstrated heightened epidermal proliferation and inflammatory infiltration in the PD-L1[KO] group, accompanied by elevated tissue expression of proliferating cell nuclear antigen (PCNA), Nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) p50, and F4/80 in comparison to IMQ-treated and WT mice. The absence of PD-L1 in IMQ-induced mice was found to intensify the immune response, as evidenced by heightened expression of phosphorylated signal transducers and activators of transcription 3 (pSTAT3) and CD3 in the affected tissues compared to both IMQ-treated and WT mice. According to our findings, PD-L1 plays important roles in inhibiting inflammation, proliferation, and regulating immune responses. Targeting PD-L1 may present a promising therapeutic strategy for the management of psoriasis.},
}
@article {pmid41202215,
year = {2025},
author = {Wang, T and Brogan, DJ and Zawalick, NM and Chen, F and Mkrtchian, Y and Komor, AC and Akbari, OS},
title = {CTRL Enables Gene-Specific RNA Regulation Using CRISPR-Cas7-11.},
journal = {ACS synthetic biology},
volume = {},
number = {},
pages = {},
doi = {10.1021/acssynbio.5c00658},
pmid = {41202215},
issn = {2161-5063},
abstract = {Advancements in synthetic biology have enabled the development of precision gene expression technologies for comprehensive investigations of biological and biochemical networks. Here, we describe the development of a refined and innovative tool, CRISPR-Cas Transgenic Repressible eLement (CTRL), which utilizes the direct repeat processing activity of the recently discovered CRISPR-Cas7-11 effector to site-specifically target synthetic mRNA molecules. We demonstrate that CTRL exhibits high efficiency, tunable regulation of expression, and gene-specific repression of mRNA and protein expression. We engineered multiple permutations of the Cas7-11 effector that differ in their ability to reduce gene expression, suggesting flexibility for the application of choice. CTRL is a novel variation on gene repression technology that exhibits broad applicability across multiple model systems.},
}
@article {pmid41202131,
year = {2025},
author = {Liu, Y and Ban, Y and Gao, D},
title = {Oligo-CALL: A next-generation barcoding platform for studying resistance to targeted therapy.},
journal = {Science advances},
volume = {11},
number = {45},
pages = {eadw9990},
doi = {10.1126/sciadv.adw9990},
pmid = {41202131},
issn = {2375-2548},
mesh = {Humans ; *Drug Resistance, Neoplasm/genetics ; Cell Line, Tumor ; CRISPR-Cas Systems ; *Lung Neoplasms/genetics/drug therapy/pathology ; Single-Cell Analysis ; Molecular Targeted Therapy ; Proto-Oncogene Proteins p21(ras)/genetics/antagonists &amp; inhibitors ; },
abstract = {Understanding therapy resistance requires deconvolving heterogeneous cell populations and tracking clonal trajectories. While CRISPR-based cellular barcoding is powerful for lineage tracing, many platforms suffer from low efficiency and limited compatibility with single-cell transcriptomics. We developed Oligo-CALL (Oligonucleotide-inducible CRISPR transcriptional activator-Assisted Lineage Labeling), an advanced barcoding system enabling precise lineage tracing, live clone isolation, and seamless integration with single-cell RNA sequencing. Applied to lung cancer cells treated with a KRAS[G12C] inhibitor, Oligo-CALL identified clones consistently enriched posttreatment, supporting a model of predestined resistance. Oligo-CALL achieved >95% efficiency in linking lineage identity to transcriptomes, uncovering diverse clone-specific pathways with underlying resistance. Paired analysis of barcode-matched clones from naïve and resistant populations revealed transient and fixed resistance phenotypes. Notably, DNA repair pathways are recurrently altered in resistant clones, and inhibition of poly(adenosine 5'-diphosphate-ribose) polymerase synergizes with KRAS G12C inhibition to overcome resistance. Together, Oligo-CALL provides a versatile platform for dissecting lineage evolution and molecular dynamics of targeted therapy resistance.},
}
@article {pmid41202069,
year = {2025},
author = {Verkuyl, C and Belotserkovsky, A and Zerbes, T and Williams, D and Krishnan, MR and Zhu, S and Grunnesjӧ, S and Eid, S and Zhang, C and Zhao, W and Xu, L and Lin, E and O'Shea, T and Draper, B and Jungman, A and Most, P and Schmitt-Ulms, G},
title = {Toward an all-in-one recombinant adeno-associated virus vector for functionally ablating the prion gene using CRISPR-Cas technology.},
journal = {PloS one},
volume = {20},
number = {11},
pages = {e0336578},
doi = {10.1371/journal.pone.0336578},
pmid = {41202069},
issn = {1932-6203},
mesh = {*Dependovirus/genetics ; *CRISPR-Cas Systems/genetics ; Animals ; *Genetic Vectors/genetics ; Mice ; Humans ; Gene Editing/methods ; Genetic Therapy/methods ; Prion Diseases/genetics/therapy ; RNA, Guide, CRISPR-Cas Systems/genetics ; HEK293 Cells ; },
abstract = {Any strategy that can selectively and persistently lower the brain levels of the cellular prion protein (PrPC) is expected to extend survival in prion diseases. Recent advances in the virus-mediated delivery of gene therapies prompted us to explore if a recombinant adeno-associated virus (rAAV) vector delivering a CRISPR-Cas-based gene editor can be devised that induces a functional knockout of the prion gene. Whereas the eventual objective is to assess the therapeutic potency of an optimized vector in prion-infected mice, in this proof-of-concept study, we evaluated tools and methods that are suited to achieve this goal. The result of these efforts is a first-generation all-in-one rAAV vector that codes for a prion gene-specific guide RNA and a small Cas9 endonuclease, whose expression is controlled by a truncated neural cell adhesion molecule 1 (NCAM1) promoter that is active in PrPC expressing cells. We also constructed a second rAAV vector coding for a prion gene-specific 'traffic light reporter' (TLR). The TLR can be used to monitor prion gene-editing efficacy by coding for red and green fluorescent proteins separated by a segment of the prion gene that is targeted by the gene editor. For the purification of AAVs, we adopted a robust and scalable rAAV vector assembly pipeline and undertook proof-of-concept prion gene editing experiments in human cells and mice, which to date yielded prion gene editing rates of approximately 20% and 5%, respectively. Finally, we compared brain distributions of rAAV vectors following intrathalamic versus retro-orbital injection, and selected the 9P31 capsid for future studies based on a 7.5-fold higher heterologous gene expression level as compared to the PHP.eB capsid.},
}
@article {pmid41201953,
year = {2025},
author = {Fan, J and Huang, L and Chen, J and Jiang, Y and Du, X and Wang, L and Ding, K and Huang, J and Qiu, S and Liu, H and Song, H},
title = {Highly Sensitive Field Detection Technology for Anthrax Based on the CRISPR/Cas13a System.},
journal = {Microbial biotechnology},
volume = {18},
number = {11},
pages = {e70240},
doi = {10.1111/1751-7915.70240},
pmid = {41201953},
issn = {1751-7915},
support = {32141003//National Science Foundation of China/ ; 32300080//National Science Foundation of China/ ; },
mesh = {*Bacillus anthracis/genetics/isolation &amp; purification ; *Anthrax/diagnosis/microbiology ; *CRISPR-Cas Systems ; *Nucleic Acid Amplification Techniques/methods ; *Molecular Diagnostic Techniques/methods ; Sensitivity and Specificity ; Humans ; Point-of-Care Testing ; },
abstract = {In this study, we established a highly sensitive on-site detection technology for Bacillus anthracis. Firstly, we integrated Multiple Enzyme Isothermal Rapid Amplification (MIRA) with the clustered regularly interspaced short palindromic repeats (CRISPR) /associated protein 13a (CRISPR/Cas13a) detection system to develop a highly sensitive CRISPR/Cas13a assay. After testing crRNA selection, MIRA primers, reaction temperature, and CRISPR detection conditions, the CRISPR/Cas13a detection system employing dual crRNAs achieved a detection limit of 1000 copies/mL for B. anthracis. Quantitative analysis was additionally attempted. Compared with other common respiratory pathogens, the assay demonstrated high specificity. In clinically simulated samples, all 20 positive specimens were correctly identified, and all 13 negatives were unambiguously classified as negative. Based on these findings, we established a CRISPR point-of-care testing technology. By developing a CRISPR point-of-care testing device together with a tested lyophilised reagent system, the device achieved a detection limit of 250 copies/mL and delivered results within 30 min. All positive samples were accurately identified, and every negative sample was classified as negative. Consequently, this study presents a highly sensitive and portable technology for on-site detection of B. anthracis. It holds significant value for on-site detection of emerging infectious diseases.},
}
@article {pmid41200999,
year = {2025},
author = {Moreira, R and Nascimento, H and Martins, TMDM and Barbieri, G and Pires, P and Carvalho, LN and Rosa, LR and Almeida, A and Pessuti, CL and Ferrer, H and Gomes, J&#xc1;P and Goulart, E and Raia, S and Belfort, R},
title = {Porcine Corneal Tissue and Xenozoonotic Risks: A Review of the Current Evidence.},
journal = {Xenotransplantation},
volume = {32},
number = {6},
pages = {e70068},
doi = {10.1111/xen.70068},
pmid = {41200999},
issn = {1399-3089},
support = {//by FAPESP/ ; },
mesh = {Animals ; *Transplantation, Heterologous/methods/adverse effects ; Swine ; Humans ; *Corneal Transplantation/methods/adverse effects ; *Cornea/microbiology/virology ; Heterografts/microbiology ; },
abstract = {Corneal opacities affect millions worldwide, with corneal transplantation as the primary treatment. However, donor shortages remain a challenge, leaving thousands waiting for transplants. Xenotransplantation using porcine corneas has emerged as a promising alternative due to anatomical and physiological similarities with human corneas. Advances in CRISPR/Cas technology enable genetic modifications to address immune rejection and zoonotic risks. A key concern is xenozoonosis, the transmission of infectious agents from pigs to humans. Pathogens such as porcine endogenous retroviruses (PERVs), porcine cytomegalovirus (PCMV), and bacteria pose potential risks. While PERVs can infect human cells in vitro, no transmission has been documented in vivo. Regulatory bodies, including the WHO and IXA, have established guidelines for monitoring and clinical trials. The first human corneal xenotransplantation trials in South Korea and China are underway. Enhanced biosecurity measures in tissue banks have reduced microbial contamination, improving safety. Corneal xenotransplantation presents lower risks compared to solid organ xenotransplants. With ongoing research, stringent regulations, and improved pathogen-free animal models, this technique could become a viable clinical option. Future human trials will provide crucial insights into its long-term safety and effectiveness.},
}
@article {pmid41199512,
year = {2025},
author = {Friess, L and van Sinderen, D and Lee, C},
title = {A CRISPRi Gene Regulation System for Bifidobacteria.},
journal = {Microbial biotechnology},
volume = {18},
number = {11},
pages = {e70260},
doi = {10.1111/1751-7915.70260},
pmid = {41199512},
issn = {1751-7915},
support = {883766//Horizon 2020 Framework Programme/ ; SFI/12/RC/2273-412 P1/SFI_/Science Foundation Ireland/Ireland ; SFI/12/RC/2273 P2/SFI_/Science Foundation Ireland/Ireland ; //APC Microbiome Institute/ ; },
mesh = {*CRISPR-Cas Systems ; Streptococcus thermophilus/genetics/enzymology ; *Gene Expression Regulation, Bacterial ; *Bifidobacterium/genetics ; Plasmids ; *Bifidobacterium breve/genetics ; },
abstract = {This work describes the development of a CRISPR interference (CRISPRi) system for targeted gene repression in bifidobacteria. We first validated the CRISPRi-based approach using Bifidobacterium breve strains engineered to express nuclease-dead orthologs of Cas9 and demonstrated that the CRISPR-Cas system from Streptococcus thermophilus is efficient at targeting both reporter and endogenous genes through the use of single guide RNAs corresponding to the gene of interest. We also developed a one-plasmid system for targeted gene repression in bifidobacteria and demonstrated its utility by targeting genes involved in nucleotide metabolism and carbohydrate metabolism in several species of bifidobacteria. Efficient gene repression was achieved across all tested bifidobacterial species without the requirement for extensive optimization of transformation parameters or sequence optimization to avoid restriction modification systems thus removing the key barriers to genetic manipulation in this genus. This CRISPRi system provides a novel approach to functional genomics in bifidobacteria which facilitates future mechanistic studies in these commercially important microbes.},
}
@article {pmid41199069,
year = {2025},
author = {Singh, S and Chaudhary, R and Tiwari, S},
title = {CRISPR/Cas9-mediated gene targeting at BBM2 locus demonstrates HDR-assisted precise knock-in in banana cv. Grand Naine.},
journal = {Plant cell reports},
volume = {44},
number = {12},
pages = {265},
pmid = {41199069},
issn = {1432-203X},
support = {BRIC-NABI Core Research Grant//BRIC-National Agri-Food and Biomanufacturing Institute (BRIC-NABI)/ ; BIRAC/Tech Transfer/08/I2/QUT-BBF//Biotechnology Industry Research Assistance Council/ ; },
mesh = {*Musa/genetics ; *CRISPR-Cas Systems/genetics ; *Gene Knock-In Techniques/methods ; Gene Editing/methods ; Green Fluorescent Proteins/genetics/metabolism ; Plants, Genetically Modified ; *Gene Targeting/methods ; *Plant Proteins/genetics/metabolism ; *Recombinational DNA Repair/genetics ; },
abstract = {The present study demonstrates the first CRISPR/Cas-mediated precise knock-in of the eGFP gene at the BABYBOOM2 (GN-BBM2) locus in banana cv. Grand Naine, facilitating the detection of editing events in early embryogenic developmental stages. Genome editing has accelerated crop improvement programs by introducing targeted and precise genetic modifications. Among different tools, CRISPR/Cas-based genome editing has been widely used for enabling mutations through double-stranded breaks (DSBs), repaired either by non-homologous end joining (NHEJ) for gene knockouts or homology-directed repair (HDR) to generate knock-in events. While gene knockouts are well established in banana, efficient knock-in remains a major challenge due to low HDR activity, sterility, and the vegetatively propagated nature of banana. In the present study, we report the first successful CRISPR/Cas-based gene knock-in editing in banana by targeting the BABYBOOM2 (BBM2) gene, which encodes a transcription factor involved in somatic embryogenesis. The enhanced green fluorescent protein (eGFP) gene was precisely inserted at the BBM2 locus in banana cv. Grand Naine to enable visual detection during embryogenesis. In vitro validation showed ~ 95% target cleavage efficiency of the selected gRNA. The PCR-based screening and shift-in amplicon size analyses confirmed three edited lines (#3, #11, and #14) harboring eGFP knock-in at the targeted locus. Sequencing of the amplicon from these lines further confirmed the precise knock-in events. Hence, this study establishes a foundation for precise knock-in-based genome modification in banana and opens new avenues for targeted trait improvement in this important clonally propagated crop.},
}
@article {pmid41198952,
year = {2025},
author = {Makarova, KS and Shmakov, SA and Wolf, YI and Mutz, P and Altae-Tran, H and Beisel, CL and Brouns, SJJ and Charpentier, E and Cheng, D and Doudna, J and Haft, DH and Horvath, P and Moineau, S and Mojica, FJM and Pausch, P and Pinilla-Redondo, R and Shah, SA and Siksnys, V and Terns, MP and Tordoff, J and Venclovas, &#x10c; and White, MF and Yakunin, AF and Zhang, F and Garrett, RA and Backofen, R and van der Oost, J and Barrangou, R and Koonin, EV},
title = {An updated evolutionary classification of CRISPR-Cas systems including rare variants.},
journal = {Nature microbiology},
volume = {},
number = {},
pages = {},
pmid = {41198952},
issn = {2058-5276},
support = {Intramural funds//U.S. Department of Health &amp; Human Services | National Institutes of Health (NIH)/ ; },
abstract = {The known diversity of CRISPR-Cas systems continues to expand. To encompass new discoveries, here we present an updated evolutionary classification of CRISPR-Cas systems. The updated CRISPR-Cas classification includes 2 classes, 7 types and 46 subtypes, compared with the 6 types and 33 subtypes in our previous survey 5 years ago. In addition, a classification of the cyclic oligoadenylate-dependent signalling pathway in type III systems is presented. We also discuss recently characterized alternative CRISPR-Cas functionalities, notably, type IV variants that cleave the target DNA and type V variants that inhibit the target replication without cleavage. Analysis of the abundance of CRISPR-Cas variants in genomes and metagenomes shows that the previously defined systems are relatively common, whereas the more recently characterized variants are comparatively rare. These low abundance variants comprise the long tail of the CRISPR-Cas distribution in prokaryotes and their viruses, and remain to be characterized experimentally.},
}
@article {pmid41198678,
year = {2025},
author = {Nukaga, S and Shiraishi, K and Hamabe, K and Mochizuki, A and Hamaguchi, Y and Ogawa, E and Le, NT and Shimada, Y and Ono, H and Nishinakamura, H and Kobayashi, Y and Hamamoto, J and Ui, A and Araki, M and Sagae, Y and Ohgino, K and Sugihara, K and Endo, S and Miyakoshi, J and Shiraishi, Y and Yasuda, H and Okuno, Y and Yoshida, T and Goto, Y and Ohe, Y and Watanabe, SI and Yatabe, Y and Nishikawa, H and Hamamoto, R and Kohno, T and Nakaoku, T},
title = {Mutation of CMTR2 in Lung Adenocarcinoma Alters RNA Alternative Splicing and Reveals Therapeutic Vulnerabilities.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {9754},
pmid = {41198678},
issn = {2041-1723},
support = {JP23ak0101205//Japan Agency for Medical Research and Development (AMED)/ ; JP24ama221233//Japan Agency for Medical Research and Development (AMED)/ ; JP24ck0106905//Japan Agency for Medical Research and Development (AMED)/ ; 25K02539//MEXT | Japan Society for the Promotion of Science (JSPS)/ ; 22KJ3158//MEXT | Japan Society for the Promotion of Science (JSPS)/ ; 20H00545//MEXT | Japan Society for the Promotion of Science (JSPS)/ ; JPMJCR1689//MEXT | Japan Science and Technology Agency (JST)/ ; JPMJCR18Y4//MEXT | Japan Science and Technology Agency (JST)/ ; },
mesh = {Humans ; *Adenocarcinoma of Lung/genetics/drug therapy ; *Lung Neoplasms/genetics/drug therapy/pathology ; *Alternative Splicing/genetics ; *Mutation ; Cell Line, Tumor ; *Methyltransferases/genetics/metabolism ; Gene Expression Regulation, Neoplastic ; CRISPR-Cas Systems ; },
abstract = {RNA splicing dysregulation has emerged as a hallmark of cancer and a promising therapeutic target; however, its full landscape in human solid cancer remains poorly characterized. To address this, we perform alternative splicing analyses using RNA-sequencing data from 751 lung adenocarcinoma samples from our cohort integrated with 519 samples from The Cancer Genome Atlas. Visualization of splicing patterns using t-distributed stochastic neighbor embedding reveals substantial inter-tumor heterogeneity driven by distinct molecular subtypes and histological differentiation. We identify a unique molecular subtype associated with inactivating mutations in CMTR2, which encodes Cap-specific mRNA (nucleoside-2'-O-)-methyltransferase 2. CMTR2 mutations are observed in 3.8% of cases and are predominantly truncating mutations, which form an isolated cluster within the splicing landscape. Intrinsic and CRISPR-Cas9-engineered CMTR2 mutations disrupt alternative splicing and sensitize cancer cells to sulfonamide-based RNA splicing modulators and immune checkpoint blockade therapy. Retrospective patient data confirm the increased sensitivity of CMTR2-deficient tumors to immune checkpoint blockade therapy. These findings uncover a previously unrecognized RNA splicing deficiency in human cancers and define a molecular subtype of lung adenocarcinoma driven by RNA splicing dysregulation, suggesting targets for therapeutic intervention in lung cancer.},
}
@article {pmid41197724,
year = {2025},
author = {Ito, C and Yamamoto, M and Yokota, N and Nakamura, N and Shiomi, M and Kizu, M and Ichinose, M and Fujii, S and Fujii, T and Fukunaga, R},
title = {Dissecting stress-activated protein kinase (SAPK)-signaling pathways using multiplex gene knockout HeLa cells.},
journal = {The Journal of biological chemistry},
volume = {},
number = {},
pages = {110901},
doi = {10.1016/j.jbc.2025.110901},
pmid = {41197724},
issn = {1083-351X},
abstract = {The stress-activated protein kinase (SAPK) family consists of three JNK and four p38 members. To explore the isotype-specific or overlapping roles of SAPK members, HeLa-derived multiplex SAPK-knockout (KO) cells, such as JNK1/2/3-triple KO, p38α/β/γ/δ-quadruple KO, and JNK1/2/3/p38α/β/γ/δ-septuple KO cells were generated using the CRISPR/Cas9 method. Also, 'sole survivor' hextuple KO (ss-HKO) cells, in which only one of seven SAPK genes remains intact, were generated. Western blot analyses using phospho-specific antibodies for SAPK substrates showed that serum or anisomycin-induced phosphorylation of MAPKAPK2, MSK1, Mnk1, and CREB/ATF-1 largely depended on p38, while anisomycin-induced phosphorylation of c-Jun/JunD depended on JNK. Similar analyses using the ss-HKO cell lines revealed that JNK1 rather than JNK2 contributed to the c-Jun/JunD phosphorylation, while p38α was the primary species phosphorylating the examined p38 substrates. Expression analyses of three typical immediate-early genes, c-Jun, EGR1, and c-Fos, demonstrated that JNK1 and 2 are responsible for c-Jun expression induced by interleukin-1β, tumor necrosis factor-α, ultraviolet-C (UV), and heat shock (HS), whereas p38 is predominant in EGR1 expression induced by UV and HS, and in c-Fos expression induced by the cytokines, UV, and HS. On the other hand, neither JNK nor p38 contributed significantly to the cytokine-induced EGR1 expression, suggesting complicated SAPK-signaling mechanisms that regulate immediate-early gene expression. Together, these results demonstrate the utility of the comprehensive multigene KO and sole-survivor KO strategy in dissecting intracellular signaling pathways consisting of multiple family members.},
}
@article {pmid41193960,
year = {2025},
author = {Van Goethem, MW and Vikram, S and Cowan, DA and Makhalanyane, TP},
title = {Comparative genomics reveals adaptive traits in novel Antarctic lithic cyanobacteria.},
journal = {BMC genomics},
volume = {26},
number = {1},
pages = {994},
pmid = {41193960},
issn = {1471-2164},
abstract = {BACKGROUND: Terrestrial polar cyanobacteria persist at the cold limits of life, enduring a suite of extreme stressors including sub-zero temperatures, frequent freeze–thaw cycles, oligotrophic soils, variable light regimes with long periods of darkness, and desiccation. To survive, cyanobacteria have evolved diverse physiological strategies. A key adaptation among Antarctic terrestrial cyanobacteria is niche colonization: inhabiting the undersides of translucent quartz rocks (hypoliths) and the interior spaces of porous rocks (endoliths), which buffer environmental extremes and sustains the potential for photoautotrophic carbon fixation. However, the full genomic repertoire facilitating their resilience is incomplete.<br><br>RESULTS: We cultivated cyanobacteria from endolithic and hypolithic niches in Victoria Valley, Eastern Antarctica, and recovered four near-complete genomes (100% completeness,&#x2009;<&#x2009;1% contamination). Three hypolithic genomes showed near-identical sequence similarity (whole genome average nucleotide identity&#x2009;=&#x2009;99.98%) and phylogenomic proximity to the genus Coleofasciculus, yet represent a novel species, Coleofasciculus caryii H7-2. The fourth genome, derived from an endolith, showed moderate similarity to Aliterella antarctica (whole genome average nucleotide identity&#x2009;=&#x2009;79.1%), and is proposed as a new species, Aliterella bergstromii E5.1. C. caryii H7-2 possessed a larger genome (~&#x2009;6.1 Mbp) than A. bergstromii E5.1 (~&#x2009;5.4 Mbp). Both genomes encoded complete pathways for carbon fixation via oxygenic photosynthesis (RuBisCO and phosphoribulokinase), extensive phycobilisomes, and multiple photoprotective mechanisms. Predicted optimal growth temperatures were 21.7&#xa0;&#xb0;C and 23.2&#xa0;&#xb0;C, respectively. Shared stress-mitigation genes included those for osmotic, thermal and oxidative (superoxide dismutase) stress response. All genomes contained biosynthetic gene clusters associated with stress-adaptive secondary metabolites, including heterocyst glycolipids, siderophores, phenazines, compounds related to nostopeptolide and merocyclophane. The C. caryii H7-2 genome encoded multiple CRISPR-Cas systems, suggesting adaptive immunity and historical phage exposure. In contrast, A. bergstromii E5.1 harboured a single prophage and an array of 24 plasmids.<br><br>CONCLUSIONS: These finding reveal that the newly-described cyanobacteria possess a rich genomic repertoire of adaptations to withstand Antarctic extremes, emphasizing the resilience and ecological importance of lithobiontic cyanobacteria in polar deserts.<br><br>SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s12864-025-12203-7.},
}
@article {pmid41191214,
year = {2025},
author = {Choi, S and Lee, JM and Kim, KK},
title = {Biomolecular condensates: molecular structure, biological functions, diseases, and therapeutic targets.},
journal = {Molecular biomedicine},
volume = {6},
number = {1},
pages = {99},
pmid = {41191214},
issn = {2662-8651},
support = {RS-2025-00513647//National Research Foundation of Korea/ ; 20240678//Bisa Research Grant of Keimyung University/ ; },
mesh = {Humans ; *Biomolecular Condensates/chemistry/metabolism ; Animals ; Neurodegenerative Diseases/metabolism ; },
abstract = {Cells constantly encounter environmental and physiological fluctuations that challenge homeostasis and threaten viability. In response to these cues, specific proteins and nucleic acids engage in multivalent interactions and undergo phase separation to form membraneless assemblies known as biomolecular condensates. Nuclear condensates include paraspeckles, nuclear speckles, and Cajal bodies, while cytoplasmic condensates include stress granules, processing bodies, RNA transport granules, U-bodies, and Balbiani bodies. These assemblies regulate transcription, splicing fidelity, RNA stability, translational reprogramming, and integration of signaling pathways, thereby serving as dynamic platforms for metabolic regulation and physiological adaptation. However, dysregulation of these condensates has been increasingly recognized as a central pathogenic mechanism in neurodegenerative diseases, cancers, and viral infections, contributing to toxic protein aggregation, nucleic acid dysregulation, and aberrant cell survival signaling. This review provides a comprehensive synthesis of the molecular mechanisms governing condensation, delineates the diverse types and functions of major biomolecular condensates, and examines therapeutic approaches based on their pathophysiological relevance to disease development and progression. Furthermore, we highlight the cutting-edge technologies, including CRISPR/Cas-based imaging, optogenetic manipulation, and AI-driven phase separation prediction tools, which enable the real-time monitoring and precision targeting of cytoplasmic biomolecular condensates. These insights underscore the emerging potential of biomolecular condensates as both biomarkers and therapeutic targets, paving the way for precision medicine approaches in condensate-associated diseases.},
}
@article {pmid41058551,
year = {2025},
author = {Dear, HS and Franks, NE and Markley, EE and Holtz, AM and Song, JY and Johnson, CN and Medina-Cabrera, PI and Hernandez, D and Joel, P and Pasca di Magliano, M and Wellik, DM and Allen, BL},
title = {GliFHV mice: a tool to investigate GLI processing and localization.},
journal = {Development (Cambridge, England)},
volume = {152},
number = {21},
pages = {},
doi = {10.1242/dev.204251},
pmid = {41058551},
issn = {1477-9129},
support = {R01 CA275182/CA/NCI NIH HHS/United States ; R01 CA275182/CA/NCI NIH HHS/United States ; //Rogel Cancer Center, University of Michigan/ ; },
mesh = {Animals ; Mice ; *Zinc Finger Protein GLI1/metabolism/genetics ; Cilia/metabolism ; Fibroblasts/metabolism ; Hedgehog Proteins/metabolism ; Zinc Finger Protein Gli3/metabolism/genetics ; Gene Editing ; Signal Transduction ; Alleles ; CRISPR-Cas Systems ; Female ; Mice, Transgenic ; Nerve Tissue Proteins ; },
abstract = {GLI proteins (GLI1-3) are the transcriptional effectors of mammalian hedgehog (HH) signaling. However, studies of GLI function have been hampered by the lack of robust GLI antibodies. To address this, we utilized CRISPR-based gene editing to generate endogenous epitope-tagged Gli alleles for each Gli gene (Gli1FLAG, Gli2HA, Gli3V5). Through breeding, we established a novel mouse model, Gli1FLAG/FLAG;Gli2HA/HA;Gli3V5/V5, referred to as GliFHV. Importantly, GliFHV animals are viable and fertile with no overt phenotypes. Sanger and long-range DNA sequencing confirmed proper editing of each Gli allele, while qPCR and western blot analysis confirmed similar gene expression and protein levels, respectively, between wild-type and GliFHV animals. We utilized these mice to assess GLI localization in the developing limb, finding that all three GLIs localize to primary cilia with distinct distributions. Finally, we generated immortalized GliFHV mouse embryonic fibroblasts, demonstrating that these cells are HH responsive and that GLIs localize to primary cilia and nuclei in a HH-dependent fashion. These animals and cell lines provide a valuable resource for analyses of GLI processing, localization and function throughout embryogenesis, postnatal development, and in adults.},
}
@article {pmid40944362,
year = {2025},
author = {Hillebrand, GH and Carlin, SC and Giacobe, EJ and Stephenson, HA and Collins, J and Hooven, TA},
title = {A Cas12a Toolbox for Rapid and Flexible Group B Streptococcus Genomic Editing and CRISPRi.},
journal = {Molecular microbiology},
volume = {124},
number = {5},
pages = {449-461},
pmid = {40944362},
issn = {1365-2958},
support = {P20GM125504//Centers of Biomedical Research Excellence/ ; P20 GM125504/GM/NIGMS NIH HHS/United States ; R01 AI182835/AI/NIAID NIH HHS/United States ; //UPMC Children's Hospital of Pittsburgh Research Advisory Council/ ; R01AI177991/NH/NIH HHS/United States ; R01 AI177991/AI/NIAID NIH HHS/United States ; R21AI178067/NH/NIH HHS/United States ; R01AI182835/NH/NIH HHS/United States ; R21 AI178067/AI/NIAID NIH HHS/United States ; R01AI177991/NH/NIH HHS/United States ; R01AI182835/NH/NIH HHS/United States ; R21AI178067/NH/NIH HHS/United States ; },
mesh = {*Streptococcus agalactiae/genetics ; *Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; Plasmids/genetics ; Genome, Bacterial ; *Bacterial Proteins/genetics/metabolism ; *CRISPR-Associated Proteins/genetics/metabolism ; *Endodeoxyribonucleases/genetics/metabolism ; Mutagenesis ; Enterococcus faecium/genetics ; },
abstract = {Streptococcus agalactiae (group B Streptococcus; GBS) is a leading cause of neonatal sepsis and meningitis. Despite advances in molecular microbiology, GBS genome engineering remains laborious due to inefficient mutagenesis protocols. Here, we report a versatile and rapid Cas12a-based toolkit for GBS genetic manipulation. We developed two shuttle plasmids-pGBSedit for genome editing and pGBScrispri for inducible CRISPR interference-derived from an Enterococcus faecium system and optimized for GBS. Using these tools, we achieved targeted gene insertions, markerless deletions, and efficient, template-free mutagenesis via alternative end-joining repair. Furthermore, a catalytically inactive dCas12a variant enabled inducible gene silencing, with strand-specific targeting effects. The system demonstrated broad applicability across multiple GBS strains and minimal off-target activity, as confirmed by whole-genome sequencing. In benchmarking, template-less Cas12a mutagenesis yielded sequence-confirmed mutants in ~7 days and homology-directed edits in ~7-14 days; aTC-resistant colonies arose at ~10[-4] of uninduced CFU, and 27%-65% of resistant clones carried the intended homology-directed edit depending on locus and homology arm length (e.g., ~27% markerless deletion; ~35% insertion; 65% with 1 kb arms). These workflows provide a rapid alternative to temperature-sensitive plasmid mutagenesis protocols that typically require ≥ 4 weeks. This Cas12a-based platform offers an efficient, flexible, and scalable approach to genetic studies in GBS, facilitating functional genomics and accelerating pathogenesis research.},
}
@article {pmid40675807,
year = {2025},
author = {Bykonya, AG and Guschin, DY and Barlev, NA},
title = {Modern approaches to engineering human reporter cell lines using CRISPR within Safe Harbor loci and endogenous genes.},
journal = {Critical reviews in biotechnology},
volume = {45},
number = {8},
pages = {1816-1832},
doi = {10.1080/07388551.2025.2515834},
pmid = {40675807},
issn = {1549-7801},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Genes, Reporter/genetics ; Cell Line ; *Gene Editing/methods ; *Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {Reporter systems are gaining increasing popularity in modern molecular biology as they provide reliable and clear readouts for various types of assays, both in cellulo and in vivo. The generation of reporter cell lines is instrumental for screening activators and inhibitors of signaling pathways to develop new therapeutic approaches. Reporter cell lines are those with stably integrated reporter constructs containing signaling genes (often luciferase or fluorescent proteins), enabling the visualization and tracking of protein expression. Although seemingly harmless and straightforward, untargeted genomic integration of reporter genes may severely affect the expression of neighboring genes, causing unwanted and unpredictable effects. Unlike the untargeted approach, the CRISPR/Cas9 system provides a more precise method of reporter integration, especially when reporters are integrated into Safe Harbor loci. This ensures minimal influence on neighboring genomic regions. This review discusses recent advancements in creating reporter lines using the CRISPR/Cas9 system and experimental approaches for identifying suitable Safe Harbor loci.},
}
@article {pmid41191093,
year = {2025},
author = {Sun, Y and Han, H and Zhao, R and Liu, X and Liu, Z and Liang, J and Wang, B and Zhao, B and Pang, Y and Xiao, R},
title = {CRISPR-assisted MoS2@TQD nanosheets-based lateral flow assay for dual-mode and sensitive diagnosis of SARS-CoV-2.},
journal = {Mikrochimica acta},
volume = {192},
number = {12},
pages = {792},
pmid = {41191093},
issn = {1436-5073},
mesh = {Humans ; *SARS-CoV-2/isolation &amp; purification/genetics ; *COVID-19/diagnosis ; *Molybdenum/chemistry ; *Quantum Dots/chemistry ; Limit of Detection ; *Disulfides/chemistry ; Colorimetry/methods ; CRISPR-Cas Systems ; Nanostructures/chemistry ; *Clustered Regularly Interspaced Short Palindromic Repeats ; COVID-19 Testing/methods ; },
abstract = {Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) assembled lateral flow assay (LFA) have been developed for rapid virus diagnosis, however, the single-mode visual half-quantitative characteristics limit their applications in accurate quantitative diagnosis. Herein, a CRISPR assisted MoS2@TQD (MoS2 nanosheet assembled ternary quantum dot) based colorimetric and fluorescent dual-mode LFA was fabricated for the ultrasensitive and flexible detection of the SARS-CoV-2. Compared with single QDs, MoS2@TQD can provide stable and superior colorimetric/fluorescent signals in various bio-samples. RT-RAA/CRISPR-Cas13 was adopted to couple with MoS2@TQD so that the detection sensitivity can be further enhanced. Based on the dual-mode LFA, the colorimetric visual detection limit was 400 copies/mL, and for fluorescence quantification mode, as low as 250 copies/mL can be obtained. For clinic diagnosis, 35 SARS-CoV-2 positive throat swab samples and 18 negative samples were detected by this LFA, 100% accuracy and 100% consistency with quantitative polymerase chain reaction (qPCR) results can be obtained. It indicated that MoS2@TQD-based dual-mode LFA can be a rapid, sensitive, and accurate detection platform for SARS-CoV-2 and other infectious pathogens in the point-of-care diagnosis.},
}
@article {pmid41188812,
year = {2025},
author = {Hussain, T and Awasthi, S and Shahid, F and Yi, SS and Sahni, N and Aldaz, CM},
title = {Therapeutic potential of PRMT1 as a critical survival dependency target in multiple myeloma.},
journal = {BMC cancer},
volume = {25},
number = {1},
pages = {1704},
pmid = {41188812},
issn = {1471-2407},
support = {R35GM133658/GM/NIGMS NIH HHS/United States ; R35GM137836/NH/NIH HHS/United States ; 7016-18//Leukemia and Lymphoma Society/ ; },
mesh = {*Protein-Arginine N-Methyltransferases/antagonists &amp; inhibitors/genetics/metabolism ; Humans ; *Multiple Myeloma/drug therapy/genetics/pathology/metabolism ; Cell Survival/drug effects ; Cell Line, Tumor ; *Repressor Proteins/antagonists &amp; inhibitors/genetics/metabolism ; Arginine/metabolism ; Gene Expression Regulation, Neoplastic/drug effects ; CRISPR-Cas Systems ; Cell Cycle/drug effects ; DNA Damage ; },
abstract = {Multiple myeloma (MM) is a neoplasm of antibody-producing plasma cells and is the second most prevalent hematological malignancy worldwide. Development of drug resistance and disease relapse significantly impede the success of MM treatment, highlighting the critical need to discover novel therapeutic targets. In a custom CRISPR/Cas9 screen targeting 197 DNA damage response-related genes, Protein Arginine N-Methyltransferase 1 (PRMT1) emerged as a top hit, revealing it as a potential therapeutic vulnerability and survival dependency in MM cells. PRMT1, a major Type I PRMT enzyme, catalyzes the asymmetric transfer of methyl groups to arginine residues, influencing gene transcription and protein function through post-translational modification. Dysregulation or overexpression of PRMT1 has been observed in various malignancies including MM and is linked to chemoresistance. Treatment with the Type I PRMT inhibitor GSK3368715 resulted in a dose-dependent reduction in cell survival across a panel of MM cell lines. This was accompanied by reduced levels of asymmetric dimethylation of arginine (ADMA) and increased arginine monomethylation (MMA) in MM cells. Cell cycle analysis revealed an accumulation of cells in the G0/G1 phase and a reduction in the S phase upon GSK3368715 treatment. Additionally, PRMT1 inhibition led to a significant downregulation of genes involved in cell proliferation, DNA replication, and DNA damage response (DDR), likely inducing genomic instability and impairing tumor growth. This was supported by Reverse Phase Protein Array (RPPA) analyses, which revealed a significant reduction in levels of proteins associated with cell cycle regulation and DDR pathways. Overall, our findings indicate that MM cells critically depend on PRMT1 for survival, highlighting the therapeutic potential of PRMT1 inhibition in treating MM.},
}
@article {pmid41188277,
year = {2025},
author = {Yeh, TY and Chiu, IM and Huang, YJ and Wu, SC and Hsieh, CC and Sung, HH and Hwu, WL and Wang, GJ and Ju, JC},
title = {Generation of exogenous kidneys via CRISPR/Cas9 mediated blastocyst complementation targeting Osr1 gene in mice.},
journal = {Scientific reports},
volume = {15},
number = {1},
pages = {38648},
pmid = {41188277},
issn = {2045-2322},
support = {108-2313-B-039-002//Ministry of Science and Technology, Taiwan/ ; 108-2313-B-039-002//Ministry of Science and Technology, Taiwan/ ; 108-2313-B-039-002//Ministry of Science and Technology, Taiwan/ ; 108-2313-B-039-002//Ministry of Science and Technology, Taiwan/ ; },
mesh = {Animals ; *CRISPR-Cas Systems ; Mice ; *Blastocyst/metabolism/cytology ; *Kidney/metabolism/embryology ; Mice, Knockout ; Female ; Gene Targeting ; RNA, Guide, CRISPR-Cas Systems/genetics ; Green Fluorescent Proteins/genetics/metabolism ; Gene Editing ; },
abstract = {Blastocyst complementation has been reported to produce exogenous mouse organs including the pancreas, lungs, and kidneys, but the complemented kidneys still failed to rescue the host animals. In the present study, we generated mouse complemented kidneys through a two-step procedure: using CRISPR/Cas9 ribonucleoproteins (RNP) to knockout Osr1 alleles, followed by injecting mouse embryonic stem (ES) cells that express enhanced green fluorescent protein (EGFP). When two different sgRNAs targeting the exon 2 of Osr1 were microinjected into the pronucleus of a mouse zygote, 34% of the embryos had deletions on both alleles, and these Osr1-knockouts died with no mesonephric duct development shown by histochemical staining. With three sgRNA injections, the knockout efficiencies increased, and mesonephric duct development with EGFP-positive cells was observed in ES cell-injected E12.5 embryos. Most of the ES cell-injected Osr1-knockout embryos degenerated from E13.5 to E15.5. Four of the 264 ES cell-injected embryos were born alive and survived to the second day, with strong EGFP signals observed in both the kidneys and the heart. Therefore, complementation of the Osr1-knockout blastocyst is a potential method to produce exogenous kidneys, although further modification is still needed to increase the efficiency.},
}
@article {pmid41187082,
year = {2025},
author = {Avaro, AS and Mirjalili, S and Griffiths, AD and Santiago, JG},
title = {Microfluidic networks using isotachophoresis.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {122},
number = {45},
pages = {e2511724122},
doi = {10.1073/pnas.2511724122},
pmid = {41187082},
issn = {1091-6490},
support = {//Swedish e-Science Research Centre (SeRC)/ ; //Kungliga Tekniska H&#xf6;gskolan (KTH)/ ; },
abstract = {The development of microfluidic technologies has enabled chemical and biological analysis systems with increased functionality, complexity, and parallelization. These functionalities often drive the creation and control of complex and dynamic fluidic architectures. Introduced here is a class of microfluidic network based on isotachophoresis (ITP), an electrokinetic process that can extract and purify samples, selectively transport, mix, and aliquot (split) samples in a system with no moving parts. Presented is a theoretical framework to describe these networks. The framework relies on the coupling between a one-dimensional description of ITP and two-dimensional, transient graphs to describe the dynamic evolution of ITP networks. We leverage this framework to create numerical simulations of branched ITP circuits. We build, control, and experimentally study a variety of ITP networks. These systems automatically split and merge ITP zones, enabling complex sample manipulation with minimal external control. The model captures the experimentally observed sample dynamics. We demonstrate an example system where an ITP network is used to control and quantify parallel CRISPR-Cas enzymatic reactions. The methods described here are generally applicable to highly complex topologies and may offer a basis for easily reconfigurable, electric field-driven microfluidic systems. Networks generally offer broad potential for automated chemical and biochemical analysis and lab-on-a-chip integration.},
}
@article {pmid41171899,
year = {2025},
author = {Gil-Campillo, C and Mignolet, J and Domínguez-San Pedro, A and Rapún-Araiz, B and Janssen, AB and de Bakker, V and Veening, JW and Garmendia, J},
title = {CRISPRi-seq in Haemophilus influenzae reveals genome-wide and medium-specific growth determinants.},
journal = {PLoS pathogens},
volume = {21},
number = {10},
pages = {e1013650},
pmid = {41171899},
issn = {1553-7374},
mesh = {*Haemophilus influenzae/genetics/growth &amp; development ; *Genome, Bacterial ; *CRISPR-Cas Systems/genetics ; Humans ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Gene Expression Regulation, Bacterial ; Genomics/methods ; },
abstract = {Work in the human pathobiont Haemophilus influenzae has pioneered functional genomics in bacteria such as genome-wide transposon mutagenesis combined with deep sequencing. These approaches unveiled a large set of likely essential genes, but functional studies are hampered due to a limited molecular toolbox. To bridge this gap, we engineered a titratable anhydrotetracycline-inducible CRISPRi (Clustered Regularly Interspaced Short Palindromic Repeats interference) platform for efficient regulation of gene expression in H. influenzae. Genome-wide fitness analyses in two different in vitro culture media by CRISPRi-seq revealed growth medium-dependent fitness cost for a panel of H. influenzae genes. We demonstrated that CRISPRi-programmed fitness defects can be rescuable, and we refined previous Tn-seq based essentialome studies. Finally, we introduce HaemoBrowse, an extensive user-friendly online resource for visual inspection of H. influenzae genome annotations, including sgRNA spacers. The inducible CRISPRi platform described here represents a valuable tool enabling functional genomics and the study of essential genes, thereby contributing to the identification of therapeutic targets for developing drugs and vaccines against H. influenzae.},
}
@article {pmid41109141,
year = {2026},
author = {Song, L and Bu, S and Ding, Y and Chai, Y and Fu, Y and Zhang, P and Yuan, R},
title = {CRISPR-enhanced low-potential ECL sensor based on AuPt-functionalized porphyrin AlOG for ultrasensitive aflatoxin B1 detection.},
journal = {Biosensors &amp; bioelectronics},
volume = {292},
number = {},
pages = {118089},
doi = {10.1016/j.bios.2025.118089},
pmid = {41109141},
issn = {1873-4235},
mesh = {*Aflatoxin B1/analysis/isolation &amp; purification/chemistry ; *Biosensing Techniques/methods ; CRISPR-Cas Systems ; Limit of Detection ; Luminescent Measurements/methods ; Electrochemical Techniques/methods ; *Porphyrins/chemistry ; Gold/chemistry ; Metal Nanoparticles/chemistry ; Aluminum/chemistry ; Food Contamination/analysis ; },
abstract = {Herein, an ultrasensitive low-potential electrochemiluminescence (ECL) sensor was developed by integrating an AuPt nanoparticle-functionalized porphyrin aluminum-organic gel (P-AlOG@AuPt) emitter with an enhanced CRISPR/Cas12a amplification module for the detection of aflatoxin B1 (AFB1) related to food safety. Impressively, the P-AlOG@AuPt emitter exhibited strong ECL emission and high signal-to-noise ratio at a low triggering potential (-0.8 V), addressing key limitations of previous high-potential aluminum-organic gels. Meanwhile, the in situ enriched AuPt as a coreaction accelerator catalyzed the reduction of coreactant S2O8[2-] to produce abundant SO4[•-] for facilitating its interactions with P-AlOG@AuPt[•-], resulting in a significant ECL enhancement. Furthermore, a synergistic catalytic hairpin assembly (CHA) pre-amplification and tetrahedral DNA nanostructure (TDN)-anchored ssDNA reporters significantly improved the cleavage efficiency of CRISPR/Cas12a, boosting the detection sensitivity of the ECL sensor for AFB1. The established ECL sensor exhibited ultrasensitive detection of AFB1 with a linear range of 0.1 pg mL[-1] - 500 ng mL[-1] and detection limit of 0.044 pg mL[-1], outperforming most reported ECL- and CRISPR-based AFB1 sensors. The practical usability of the proposed ECL sensor was verified by applying it for the quantification of AFB1 in rice samples. This work offers a promising low-potential ECL sensing platform for practical mycotoxin monitoring and broader biosensing applications.},
}
@article {pmid41052497,
year = {2026},
author = {Huang, X and Wang, A and Lin, Z and Xu, Y and Zheng, J},
title = {Novel electrochemiluminescence resonance energy transfer biosensor driven by CRISPR-Cas12a system for ctDNA detection.},
journal = {Biosensors &amp; bioelectronics},
volume = {292},
number = {},
pages = {118067},
doi = {10.1016/j.bios.2025.118067},
pmid = {41052497},
issn = {1873-4235},
mesh = {*Biosensing Techniques/methods ; *CRISPR-Cas Systems/genetics ; Humans ; *Circulating Tumor DNA/genetics/blood/isolation &amp; purification ; *Lung Neoplasms/genetics/blood/diagnosis ; *Luminescent Measurements/methods ; Limit of Detection ; Electrochemical Techniques/methods ; Gold/chemistry ; *Carcinoma, Non-Small-Cell Lung/genetics/blood/diagnosis ; Energy Transfer ; Mutation ; },
abstract = {Efficient energy donor-acceptor pairing and distance control are critical in resonance energy transfer-based electrochemiluminescence (ECL-RET) biosensor. In this work, a novel ECL-RET system with Ru(phen)3[2+] as ECL energy donor and Au nanocages as energy acceptor was constructed based on the characteristic that Ru(phen)3[2+] can be stably embedded into double-stranded DNA. On this basis, a homogeneous ECL biosensor based on target activated CRISPR-Cas12a system and driving ECL-RET effect was developed. The ECL biosensor has been successfully applied to the detection of circulating tumor DNA (ctDNA) and demonstrated efficient recognition of L858R mutation in ctDNA of non-small cell lung cancer patients. The presented ECL-RET biosensor achieves a wide linear detection range from 10 fM to 1 nM and an excellent detection limit as low as 3.0 fM for ctDNA due to its combination of the highly efficient and specific cleavage of the CRISPR-Cas12a system.},
}
@article {pmid40883986,
year = {2025},
author = {Jenny Gao, and Bamidele, N and Pires-Ferreira, D and Zheng, C and Destefano, A and Cheng, H and Tang, Q and Cao, Y and Xie, J and Gao, G and Gruntman, A and Sontheimer, E and Flotte, TR and Xue, W},
title = {A compact base editor rescues AATD-associated liver and lung disease in mouse models.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {33},
number = {11},
pages = {5817-5828},
pmid = {40883986},
issn = {1525-0024},
support = {UH3 HL147367/HL/NHLBI NIH HHS/United States ; R01 CA275945/CA/NCI NIH HHS/United States ; P01 HL158506/HL/NHLBI NIH HHS/United States ; R01 GM150273/GM/NIGMS NIH HHS/United States ; F30 HL176024/HL/NHLBI NIH HHS/United States ; },
mesh = {Animals ; Mice ; Disease Models, Animal ; *Gene Editing/methods ; *alpha 1-Antitrypsin/genetics ; *alpha 1-Antitrypsin Deficiency/genetics/therapy/complications ; *Liver Diseases/therapy/genetics/etiology ; *Lung Diseases/therapy/genetics/etiology ; Mice, Transgenic ; Humans ; *Genetic Therapy/methods ; Mutation ; CRISPR-Cas Systems ; Dependovirus/genetics ; Genetic Vectors/genetics ; },
abstract = {Alpha-1 antitrypsin deficiency (AATD) is commonly caused by a G-to-A mutation in the SERPINA1 gene (the PiZ mutation). The mutant PiZ AAT protein is sequestered in hepatocytes, causing lung emphysema due to insufficient AAT protein to inhibit neutrophil elastase in the lung. Here, we show that a compact adenine base editor (ABE) with an evolved Cas9 nickase derived from Neisseria meningitidis (eNme2.C) can be packaged in a single AAV and correct the PiZ mutation in mouse models of AATD. An all-in-one eNme2.C-TadA8e/guide 2 plasmid achieved approximately 20% on-target editing in PiZ reporter cells. TadA9e demonstrated sequence-specific reduction in bystander editing compared to TadA8e, without significantly affecting the on-target edit. In PiZ transgenic mice, eNme2.C-TadA9e AAV showed approximately 23% editing efficiency after 8 weeks and reduced liver disease burden in treated mice. In a new AAT-null;PiZ transgenic mouse model, ABE restored serum levels of AAT to beyond the 570 μg/mL therapeutic level. Moreover, ABE treatment was able to significantly correct lung functions in AAT-null;PiZ animals with emphysema. This study demonstrates the feasibility of an eNme2.C-based ABE in a single AAV to treat both AATD-associated liver and lung disease.},
}
@article {pmid40842155,
year = {2025},
author = {Ibel, A and Bhardwaj, R and Yilmaz, DE and Kong, S and Wendlinger, S and Cordero, C and Papaioannou, D and Papazian, M and Schönauer, R and Meng, Q and Eckardt, KU and Hassan, F and Volpe, I and Klämbt, V and Halbritter, J and Fedeles, S and Krappitz, M and Kaminski, MM},
title = {In vivo base editing reduces liver cysts in autosomal dominant polycystic kidney disease.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {33},
number = {11},
pages = {5373-5382},
doi = {10.1016/j.ymthe.2025.08.026},
pmid = {40842155},
issn = {1525-0024},
mesh = {*Polycystic Kidney, Autosomal Dominant/genetics/therapy/pathology ; Animals ; *Gene Editing/methods ; Mice ; Humans ; TRPP Cation Channels/genetics ; *Cysts/genetics/therapy/pathology/etiology ; *Liver Diseases/genetics/therapy/etiology/pathology ; Disease Models, Animal ; CRISPR-Cas Systems ; Genetic Therapy/methods ; Liver/pathology/metabolism ; Mutation, Missense ; },
abstract = {Autosomal dominant polycystic kidney disease (ADPKD) is the most prevalent genetic kidney disorder, affecting over 10 million individuals worldwide. Cystic expansion typically progresses to kidney failure and also involves the liver with limited treatment options. Pathogenic variants in PKD1 or PKD2 account for 85%-90% of cases. Genetic re-expression of Pkd1 or Pkd2 has been shown to partially reverse key characteristics of the disease phenotype in mice. Despite advancements in understanding the genetic basis, it remains unclear whether correcting pathogenic variants can effectively prevent, modify, or reverse the disease. Additionally, the feasibility of genome editing as a treatment remains largely unexplored. In this study, we employed CRISPR base editing to correct representative pathogenic PKD1 variants selected from a patient cohort, achieving precise and efficient editing in vitro. Correction of a murine missense variant (c.6646C>T (R2216W)) in primary renal epithelial cells increased polycystin-1 expression and reduced the endoplasmic reticulum stress marker sXBP1. In vivo, base editor delivery to the c.6646C>T (R2216W) knockin mouse enabled correction of the pathogenic variant, resulting in a significant reduction in liver cysts. These findings provide the first evidence that genome editing may ameliorate key features of ADPKD, opening promising therapeutic perspectives for affected patients and their families.},
}
@article {pmid40833860,
year = {2025},
author = {Yazdani, R and Nasr Esfahani, MH and Eghbalsaied, S and Karamali, F},
title = {Extracellular Vesicle-Liposome Hybrid Nanoparticles Delivery of CRISPR/Cas9 Induces a Unique DNA Repair Pattern in the HGF Gene of Stem Cells from Apical Papilla.},
journal = {DNA and cell biology},
volume = {44},
number = {11},
pages = {618-627},
doi = {10.1177/10445498251370554},
pmid = {40833860},
issn = {1557-7430},
mesh = {*CRISPR-Cas Systems/genetics ; *Liposomes/chemistry ; *Nanoparticles/chemistry ; *Extracellular Vesicles/metabolism/chemistry ; *Hepatocyte Growth Factor/genetics ; Humans ; *Stem Cells/metabolism ; Gene Editing/methods ; },
abstract = {Extracellular vesicles (EVs) have been investigated due to their natural biocompatibility and targeting capabilities. The specific approach of combining EVs with liposomes to create hybrid nanoparticles (ELNPs) for the delivery of the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas9) system for deletion of the HGF gene in stem cells, but their effectiveness in encapsulating large nucleic acids is limited due to their small size. This study aimed to knock out the HGF gene by the CRISPR/Cas9 system by ELNPs, and it was expected that the efficiency of the CRISPR/Cas9 system transfer would increase compared to the usual methods of using lipofectamine in stem cells from apical papilla (SCAPs). In this study, gRNA suitable for the HGF gene is designed first, and after insertion into the CRISPR/Cas9 vector, it enters Lipofectamine 2000. In the next step, ELNPs are prepared after collecting EVs and hybridizing them with liposomes containing CRISPR/Cas9 vector. Then, these integrated nanoparticles were presented to SCAPs, and the removal of HGF gene expression was evaluated at the level of RNA and protein. This study showed that the CRISPR/Cas9 system can be efficiently transferred to SCAP cells using ELNPs. Genomic DNA sequencing analyses of SCAP cells showed a unique pattern of mutation, highly likely mediated through EVs. Quantitative PCR and protein staining further showed a decrease in HGF gene expression in the knockout cells. Moreover, cell proliferation analysis showed a decrease in cell proliferation in KO-HGF adipose cells compared to the nonedited counterpart. In summary, this study highlights the supportive role of EVs in facilitating cell transfection and promoting a dominant DNA repair pattern, likely through an RNA-mediated mechanism, rather than the random insertions and deletions typically induced during CRISPR editing of the HGF gene in SCAPs.},
}
@article {pmid40808259,
year = {2025},
author = {Mamia, K and Kolbeinsdottir, S and Labun, K and Li, Z and Komisarczuk, A and Keskitalo, S and Reint, G and Loe Haugen, F and Olaug Lindestad, B and Skundberg Jensen, S and Gjerdingen, TJ and Tuhkala, A and Wieczorek Ervik, C and Kopcil, P and Fatkhutdinov, N and Bronken Martinsen, KH and Erichsen, HC and Szymanska, M and Tölö, E and Glumoff, V and Saarela, J and Melbye Michelsen, T and Schalin-Jäntti, C and Olweus, J and Leinonen, E and Varjosalo, M and Valen, E and Hautala, T and Enge, M and Martelius, T and Dahal-Koirala, S and Haapaniemi, E},
title = {Precision T cell correction platform for inborn errors of immunity.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {33},
number = {11},
pages = {5624-5643},
doi = {10.1016/j.ymthe.2025.08.018},
pmid = {40808259},
issn = {1525-0024},
mesh = {*Gene Editing/methods ; *T-Lymphocytes/metabolism/immunology ; Humans ; CRISPR-Cas Systems ; Animals ; Mice ; STAT1 Transcription Factor/genetics ; Genetic Therapy/methods ; Disease Models, Animal ; },
abstract = {CRISPR-Cas9 gene editing is a promising tool to correct pathogenic variants for autologous cell therapies targeting inborn errors of immunity (IEI). Current strategies, such as gene knockout or cDNA knockin, address many single-gene defects but can disrupt gene expression, highlighting the need for precise correction platforms. While transplanting corrected autologous hematopoietic stem cells is a curative approach, it is unsuitable for patients with advanced disease, inflammation, or acute infections. As correcting T cells is an alternative therapeutic strategy for lymphoid IEIs, we present an efficient T cell single-nucleotide variant (SNV) correction platform based on homology-directed repair (HDR). By using STAT1 gain-of-function, cartilage hair hypoplasia, deficiency of ADA2, and autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy as IEI models, we demonstrate that our platform achieves up to 80% correction, with resultant functional correction of the disease phenotype in the selected models. Furthermore, we performed safety profiling using GUIDE-seq, single-cell RNA sequencing, long-read genome sequencing, and proteomics analysis and detected no genomic, transcriptomic, or proteomic aberrations. This study establishes HDR-based SNV editing as a portable method for developing clinical autologous T cell therapies and represents a promising step toward a broad-spectrum gene correction platform for treating diverse monogenic immune disorders.},
}
@article {pmid40739756,
year = {2025},
author = {Skov, TW and Wolff, JH and Haslund, D and Revenfeld, AL and van de Venn, L and Dorset, SR and Ravendran, S and Jørgensen, SE and Thomsen, MK and Holm, M and Corn, JE and Møller, BK and Mogensen, TH and Bak, RO and Mikkelsen, JG},
title = {Treatment of GATA2 deficiency by allele-specific CRISPR-Cas9-directed gene correction in hematopoietic stem cells.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {33},
number = {11},
pages = {5644-5660},
doi = {10.1016/j.ymthe.2025.07.038},
pmid = {40739756},
issn = {1525-0024},
mesh = {*CRISPR-Cas Systems ; *Hematopoietic Stem Cells/metabolism ; Humans ; Alleles ; *Gene Editing/methods ; *GATA2 Transcription Factor/genetics/deficiency ; *GATA2 Deficiency/therapy/genetics ; Dependovirus/genetics ; *Genetic Therapy/methods ; RNA, Guide, CRISPR-Cas Systems/genetics ; Animals ; Mice ; Genetic Vectors/genetics ; Recombinational DNA Repair ; },
abstract = {GATA2 deficiency is a rare inborn error of immunity caused by monoallelic variants in the GATA2 gene, leading to dysfunction of hematopoietic stem and progenitor cells (HSPCs). Here, we investigate a potential therapeutic strategy for GATA2 deficiency based on CRISPR-Cas9-based gene correction, utilizing recombinant adeno-associated virus serotype 6 (rAAV6) as a template for homology-directed repair (HDR). For a 7-base pair (bp) deletion giving rise to GATA2 deficiency, we identify a single guide RNA (sgRNA) supporting allele-specific cleavage in the disease allele. Initially, we observe high cytotoxicity in HSPCs upon Cas9/sgRNA ribonucleoprotein nucleofection and rAAV6 transduction, but this is mitigated by co-administering mRNA-based modulators of the DNA damage response combined with a 10-fold reduction in rAAV6 dose. Using this protocol, we achieve efficient HDR (>80%) in HSPCs derived from a patient carrying the 7-bp deletion and show increased engraftment potential after GATA2 correction. Using DISCOVER-seq, we find limited off-target activity. However, with PCR-free long-read sequencing, we detect frequent large aberrations at the on-target site in HSPCs, primarily attributed to the integration of AAV concatemers identified in 15% of the targeted alleles. Our findings describe the effect of gene correction on GATA2 deficiency and highlight potential on-target aberrations following HDR-mediated gene correction.},
}
@article {pmid40652526,
year = {2025},
author = {Oksenych, V and Petakh, P and Kainov, D and Kamyshnyi, O},
title = {A CRISPR-Cas9-based tool for dose-dependent DNA damage detection.},
journal = {The FEBS journal},
volume = {292},
number = {21},
pages = {5625-5628},
doi = {10.1111/febs.70186},
pmid = {40652526},
issn = {1742-4658},
mesh = {*CRISPR-Cas Systems/genetics ; *Saccharomyces cerevisiae/genetics ; Saccharomyces cerevisiae Proteins/genetics/metabolism ; *DNA Breaks, Double-Stranded ; *DNA Damage/genetics ; Retroelements/genetics ; Protein Serine-Threonine Kinases/genetics/metabolism ; Intracellular Signaling Peptides and Proteins ; },
abstract = {Auboiron et al. developed a CRISPR-Cas9-based system in yeast Saccharomyces cerevisiae that allows precise, dose-dependent induction of DNA double-strand breaks by targeting Ty retrotransposons. This system combines sequence specificity with control over break numbers (×1, ×15, or ×59), enabling a more detailed and precise study of DNA damage response. It revealed that the key DNA damage response kinase, Tel1, localizes to the nuclear periphery and forms multiple foci after induction of DNA double-strand breaks. The system also exposed limitations in Cas9 availability at higher break levels and offers a scalable platform for studying genome stability across organisms.},
}
@article {pmid40631397,
year = {2025},
author = {Auboiron, M and Coiffard, J and Kumanski, S and Santt, O and Pardo, B and Moriel-Carretero, M},
title = {A CRISPR-Cas9-based system for the dose-dependent study of DNA double-strand break sensing and repair.},
journal = {The FEBS journal},
volume = {292},
number = {21},
pages = {5629-5658},
doi = {10.1111/febs.70143},
pmid = {40631397},
issn = {1742-4658},
support = {Laureate 2018//ATIP-Avenir Program/ ; ANR-21-CE12-0004-01//Agence Nationale de la Recherche/ ; ARCPJA22020060002119//Fondation ARC pour la Recherche sur le Cancer/ ; PLBIO19-098 INCA_13832//Institut National Du Cancer/ ; },
mesh = {*DNA Breaks, Double-Stranded ; *Saccharomyces cerevisiae/genetics/metabolism ; *Saccharomyces cerevisiae Proteins/genetics/metabolism ; *CRISPR-Cas Systems/genetics ; *DNA Repair/genetics ; Protein Serine-Threonine Kinases/genetics/metabolism ; Intracellular Signaling Peptides and Proteins/genetics/metabolism ; RNA, Guide, CRISPR-Cas Systems/genetics ; DNA Transposable Elements/genetics ; },
abstract = {The integrity of DNA is put at risk by different lesions, among which double-strand breaks (DSBs) occur at a lower frequency but have the most life-threatening consequences. The study of DSB repair requires tools that can induce the accumulation of these breaks and includes the use of chemical genotoxins, ionizing radiation, or the expression of sequence-specific nucleases. While genotoxins and irradiation allow for dose-dependent studies, nuclease expression permits assessments at precise locations. In this work, we have leveraged the repetitive nature of the Ty transposon elements in the genome of Saccharomyces cerevisiae and the cutting activity of the RNA-guided Cas9 nuclease to create a tool that combines sequence specificity and dose-dependency. In particular, we can achieve the controlled induction of 0, 1, 15, or 59 DSBs in cells with an otherwise identical genetic background. We make the first application of this tool to better understand the behavior of the apical kinase of the DNA damage response Tel1 in the nuclear space. We found that Tel1 is capable of forming nuclear foci, which are clustered by condensin when DSBs occur in Ty elements. In striking contrast with other DSB-related protein foci, Tel1 foci are in tight contact with the nuclear periphery, therefore suggesting a role for the nuclear membrane in their congregation.},
}
@article {pmid40501348,
year = {2025},
author = {Rafiq, S and Assad, A},
title = {RNAS-sgRNA: Recurrent Neural Architecture Search for Detection of On-Target Effects in Single Guide RNA.},
journal = {Journal of computational biology : a journal of computational molecular cell biology},
volume = {32},
number = {11},
pages = {1041-1059},
doi = {10.1089/cmb.2025.0031},
pmid = {40501348},
issn = {1557-8666},
mesh = {*RNA, Guide, CRISPR-Cas Systems/genetics ; Humans ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; *Neural Networks, Computer ; Computational Biology/methods ; Algorithms ; },
abstract = {Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 is a leading genomic editing tool, but its effectiveness is limited by considerable heterogeneity in target efficiency among different single guide RNAs (sgRNA). This study presents RNAS-sgRNA, a hybrid model that integrates neural architecture search (NAS) with recurrent neural networks (RNN) to evaluate the on-target efficacy of CRISPR/Cas9 sgRNA. The RNAS-sgRNA model automates architectural discovery, improving sgRNA sequence categorization without considerable manual adjustment. The NAS component improves the RNN architecture, which analyzes sgRNA sequences represented as binary matrices and produces a classification score. Upon evaluation across several datasets, RNAS-sgRNA exhibits substantial performance enhancements with multiple cell lines, comparing its area under the receiver operating characteristic curve (AUROC) performance to the baseline CRISPRpred(SEQ) and DeepCRISPR models. RNAS-sgRNA demonstrated substantial improvements in AUROC performance in several cell lines compared with existing models. Notable improvements include enhancements of 8.62% for HCT116, 121.57% for HEK293T, 13.40% for HeLa, and 20.78% for HL60 cell lines, resulting in an overall improvement of 13.46%. Compared with DeepCRISPR, the model achieved additional AUROC gains in all cell lines tested, with an average improvement of 14.74%. The study also highlighted the ability of the model to deliver superior performance on smaller datasets through transfer learning, underscoring its potential applications in personalized medicine and genetic research. RNAS-sgRNA introduces a novel integration of NAS with RNN to evaluate the efficacy of CRISPR/Cas9 sgRNA. Unlike traditional methods that require significant manual adjustments, this model automates architectural discovery, optimizing the RNN structure for sgRNA sequence analysis. Furthermore, the application of transfer learning to fine-tune the pretrained model on small cell-line datasets represents a pioneering approach in the domain. The model's demonstrated ability to significantly outperform existing algorithms, including CRISPRpred(SEQ) and DeepCRISPR, across multiple cell lines highlights its innovative contribution to genomic editing research and personalized medicine.},
}
@article {pmid41184760,
year = {2025},
author = {Guo, F and Wang, D and Wu, H and Zhao, X and Xu, F and Cui, Y and Shao, Y and Zhang, Y and Bu, W and Wen, T},
title = {Genomic characteristics, antimicrobial resistance profiles and virulence factors of Gallibacterium anatis isolates from layer chickens in Northern China.},
journal = {BMC microbiology},
volume = {25},
number = {1},
pages = {706},
pmid = {41184760},
issn = {1471-2180},
support = {31802193//National Natural Science Foundation of China/ ; XMS202505//the R &amp; D Foundation of Institute of Animal Husbandry and Veterinary Medicine/ ; KJCX20230427//the Special Program on Science and Technology Innovation Capacity Building of BAAFS/ ; },
mesh = {Animals ; *Chickens/microbiology ; China ; *Virulence Factors/genetics ; *Poultry Diseases/microbiology ; Phylogeny ; *Pasteurellaceae/genetics/drug effects/pathogenicity/isolation &amp; purification/classification ; *Genome, Bacterial ; Anti-Bacterial Agents/pharmacology ; *Pasteurellaceae Infections/veterinary/microbiology ; Drug Resistance, Multiple, Bacterial/genetics ; Virulence ; Genomics ; *Drug Resistance, Bacterial ; },
abstract = {BACKGROUND: Gallibacterium anatis is an emerging pathogen causing substantial economic losses in global poultry production. Despite its growing clinical significance, the genomic basis of pathogenicity and antimicrobial resistance in this species remains poorly understood, particularly in China.<br><br>RESULTS: We sequenced and analyzed five clinical G. anatis isolates from Chinese layer chickens alongside 31 global strains, their genomes ranged from 2.25 to 2.81&#xa0;Mb with 39.8% average GC content. Phylogenetic analysis revealed that Chinese isolates cluster according to historical breeding stock importation patterns, reflecting international trade influences on pathogen distribution. Contemporary isolates showed extensive multidrug resistance compared to the antimicrobial-sensitive historical strain F149, with resistance profiles correlating directly with documented antibiotic usage in Chinese poultry production. Virulence analysis identified universal conservation of the RTX toxin system (97-100% prevalence) across all strains, contrasting sharply with variable distribution of other factors including fimbriae (30-35%) and secretion systems (50-60%). All strains harbored CRISPR-Cas systems, predominantly types I and III, indicating strain-specific phage defense adaptations.<br><br>CONCLUSIONS: The RTX toxin system represents a core virulence mechanism and potential universal vaccine target for G. anatis. The rapid evolution from antimicrobial-sensitive to extensively resistant phenotypes demonstrates how global poultry trade accelerates both pathogen spread and resistance development. These findings provide molecular insights for targeted interventions against an increasingly problematic poultry pathogen.},
}
@article {pmid41184307,
year = {2025},
author = {White, N and Chalk, JA and Hu, YT and Pins, SM and Joseph, CR and Antoniou, P and Wimberger, S and Svensson, S and Caetano-Silva, SP and Mudde, ACA and Rai, R and Selvaraj, S and Feist, WN and Romito, M and Sienski, G and Nitsch, R and Booth, C and Santilli, G and Cavazza, A and Porteus, MH and Maresca, M and Thrasher, AJ and Turchiano, G},
title = {Unveiling the cut-and-repair cycle of designer nucleases in human stem and T cells via CLEAR-time dPCR.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {9571},
pmid = {41184307},
issn = {2041-1723},
support = {217112/Z/19/Z//Wellcome Trust (Wellcome)/ ; },
mesh = {Humans ; *DNA Repair/genetics ; *T-Lymphocytes/metabolism ; Gene Editing/methods ; DNA Breaks, Double-Stranded ; CRISPR-Cas Systems ; Induced Pluripotent Stem Cells/metabolism ; *Endonucleases/metabolism/genetics ; Mutation ; },
abstract = {DNA repair mechanisms in human primary cells, including error-free repair, and, recurrent nuclease cleavage events, remain largely uncharacterised. We elucidate gene-editing related repair processes using Cleavage and Lesion Evaluation via Absolute Real-time dPCR (CLEAR-time dPCR), an ensemble of multiplexed dPCR assays that quantifies genome integrity at targeted sites. Utilising CLEAR-time dPCR we track active DSBs, small indels, large deletions, and other aberrations in absolute terms in clinically relevant edited cells, including HSPCs, iPSCs, and T-cells. By quantifying up to 90% of loci with unresolved DSBs, CLEAR-time dPCR reveals biases inherent to conventional mutation screening assays. Furthermore, we accurately quantify DNA repair precision, revealing prevalent scarless repair after blunt and staggered end DSBs and recurrent nucleases cleavage. This work provides one of the most precise analyses of DNA repair and mutation dynamics, paving the way for mechanistic studies to advance gene therapy, designer editors, and small molecule discovery.},
}
@article {pmid41184277,
year = {2025},
author = {Nguyen, C and Omotayo, AI and Sanz Juste, S and Feng, X and López Del Amo, V},
title = {A temperature-sensitive CRISPR-Cas12a system for sterile insect technique.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {9677},
pmid = {41184277},
issn = {2041-1723},
mesh = {Animals ; *CRISPR-Cas Systems/genetics ; Male ; Female ; Temperature ; RNA, Guide, CRISPR-Cas Systems/genetics ; Animals, Genetically Modified ; *Infertility, Male/genetics ; *CRISPR-Associated Proteins/genetics/metabolism ; *Endodeoxyribonucleases/genetics/metabolism ; Gene Editing/methods ; *Bacterial Proteins/genetics/metabolism ; },
abstract = {The sterile insect technique (SIT) reduces population numbers by releasing sterile males that produce non-viable progeny. Specifically, CRISPR/Cas9-based precision-guided SIT (pgSIT) generates sterile males through genetic crosses of two transgenic lines: a Cas9 strain and a guide RNA (gRNA) strain targeting male sterility and female viability or infertility. However, pgSIT requires separate maintenance of the two lines and sorting to obtain sterile males, creating possible challenges for scaling. To overcome this, we propose using Cas12a nuclease, which is inoperative at lower temperatures but active at higher temperatures. Here, we develop a Cas12a-based pgSIT system involving a single strain containing both the Cas12a nuclease and gRNAs to induce male sterility and female lethality. This strain can be maintained as a mixed stock of both sexes and only activated by increasing temperature, producing sterile males after just one generation. By reducing the challenges that arise with maintaining two separate lines, this system could offer a scalable alternative for vector control in combating vector-borne diseases.},
}
@article {pmid41106454,
year = {2025},
author = {Xie, D and Wang, J and Huang, L and Zhao, Z and Wang, R and Long, H and Ni, Q and Liu, H and Hu, M and Huang, J},
title = {A novel X-linked retinitis pigmentosa beagle dog model for ophthalmic research established rapidly by editing the RPGR gene in zygotes.},
journal = {Experimental eye research},
volume = {261},
number = {},
pages = {110697},
doi = {10.1016/j.exer.2025.110697},
pmid = {41106454},
issn = {1096-0007},
mesh = {Animals ; *Retinitis Pigmentosa/genetics/metabolism/physiopathology ; Dogs ; *Disease Models, Animal ; *Eye Proteins/genetics ; *Gene Editing/methods ; *Genetic Diseases, X-Linked/genetics ; Female ; *Zygote/metabolism ; Electroretinography ; CRISPR-Cas Systems ; Male ; Mutation ; },
abstract = {Inherited retinal diseases (IRDs) constitute several ocular disorders leading to progressive and severe visual impairment. While significant progress has been made in understanding the genetic basis of IRDs, preclinical animal models are crucial for advancing therapeutic development. Although well-established mouse models exist, the scarcity of large animal models represents a significant limitation. Dogs (Canis familiaris) exhibit numerous physiological and anatomical similarities to humans, rendering them as potential models. X-linked retinitis pigmentosa (XLRP) is a severe, early-onset form of IRDs, primarily caused by mutations in the retinitis pigmentosa GTPase regulator (RPGR) gene. Here, we efficiently generated a stably inherited RPGR-knockout beagle model of XLRP in one-step via CRISPR/Cas9 gene editing tool with multiple gRNAs and zygote microinjection technology. Two F0 generation beagle dogs were successfully obtained with high knockout efficiency in the RPGR gene and the F0 female exhibited characteristic XLRP phenotypes, including progressive deterioration of both rod and cone photoreceptor function, a gradual reduction in the outer nuclear layer thickness from 7 to 35 months, and attenuated retinal arteries with minimal pigmentation. Notably, the F1 male of hemizygous RPGR-knockout beagle dogs displayed more pronounced disease phenotypes by 7 months of age. In conclusion, we successfully generated a stably inherited beagle dog model of XLRP exhibiting an intermediate characteristic disease onset, which is highly suitable for therapeutic research in gene therapy and stem cell transplantation for determining optimal intervention timing and therapeutic dosing.},
}
@article {pmid41081521,
year = {2025},
author = {Wu, H and Lin, X and Tian, C and Xiong, D},
title = {Establishment of an RAA-CRISPR/Cas12a assay based on CpSge1 for rapid detection of Cryphonectria parasitica.},
journal = {Microbiology spectrum},
volume = {13},
number = {11},
pages = {e0107925},
pmid = {41081521},
issn = {2165-0497},
support = {2023YFD1401301//National Key Research and Development Program/ ; },
mesh = {*Ascomycota/genetics/isolation &amp; purification ; *Plant Diseases/microbiology ; *Nucleic Acid Amplification Techniques/methods ; *CRISPR-Cas Systems ; Sensitivity and Specificity ; Fagaceae/microbiology ; Fungal Proteins/genetics ; Recombinases/metabolism/genetics ; China ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {Chestnut blight disease caused by Cryphonectria parasitica is a serious branch disease that occurs worldwide, especially in Europe and North America. In recent years, chestnut blight disease has also been severe and even posed a great threat to the healthy development of chestnut orchards in some areas of China. Accurate and rapid detection of C. parasitica during the initial stages of the disease is helpful to take corresponding prevention and control measures in advance. In this study, we selected the CpSge1 (Gti1/Pac2 transcription factor family) of C. parasitica as the detection target and established a rapid and visual detection system of C. parasitica that combined the recombinase-aided amplification (RAA) and CRISPR/Cas12a, called CpSge1-RAA-CRISPR/Cas12a. The system allows for the specific detection of C. parasitica in approximately 60 mins, with visualization of results. The detection sensitivity of this system was found to be 1 pg/µL. We combined the RAA-CRISPR/Cas12a with a lateral flow dipstick, which also showed specific, high sensitivity, and fast characters. In conclusion, the RAA-CRISPR/Cas12a assay has great potential to be a method for early diagnosis and on-site detection of C. parasitica, especially for areas where specialized equipment is lacking.IMPORTANCEA rapid, highly sensitive, and visualized detection system of Cryphonectria parasitica was established by using the RAA-CRISPR/Cas12a method based on the C-terminal variable regions of a fungal-specific transcription factor CpSge1. The detection system was performed at a constant temperature condition of 37°C, which provides important support for the diagnosis of chestnut blight diseases in the field.},
}
@article {pmid41075360,
year = {2026},
author = {Rahimi, M and Schreiber, M and Habermann, H and Haake, L and Miskel, D and Tesfaye, D and Held-Hoelker, E and Brenig, B and Benedetti, C and Hoelker, M},
title = {Efficient genome editing of bovine in vitro derived zygotes via Cas9 RNP-electroporation using extended-stored bovine ovaries.},
journal = {Theriogenology},
volume = {250},
number = {},
pages = {117703},
doi = {10.1016/j.theriogenology.2025.117703},
pmid = {41075360},
issn = {1879-3231},
mesh = {Animals ; Cattle/embryology ; Female ; *Gene Editing/veterinary/methods ; *Electroporation/veterinary/methods ; *Zygote/physiology ; *Ovary/physiology ; *CRISPR-Cas Systems ; Fertilization in Vitro/veterinary ; CRISPR-Associated Protein 9 ; Ribonucleoproteins/genetics ; },
abstract = {Bovine embryos serve as a relevant model for human preimplantation development and are increasingly used in genome editing research. Although CRISPR-Cas9 electroporation in zygotes offers a promising approach, challenges with respect to editing efficiency and mosaicism remain a challenge. However, traditional in vitro fertilization (IVF) workflows enforce highly inconvenient electroporation times of zygotes usually around midnight. This study therefore aimed to investigate the feasibility of using bovine zygotes derived from extended-stored slaughterhouse derived ovaries (Phosphate buffered saline, 14-18 °C, 20-22 h) with particular attention to the strength of the voltage field during electroporation and the Cas9-RNP concentration with respect to embryonic development and genome editing efficiency. Oocytes obtained under these conditions demonstrated in vitro developmental competence similar to those derived from fresh ovaries, with comparable cleavage and blastocyst formation rates. Electroporation conducted 10 h post-insemination at voltages of 15V, 20V, and 25V revealed that 25V yielded the highest blastocyst editing rate (40.7 %) while maintaining acceptable developmental rates. Increasing voltage to 30V and 35V further enhanced blastocyst editing rate (66.7 % and 67.9 %, respectively), with a greater proportion of blastocyst showing a full edit. While higher voltages reduced developmental rates, the optimal voltage for electroporation was found to be 30V for bovine zygotes derived from extended stored ovaries. Using that setting, contrasting Cas9-RNP concentrations (6 μM vs. 3 μM) were tested. A concentration of 6 μM resulted in higher editing rates compared to 3 μM (81.5 % vs. 60.0 %), although the distribution of monoallelic, biallelic, and mosaic modifications did not differ significantly. Taken together, this study confirmed for the first time that zygotes from extend-stored bovine ovaries are suitable for genome editing via CRISPR Cas9-RNP electroporation. Mosaicism, however, remained prevalent, underscoring the need for strategies to improve the precision and consistency of embryo editing.},
}
@article {pmid41036840,
year = {2025},
author = {Diaz, B and Krishna, R and Schoeniger, JS and Mageeney, CM},
title = {Exploring phage-host interactions in Burkholderia cepacia complex bacterium to reveal host factors and phage resistance genes using CRISPRi functional genomics and transcriptomics.},
journal = {Microbiology spectrum},
volume = {13},
number = {11},
pages = {e0193625},
pmid = {41036840},
issn = {2165-0497},
support = {LDRD//Sandia National Laboratories/ ; InCoGenTEC//U.S. Department of Energy/ ; },
mesh = {*Bacteriophages/physiology/genetics ; *Burkholderia cepacia complex/virology/genetics ; Genomics/methods ; Transcriptome ; Gene Expression Profiling ; CRISPR-Cas Systems ; Host Specificity ; Humans ; Host-Pathogen Interactions/genetics ; },
abstract = {Complex interactions of bacteriophages with their bacterial hosts determine phage host range and infectivity. While phage defense systems and host factors have been identified in model bacteria, they remain challenging to predict in non-model bacteria. In this paper, we integrate functional genomics and transcriptomics to investigate phage-host interactions, revealing active phage resistance and host factor genes in Burkholderia cenocepacia K56-2. Burkholderia cepacia complex species are commonly found in soil and are opportunistic pathogens in immunocompromised patients. We studied infection of B. cenocepacia K56-2 with Bcep176, a temperate phage isolated from Burkholderia multivorans. A genome-wide dCas9 knockdown library targeting B. cenocepacia K56-2 was constructed, and a pooled infection experiment identified 63 novel genes or operons coding for candidate host factors or phage resistance genes. The activities of a subset of candidate host factor and resistance genes were validated via single-gene knockdowns. Transcriptomics of B. cenocepacia K56-2 during Bcep176 infection revealed that expression of genes coding for host factor and resistance candidates identified in this screen was significantly altered during infection by 4 h post-infection. Identifying which bacterial genes are involved in phage infection is important to understand the ecological niches of B. cenocepacia and its phages, and for designing phage therapies.IMPORTANCEBurkholderia cepacia complex bacteria are opportunistic pathogens inherently resistant to antibiotics, and phage therapy is a promising alternative treatment for chronically infected patients. Burkholderia bacteria are also ubiquitous in soil microbiomes. To develop improved phage therapies for pathogenic Burkholderia bacteria, or engineer phages for applications, such as microbiome editing, it's essential to know the bacterial host factors required by the phage to kill bacteria, as well as how the bacteria prevent phage infection. This work identified 65 genes involved in phage-host interactions in Burkholderia cenocepacia K56-2 and tracked their expression during infection. These findings establish a knowledge base to select and engineer phages infecting or transducing Burkholderia bacteria.},
}
@article {pmid40996288,
year = {2025},
author = {Boucher, MJ and Madhani, HD},
title = {CRISPR/Cas9-compatible plasmids enabling seven dominant genetic selection methods for the human fungal pathogen Cryptococcus neoformans.},
journal = {Microbiology spectrum},
volume = {13},
number = {11},
pages = {e0193525},
pmid = {40996288},
issn = {2165-0497},
support = {R01AI100272//National Institute of Allergy and Infectious Diseases/ ; },
mesh = {*Cryptococcus neoformans/genetics/drug effects ; *CRISPR-Cas Systems ; Humans ; *Plasmids/genetics ; *Selection, Genetic ; Drug Resistance, Fungal/genetics ; Nucleosides ; },
abstract = {Cryptococcus neoformans is the most common cause of human fungal meningitis and an important model system for studying fundamental eukaryotic biology. Genetic manipulation of this organism relies on three dominant drug resistance markers (nourseothricin acetyltransferase [NAT], neomycin phosphotransferase II [NEO], and hygromycin B phosphotransferase [HYG]) and the recyclable dominant prototrophic marker amdS. With ongoing technological advances that are expanding our ability to explore cryptococcal gene function, contemporary studies often require multiple genetic manipulations in the same strain. Additional dominant selection methods would maximize the utility of these tools by facilitating their combinatorial use. Here, we identify blasticidin S resistance via the blasticidin S deaminase (BSD) or blasticidin S resistance (BSR) markers as a novel dominant selection method for C. neoformans. We further validate phleomycin resistance via the bleomycin resistance gene (BLE) marker as an additional selection method, confirming a study that first established this marker 25 years ago (J. Hua, J. D. Meyer, and J. K. Lodge, Clin Diagn Lab Immunol 7:125-128, 2000, https://doi.org/10.1128/cdli.7.1.125-128.2000). To enable highly efficient CRISPR/Cas9-mediated genome modification, we incorporated these markers, as well as the newly established dominant prototrophic marker ptxD (M. Khongthongdam, T. Phetruen, and S. Chanarat, Microbiol Spectr 13:e01618-24, 2025, https://doi.org/10.1128/spectrum.01618-24), into a vector series that enables the construction of fused marker-sgRNA products via PCR. Altogether, this work expands the number of dominant genetic selection methods for C. neoformans to seven, including five drug selection regimes and two prototrophic methods. The vector series has been deposited at Addgene. IMPORTANCE Cryptococcus neoformans is the top-ranked World Health Organization priority fungal pathogen due to its widespread distribution and inadequate treatment options. Additionally, as a basidiomycete yeast occupying an underexplored branch of the fungal kingdom, this organism is a powerful system for deciphering core eukaryotic biology that is absent in classic model fungi. Defining functions for novel cryptococcal genes is a crucial priority, and the availability of additional genetic selection methods would facilitate these efforts. In this study, we establish blasticidin S resistance as a novel genetic selection method for C. neoformans, and we validate a previous report using phleomycin resistance as such. This work expands the number of reliable dominant selection methods to seven, providing flexibility for the introduction of sequential genetic modifications into single strains.},
}
@article {pmid40089804,
year = {2025},
author = {Zhu, J and Zhang, Y and Zhang, M and Hong, Y and Sun, C and Guo, Y and Yin, H and Lv, C and Guo, B and Wang, F and Xu, R},
title = {Natural variation and CRISPR/Cas9 gene editing demonstrate the role of a group VII ethylene response factor, HvERF62, in regulation of barley waterlogging tolerance.},
journal = {Journal of experimental botany},
volume = {76},
number = {17},
pages = {5071-5085},
doi = {10.1093/jxb/eraf101},
pmid = {40089804},
issn = {1460-2431},
support = {32301871//National Natural Science Foundation of China/ ; 2022YFD2301302//National key research and development plant/ ; },
mesh = {*Hordeum/genetics/physiology/metabolism ; *Plant Proteins/genetics/metabolism ; *CRISPR-Cas Systems ; Gene Editing ; Ethylenes/metabolism ; Genome-Wide Association Study ; Quantitative Trait Loci ; *Transcription Factors/genetics/metabolism ; Gene Expression Regulation, Plant ; },
abstract = {Waterlogging stress is a serious problem for plants throughout the world, inhibiting plant growth and development and even leading in severe circumstances to plant death. Ethylene-responsive VII (ERFVII) is a key transcription factor regulating plant waterlogging tolerance. In this study, a major quantitative trait locus influencing chlorophyll content under waterlogging stress was detected on chromosome 4H through genome-wide association study using a natural population of barley (Hordeum vulgare). Using RNA-Seq analysis, the group VII ethylene response factor HvERF62 was identified as the candidate gene. CRISPR/Cas9-guided knockout HvERF62 mutants showed sensitivity to waterlogging, with lower chlorophyll content, fewer adventitious roots, and lower root activity. Physiological and transcriptomic analyses showed that HvERF62 plays an important role in aerenchyma formation, reactive oxygen species homeostasis, and carbohydrate accumulation under waterlogging stress, and regulates waterlogging tolerance through starch and sucrose metabolism, the mitogen-activated protein kinase signaling pathway, plant hormone signal transduction, and glycolysis/gluconeogenesis signaling. Haplotype analysis showed that the amino acid sequence of the waterlogging-intolerant haplotype Hap3 was terminated prematurely. This study provides a new genetic resource and a relevant marker for identification of waterlogging-sensitive material and deepens our understanding of the molecular mechanisms of waterlogging response in barley.},
}
@article {pmid41184108,
year = {2026},
author = {Abir, AH and Benz, J and Frey, B and Bruns, H and Krönke, G and Gaipl, US and Schober, K and Mougiakakos, D and Mielenz, D},
title = {Glycolytic flux sustains human Th1 identity and effector function via STAT1 glycosylation.},
journal = {Life science alliance},
volume = {9},
number = {1},
pages = {},
doi = {10.26508/lsa.202503315},
pmid = {41184108},
issn = {2575-1077},
mesh = {Humans ; *Glycolysis ; Glycosylation ; *STAT1 Transcription Factor/metabolism/genetics ; *Th1 Cells/metabolism/immunology ; Interferon-gamma/metabolism ; Phosphorylation ; CRISPR-Cas Systems ; Cell Differentiation ; Gene Editing ; },
abstract = {T helper (Th) cell lineages are linked to metabolism, but precise mechanisms in human Th1 cells remain unclear. We addressed this question by in vitro stimulation and CRISPR/Cas9-mediated gene editing. Metabolic profiling revealed enhanced glycolytic activity in Th1 versus non-polarized cells, evidenced by increased extracellular acidification rate, ATP production via glycolysis, lactate secretion, NADH abundance, and elevated glycolysis-dependent anabolic activity. Inhibition of glycolysis reduced IFNγ production and STAT1 phosphorylation independent of JAK1/2 or SHP2 activity and STAT1 abundance, implicating glycolysis directly in sustaining STAT1-mediated Th1 functionality. O-glycosylation of STAT1 via O-glycosyltransferase was pivotal in modulating STAT1 activity. Pharmaceutical O-glycosyltransfer-ase inhibition prevented Th1 differentiation as well as STAT1 O-glycosylation. CRISPR/Cas9 mediated mutation of the O-glycosylation Ser499 and Thr510 sites diminished STAT1 Ser727 phosphorylation and IFNγ synthesis. Together, this study highlights glycolysis as key regulator of human Th1 cell identity and effector function, with STAT1 O-glycosylation selectively maintaining Th1 effector capacity. This mechanism could be explored to safeguard Th1 cells.},
}
@article {pmid41183413,
year = {2025},
author = {Chen, H and Zeng, Z and Wei, Y and Huang, H and Negahdary, M and Han, X and Lin, Y and He, L and Song, F and Wan, Y},
title = {Dynamic biosensing enables amplifier-collateral-cleavage enhancement for pathogen diagnostic.},
journal = {Biosensors &amp; bioelectronics},
volume = {293},
number = {},
pages = {118158},
doi = {10.1016/j.bios.2025.118158},
pmid = {41183413},
issn = {1873-4235},
abstract = {While CRISPR-Cas system and dynamic DNA nanotechnology have been extensively applied to mainstream biomedical domains, including gene editing, biochemical analysis, and molecular imaging-current approaches remain constrained by limitations in addressing increasingly nuanced and specialized experimental scenarios. Here, we report that CRISPR-CasΦ possesses a unique collateral-cleavage blockade characteristic: CasΦ is unable to recognize the "TTN" sequence in the loop region at the 3' end of stem-loop DNA, resulting in the blockade of collateral-cleavage activity. Leveraging this discovery, we successfully designed and customized two back-end signal amplifiers for biosensing by integrating dynamic DNA sensing studies of CasΦ. Based on these two specialized stem-loop amplifiers, we further developed the Amplifier-collateral-cleavage enhancement (ACE) method, achieving exponential signal amplification. Clinical validation using 112 urine samples demonstrated that ACE exhibits 98.8 % sensitivity and 90 % specificity. These findings highlight the potential of CasΦ dynamic sensing and establish a bridge for future integration of dynamic DNA technology and CRISPR systems.},
}
@article {pmid41124025,
year = {2025},
author = {Su, F and Zhang, H and Ren, C and Jiang, Y and Qiao, Y and Zhang, S and Liu, J and Li, S and Li, Y},
title = {MnO2@Mn3O4 Heterojunction as a Coreactant Catalyst Collaborated with T-Shaped DNA Cycling-CRISPR/Cas12a Cascade Amplification for Locus-Specific N6-Methyladenosine RNA Modification Detection.},
journal = {Analytical chemistry},
volume = {97},
number = {43},
pages = {24165-24175},
doi = {10.1021/acs.analchem.5c04791},
pmid = {41124025},
issn = {1520-6882},
mesh = {*Adenosine/analogs &amp; derivatives/analysis ; *Manganese Compounds/chemistry ; *Oxides/chemistry ; *Biosensing Techniques/methods ; Humans ; *RNA/analysis/chemistry ; Electrochemical Techniques/methods ; *CRISPR-Cas Systems ; *DNA/chemistry/genetics ; Metal Nanoparticles/chemistry ; Catalysis ; Gold/chemistry ; Nucleic Acid Amplification Techniques ; CRISPR-Associated Proteins/genetics ; Limit of Detection ; Luminescent Measurements ; HeLa Cells ; Bacterial Proteins ; Endodeoxyribonucleases ; },
abstract = {As the predominant RNA modification, N6-methyladenosine (m[6]A) is recognized to play pivotal regulatory roles in fundamental cellular functions and oncogenic processes. However, the precise analysis of site-specific m[6]A modifications continues to present significant challenges. In this work, a unique electrochemiluminescence (ECL) biosensor for the locus-specific detection of m[6]A in RNA was developed in the first utilization of a MnO2@Mn3O4 heterojunction as a coreaction catalyst in collaboration with a T-shaped DNA cycling-CRISPR/Cas12a cascade amplification strategy. The MnO2@Mn3O4 heterojunction was observed to significantly enhance coreactant catalytic activity, yielding a 7.3-fold increase in the ECL intensity of the gold nanoparticles (AuNPs)/MnO2@Mn3O4/(2,2'-bipyridine) dichlororuthenium(II) (Ru(bpy)3[2+])/Nafion/GCE compared to the AuNPs/Ru(bpy)3[2+]/Nafion/GCE. Sequentially, the T-shaped DNA cycling amplification strategy effectively converted the target m[6]A RNA into an amplified biosignal, further enhanced by a CRISPR/Cas12a signal amplification system mediated by framework nucleic acid (FNA)-based photocontrollable nucleic acid protection, ensuring the sensitivity and specificity of m[6]A RNA. The integration of the triple signal amplification strategy achieved detection limits as low as 0.05 pM (S/N = 3) for a linearity spanning from 100 fM to 100 nM. The proposed ECL biosensor has been applied in detecting site-specific m[6]A modifications in total real RNA samples extracted from HeLa cells, demonstrating its promising applications for clinical diagnosis.},
}
@article {pmid41084820,
year = {2025},
author = {Ma, AX and Zhang, Q and Zhang, MY and Liu, SL and Wang, ZG and Pang, DW},
title = {One-step naked-eye fluorescence detection of viruses using quantum dot-magnetic beads coupled with CRISPR/Cas12a.},
journal = {Chemical communications (Cambridge, England)},
volume = {61},
number = {89},
pages = {17432-17435},
doi = {10.1039/d5cc03645c},
pmid = {41084820},
issn = {1364-548X},
mesh = {*Quantum Dots/chemistry ; *CRISPR-Cas Systems ; Humans ; Fluorescence ; *CRISPR-Associated Proteins/metabolism/chemistry ; *Simplexvirus/isolation &amp; purification ; Bacterial Proteins ; Endodeoxyribonucleases ; },
abstract = {This work constructs quantum dot-magnetic beads coupled with CRISPR/Cas12a for naked-eye detection of herpes simplex virus. The system integrates the high brightness of quantum dots and trans-cleavage activity of Cas12a, enabling rapid, simple, and accurate viral detection.},
}
@article {pmid40954319,
year = {2025},
author = {Wei, D and Cheng, P and Song, Z and Liu, Y and Xu, X and Huang, X and Wang, X and Zhang, Y and Shu, W and Wei, Y},
title = {AI-guided Cas9 engineering provides an effective strategy to enhance base editing.},
journal = {Molecular systems biology},
volume = {21},
number = {11},
pages = {1563-1580},
pmid = {40954319},
issn = {1744-4292},
support = {12331018//MOST | National Natural Science Foundation of China (NSFC)/ ; 32401220//MOST | National Natural Science Foundation of China (NSFC)/ ; PT2025-03-01//Shanghai institute for Biomedical and Pharmaceutical Technologie/ ; RC2025-01//Shanghai institute for Biomedical and Pharmaceutical Technologie/ ; CXPJJH122006-1014//CHEN XIAO-PING FOUNDATION FOR THE DEVELOPMENT OF SCIENCE AND TECHNOLOGY OF HUBEI PROVINCE/ ; },
mesh = {*Gene Editing/methods ; Humans ; *CRISPR-Cas Systems/genetics ; *CRISPR-Associated Protein 9/genetics/metabolism ; *Protein Engineering/methods ; *Artificial Intelligence ; Point Mutation ; Cell Line, Tumor ; HEK293 Cells ; },
abstract = {Precise genome editing is crucial for functional studies and therapies. Base editors, while powerful, require optimization for efficiency. Meanwhile, emerging protein design methods and protein language models have driven efficient and intelligent protein engineering. In this study, we employed the Protein Mutational Effect Predictor (ProMEP) to predict the effects of single-site saturated mutations in Cas9 protein, using AncBE4max as the prototype to construct and test 18 candidate point mutations. Based on this, we further predicted combinations of multiple mutations and successfully developed a high-performance variant AncBE4max-AI-8.3, achieving a 2-3-fold increase in average editing efficiency. Introducing the engineered Cas9 into CGBE, YEE-BE4max, ABE-max, and ABE-8e improved their editing performance. The same strategy also substantially improves the efficiencies of HF-BEs. Stable enhancement in editing efficiency was also observed across seven cancer cell lines and human embryonic stem cells. In conclusion, we validated that AI models can serve as more effective protein engineering tools, providing a universal improvement strategy for a series of gene editing tools.},
}
@article {pmid40878190,
year = {2025},
author = {Tian, L and Gao, Y and Lu, Y and Xu, F and Feng, Z and Zi, L and Deng, Z and Yang, J},
title = {Modular RCA-CRISPR/Cas12a amplification on a multi-volume SlipChip for ultrafast, single-copy quantification of circRNA and miRNA in ovarian cancer.},
journal = {Lab on a chip},
volume = {25},
number = {22},
pages = {5762-5776},
doi = {10.1039/d5lc00585j},
pmid = {40878190},
issn = {1473-0189},
mesh = {Humans ; *MicroRNAs/genetics/analysis ; *RNA, Circular/genetics/analysis ; Female ; *Ovarian Neoplasms/genetics ; *Nucleic Acid Amplification Techniques/instrumentation ; *CRISPR-Cas Systems ; Cell Line, Tumor ; *Lab-On-A-Chip Devices ; *CRISPR-Associated Proteins/metabolism/genetics ; Limit of Detection ; Bacterial Proteins ; Endodeoxyribonucleases ; },
abstract = {The aberrant expression of RNAs in ovarian cancer (OC) progression highlights their potential as clinical biomarkers. However, rapid and accurate quantification of these RNAs in biosamples remains a significant challenge. In this study, we develop a modular isothermal rolling circle amplification (RCA)-activated Cas12a loop-enhanced (MIRACLE) amplification method for circRNA and miRNA quantification without the need of reverse transcription. In this design, isothermal amplification of modular DNA can be initiated by target-specific RCA primers or miRNAs, with the amplification products subsequently recognized by the Cas12a system to generate measurable signals. When integrated with a multi-volume sliding chip (SlipChip) platform, this MIRACLE method enables portable, rapid and ultra-sensitive quantification of these two types of RNA. Under optimized conditions, this platform exhibits detection limits of 0.125 copies per μL for circRNA and 0.326 copies per μL for miRNA, covering a 5-log dynamic range from 10[-1] to 10[3] copies per μL within 35 min. The platform was validated using OC cell lines and clinical blood samples. It successfully profiled OC RNA biomarkers (hsa_circ_0049101 and hsa-miR-338-3p) and effectively distinguished between early and advanced stages of OC. These results show a strong correlation with RT-qPCR (R[2] = 0.953 for circRNA and R[2] = 0.947 for miRNA). This work establishes a versatile CRISPR-microfluidic platform for cancer diagnosis. Its modular design allows for adaptation to detect other cancer-related RNA biomarkers, thereby addressing critical needs in precision oncology.},
}
@article {pmid41183015,
year = {2025},
author = {Keuthen, H and Pozhydaieva, N and Höfer, K},
title = {Precise Phage Mutagenesis with NgTET-Assisted CRISPR-Cas Systems.},
journal = {Journal of visualized experiments : JoVE},
volume = {},
number = {224},
pages = {},
doi = {10.3791/69022},
pmid = {41183015},
issn = {1940-087X},
mesh = {*CRISPR-Cas Systems/genetics ; *Bacteriophages/genetics ; *Mixed Function Oxygenases/genetics/metabolism ; Mutagenesis ; DNA, Viral/genetics/chemistry ; *Mutagenesis, Site-Directed/methods ; },
abstract = {Bacteriophages, viruses that specifically target their bacterial hosts, hold significant potential for biotechnology and medicine, especially in combating multidrug-resistant infections. However, the molecular mechanisms underlying phage infection remain largely underexplored. Precise, site-specific mutagenesis of phages is a powerful tool to elucidate gene functions and phage-host interactions. However, a major challenge in phage genome mutagenesis is the presence of phage DNA modifications that interfere with conventional genome editing tools like CRISPR-Cas. While CRISPR-Cas systems have been used successfully for targeted mutagenesis in various organisms, their effectiveness in phage mutagenesis is often limited by DNA modifications such as cytosine glycosylation. To overcome this barrier, we developed an efficient method that temporarily reduces the abundance of phage DNA modifications, enabling efficient CRISPR-Cas targeting and precise mutation introduction into phage genomes. Specifically, we use the Ten Eleven Translocation (TET) methylcytosine dioxygenase from Naegleria gruberi (NgTET), which iteratively demodifies methylated and hydroxymethylated cytosines in DNA. By oxidizing hydroxymethylated cytosines within phage DNA, NgTET prevents subsequent cytosine modification like glycosylation and significantly enhances the efficiency of Cas-mediated DNA cleavage. In conclusion, the scarless and precise genome-editing approach presented here enables the efficient introduction of point mutations while maintaining the native gene architecture in phage genomes. By preserving intact transcriptional and translational frameworks, this method minimizes unintended disruptions to complex regulatory networks. This is particularly important for investigating essential or multifunctional phage proteins. The ability to generate targeted genetic modifications without introducing extraneous sequences significantly expands the experimental toolkit for phage biology. This strategy not only facilitates detailed functional studies but also enhances the potential for rational engineering of phages for therapeutic and biotechnological applications.},
}
@article {pmid41182907,
year = {2025},
author = {Pantoja-Alonso, MA and Camas-Reyes, JA and Cano-Segura, R and Cárdenas-Aquino, MDR and Martínez-Antonio, A},
title = {A comprehensive review of genomic-scale genetic engineering as a strategy to improve bacterial productivity.},
journal = {Microbiology (Reading, England)},
volume = {171},
number = {11},
pages = {},
doi = {10.1099/mic.0.001628},
pmid = {41182907},
issn = {1465-2080},
mesh = {*Bacteria/genetics/metabolism ; *Genetic Engineering/methods ; CRISPR-Cas Systems ; *Genome, Bacterial ; Genomics/methods ; Gene Editing ; Metabolic Engineering/methods ; Synthetic Biology/methods ; },
abstract = {Bacterial genome engineering has evolved to provide increasingly precise, robust and rapid tools, driving the development and optimization of bacterial production of numerous compounds. The field has progressed from early random mutagenesis methods, labour-intensive and inefficient, to rational and multiplexed strategies enabled by advances in genomics and synthetic biology. Among these tools, CRISPR/Cas has stood out for its versatility and its ability to achieve precision levels ranging from 50% to 90%, compared to the 10-40% obtained with earlier techniques, thereby enabling remarkable improvements in bacterial productivity. Nevertheless, like its predecessors, it still demands continuous refinement to reach full maturity. In this context, the present review addresses the lack of a unified overview by summarizing historical milestones and practical applications of genomic engineering tools in bacteria. It integrates diverse approaches to provide a comprehensive perspective on the evolution and prospects of these fundamental biotechnological tools.},
}
@article {pmid41182905,
year = {2025},
author = {Lamb, CH and Riesle-Sbarbaro, S and Prescott, JB and Te Velthuis, AJW and Myhrvold, C and Nilsson-Payant, BE},
title = {Amplification-free detection of zoonotic viruses using Cas13 and multiple CRISPR RNAs.},
journal = {The Journal of general virology},
volume = {106},
number = {11},
pages = {},
doi = {10.1099/jgv.0.002169},
pmid = {41182905},
issn = {1465-2099},
mesh = {Animals ; *RNA, Viral/genetics ; Humans ; *Influenza A virus/genetics/isolation &amp; purification ; Sensitivity and Specificity ; *Orthohantavirus/genetics/isolation &amp; purification ; *CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; *Zoonoses/virology/diagnosis ; },
abstract = {Zoonotic viruses such as hantaviruses and influenza A viruses present a threat to humans and livestock. There is thus a need for methods that are rapid, sensitive and relatively cheap to detect infections with these pathogens early. Here, we use an amplification-free clustered regularly interspaced short palindromic repeats-associated protein 13 (CRISPR-Cas13)-based assay, which is simple, cheap and field-deployable, to detect the presence or absence of genomic hantavirus or influenza A virus RNA. In addition, we evaluate whether the use of multiple CRISPR RNAs (crRNAs) can improve the sensitivity of this amplification-free method. We demonstrate that for the hantaviruses Tula virus (TULV) and Andes virus (ANDV), a combination of two or three crRNAs provides the best sensitivity for detecting viral RNA, whereas for influenza virus RNA detection, additional crRNAs provide no consistent benefit. We also show that the amplification-free method can be used to detect TULV and ANDV RNA in tissue culture infection samples, ANDV from hamster lung samples and influenza A virus RNA in clinical nasopharyngeal swabs. In clinical samples, the Cas13 assay has an 85% agreement with RT-qPCR for identifying a positive sample. Overall, these findings indicate that amplification-free CRISPR-Cas13 detection of viral RNA has potential as a tool for rapidly detecting zoonotic virus infections.},
}
@article {pmid41182611,
year = {2026},
author = {Chiurillo, MA and Ahmed, M and González, C and Rosón, JN and Das, A and Lander, N},
title = {Cloning-Free Genome Editing by CRISPR/T7RNAP/Cas9 in Trypanosoma cruzi.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2982},
number = {},
pages = {59-76},
pmid = {41182611},
issn = {1940-6029},
mesh = {*Trypanosoma cruzi/genetics ; *Gene Editing/methods ; *CRISPR-Cas Systems ; *DNA-Directed RNA Polymerases/genetics ; *Viral Proteins/genetics ; Cloning, Molecular ; Humans ; *Genome, Protozoan ; Gene Knockout Techniques ; },
abstract = {The genetic manipulation of the human parasite Trypanosoma cruzi has been significantly improved since the implementation of the CRISPR/Cas9 technology for genome editing in this organism. Initially, the system was successfully used for gene knockout and endogenous C-terminal tagging in T. cruzi. Recently, an updated version of this technology has been used for gene complementation, site-directed mutagenesis, and N-terminal tagging in trypanosomatids. This cloning-free strategy, called CRISPR/T7RNAP/Cas9, is extremely useful for identifying essential genes when null mutants are not viable. Mutant cell lines obtained by this new system have been used for the functional characterization of proteins in different developmental stages of this parasite's life cycle, including infective trypomastigotes and intracellular amastigotes. In this chapter, we describe the methodology to achieve genome editing by CRISPR/T7RNAP/Cas9 in T. cruzi. Our method involves the generation of T. cruzi epimastigotes that constitutively express the T7 RNA polymerase (T7RNAP) and SpCas9, and their co-transfection with an sgRNA template and donor DNA(s) as polymerase chain reaction (PCR) products. Using this strategy, we have generated genetically modified parasites in 2-3 weeks without the need for gene cloning, cell sorting, or having to perform several transfection attempts to verify the sgRNA efficiency for targeting the gene of interest. The methodology has been organized according to three main genetic purposes: gene knockout, gene complementation of knockout cell lines, and endogenous (N- or C-terminal) tagging in T. cruzi.},
}
@article {pmid41182029,
year = {2025},
author = {Ashworth, KE and Zhang, J and D'Amata, C and Héon, E and Ballios, BG},
title = {USH2A-Mutated Human Retinal Organoids Model Rod-Cone Dystrophy.},
journal = {Investigative ophthalmology &amp; visual science},
volume = {66},
number = {14},
pages = {2},
doi = {10.1167/iovs.66.14.2},
pmid = {41182029},
issn = {1552-5783},
mesh = {Humans ; *Organoids/pathology/metabolism ; Induced Pluripotent Stem Cells/pathology/metabolism ; *Mutation ; *Extracellular Matrix Proteins/genetics/metabolism ; *Cone-Rod Dystrophies/genetics/pathology/metabolism ; Retinal Rod Photoreceptor Cells/pathology ; *Retinitis Pigmentosa/genetics/pathology ; CRISPR-Cas Systems ; },
abstract = {PURPOSE: USH2A mutations are the leading cause of autosomal recessive retinitis pigmentosa (RP), a progressive blinding disease marked by photoreceptor degeneration. Animal models fail to recapitulate the features of USH2A RP seen in humans, and its earliest pathogenic events remain unknown. Here, we established a human model of USH2A RP using retinal organoids derived from patient induced pluripotent stem cells and CRISPR-Cas9-engineered isogenic-USH2A-/- induced pluripotent stem cells.<br><br>METHODS: We assessed organoids for cellular, molecular, and morphological defects using serial live imaging and whole organoid and fixed section analyses.<br><br>RESULTS: Both patient-derived and isogenic-USH2A-/- organoids showed preferential rod photoreceptor loss followed by widespread degeneration, consistent with the clinical phenotype. Additionally, isogenic-USH2A-/- organoids showed early defects in proliferation and structure.<br><br>CONCLUSIONS: Our findings suggest that molecular changes precede overt photoreceptor loss in USH2A RP, and pathogenesis may begin before clinical symptoms emerge. By defining early and late disease features, we provide new insight on the developmental origins of USH2A RP to guide therapeutic strategies.},
}
@article {pmid41178730,
year = {2025},
author = {Avaro, AS and Santiago, JG},
title = {Engineering guidelines for CRISPR diagnostics.},
journal = {Chemical communications (Cambridge, England)},
volume = {},
number = {},
pages = {},
doi = {10.1039/d5cc04206b},
pmid = {41178730},
issn = {1364-548X},
abstract = {This Feature Article reviews engineering guidelines for the design of CRISPR assays, including experimentally validated theoretical models and recommendations for experimental research practice and reporting. First, the state of the art of CRISPR kinetics studies is reviewed. Then presented is a summary of the existence and persistence of widespread gross errors in reports of kinetic rate constants of CRISPR-Cas enzymes, as well as the fact that many CRISPR studies provide insufficient data to check for consistency or assess calibration. Proper experimental procedures including signal calibration are critical to the assessment, design, and future development of CRISPR kinetics assays and CRISPR diagnostics. This review then presents guidelines for the calibration of fluorescence-based CRISPR assays. Fluorescence is the most common detection modality, and incorrect calibration is implicated in high-profile, gross errors in the field. Also presented is a review of enzymatic kinetic rates and reporter molecule degradation as the major factor limiting CRISPR assay sensitivity. Lastly, progress in, and criticism of, microfluidic applications of CRISPR assays is summarized.},
}
@article {pmid41178318,
year = {2025},
author = {Leung, CY and Wimmer, EA and Ahmed, HMM},
title = {Synthetic biology approaches to generate temperature-sensitive alleles for the Sterile Insect Technique.},
journal = {Insect science},
volume = {},
number = {},
pages = {},
doi = {10.1111/1744-7917.70186},
pmid = {41178318},
issn = {1744-7917},
support = {ResearchcooperationLowerSaxony-Israel//Nieders&#xe4;chsisches Ministerium f&#xfc;r Wissenschaft und Kultur/ ; Project D44003//International Atomic Energy Agency/ ; },
abstract = {The Sterile Insect Technique (SIT) is an environmentally friendly, sustainable pest control approach, which uses large-scale releases of sterile insects to suppress or eradicate target populations through infertile matings. The efficiency of SIT is enhanced by male-only releases requiring genetic sexing strains (GSSs) that are classically based on selectable recessive visible markers or temperature-sensitive lethal (tsl) mutations and a rescue by a wild-type allele translocated to the male-determining chromosome. The transfer of identified or designed temperature-sensitive alleles might allow the generation of neoclassical GSSs in additional SIT target species. By using precise genome-editing tools, such as CRISPR/Cas, the creation of specific mutations in target genes and the integration of a wild-type copy is feasible without the introduction of foreign DNA. This might ease regulation of neoclassical GSSs, since they are not considered transgenic. However, integration and expression of genes at male-determining loci or chromosomes is not reliably established. Therefore, additional strategies to link temperature-sensitive phenotypes to female development are required, which could be achieved by targeting genes involved in dosage compensation or sex determination. To create temperature-sensitive alleles, rational protein design using advanced modeling and prediction tools to evaluate and tailor the effect of mutations on protein stability and temperature sensitivity can be used. In addition, emerging synthetic biology strategies such as temperature-inducible N-degrons or temperature-sensitive inteins provide powerful tools to generate temperature sensitivity. Such approaches should enable conditional control over proteins causing female lethality or sex conversion and therefore promise straightforward generic approaches to generate GSSs for male-only production in SIT target species.},
}
@article {pmid41177343,
year = {2025},
author = {Rahimian, M and Panahi, B},
title = {Genomic insights into Erwinia amylovora prophages: Diversity, defense strategies, and phage-host coevolution.},
journal = {Infection, genetics and evolution : journal of molecular epidemiology and evolutionary genetics in infectious diseases},
volume = {},
number = {},
pages = {105846},
doi = {10.1016/j.meegid.2025.105846},
pmid = {41177343},
issn = {1567-7257},
abstract = {Erwinia amylovora, the causative agent of fire blight in Rosaceae plants like apple and pear, is a major agricultural pathogen responsible for significant economic losses. This gram-negative phytopathogen has the potential to acquire antibiotic resistance and virulence genes, a situation that becomes more severe and restricts treatment options. Using an in silico approach, we analyzed 268 E. amylovora genomes and identified seven high-quality temperate prophages, all classified under Caudoviricetes, with average genome sizes of 44.2 kbp and 51 % GC content. These prophages exhibited unique genomic features, including tRNA genes (Ph-Ea644), anti-defense systems like ardc (Ph-Ea6-96), and regulatory/lysis genes (Ph-EaFC01). Comparative genomics and phylogenetic analyses grouped them into five clades, with Ph-Ea4-96, Ph-Ea3-97, and Ph-Ea2-97 being genetically identical. Functional annotation revealed streptomycin resistance genes and a CAZyme (GH23) in Ph-Ea7-3, virulence factors (e.g., alginate biosynthesis proteins), and six auxiliary metabolic genes (AMGs) linked to metabolic adaptation. Additionally, Ph-Ea644 encoded a cell wall-binding receptor protein. The prophages also carried defense systems (Gabija, CBASS) and 31 anti-CRISPR proteins (ACRs), suggesting evasion of host immunity. CRISPR-Cas analysis indicated fewer arrays and spacers in prophage-containing strains, underscoring CRISPR's role in lysogeny resistance. These findings highlight the genomic plasticity of E. amylovora prophages, their interactions with bacterial defenses, and their potential influence on pathogen evolution. This study enhances understanding of temperate phages in agricultural pathogens and underscores challenges in phage-based biocontrol strategies.},
}
@article {pmid41177179,
year = {2025},
author = {Bacci, L and Pollutri, D and Ripa, IJ and D'Andrea, M and Marchand, V and Motorin, Y and Hesse, AM and Couté, Y and Filipek, K and Penzo, M},
title = {Ribosomal protein L5 (RPL5/uL18) I60V mutation is associated to increased translation and modulates drug sensitivity in T-cell acute lymphoblastic leukemia cells.},
journal = {Biochemical pharmacology},
volume = {},
number = {},
pages = {117497},
doi = {10.1016/j.bcp.2025.117497},
pmid = {41177179},
issn = {1873-2968},
abstract = {Somatic mutations in ribosomal proteins (RPs), including RPL5, have been reported in approximately 10 % of pediatric patients with T-cell acute lymphoblastic leukemia (T-ALL). In cancer, the incorporation of mutant RPs into ribosomes often disrupts canonical ribosome function, thereby contributing to disease development. In this study, we aimed to characterize the effects of the RPL5-I60V mutation in the context of T-ALL, focusing on its impact on translation and cellular responses to a panel of compounds in vitro. Using CRISPR-Cas9, we generated a homozygous knock-in mutant in Jurkat cells and investigated its effects on ribosome biogenesis. We observed both quantitative and qualitative alterations in the production of the large ribosomal subunit. Ribosomes containing the mutant RPL5 protein exhibited intrinsically increased protein synthesis activity, which correlated with enhanced cellular proliferation. We then evaluated the response of these mutant cells to a panel of compounds targeting protein synthesis at various levels-including an MNK1 inhibitor, metformin, silvestrol, homoharringtonine, anisomycin, resveratrol, and hygromycin B-as well as cytarabine, a chemotherapeutic agent commonly used in T-ALL treatment. Our results showed that the RPL5-I60V mutation confers increased sensitivity to most of these compounds, with the exception of hygromycin B. This study advances our understanding of how oncoribosomes contribute to cancer pathogenesis and highlights the therapeutic potential of directly or indirectly targeting altered ribosomes, offering insights for the development of personalized treatment strategies.},
}
@article {pmid41176953,
year = {2025},
author = {Lankireddy, SV and Lekkala, S and Khadgi, A and Sripathi, VR and Janga, MR},
title = {Molecular biology of Cotton Leafroll Dwarf Virus (CLRDV) and potential application of CRISPR-Cas technology for developing virus-resistant cotton.},
journal = {Virology},
volume = {614},
number = {},
pages = {110730},
doi = {10.1016/j.virol.2025.110730},
pmid = {41176953},
issn = {1096-0341},
abstract = {Cotton leafroll dwarf virus (CLRDV) poses an increasing threat to global cotton production. Transmitted by the cotton aphid (Aphis gossypii) in a persistent, circulative manner, CLRDV exhibits a wide geographical distribution, with documented presence in South America, Africa, Asia, and the USA. Infection can result in either cotton blue disease (CBD) in South America or cotton leafroll dwarf disease (CLRDD) in the USA, both of which are associated with CLRDV. The considerable genetic diversity and frequent recombination events within CLRDV populations contribute to this symptom variability and complicate both diagnosis and management. While resistant cultivars have reduced disease impact in South America, these lines remain susceptible to emerging US strains, underscoring the urgent need for region-specific resistance breeding. Current molecular diagnostics rely on RT-PCR, but there is a need for rapid, field-deployable detection tools. Recent advances, such as CRISPR-Cas13a based SHERLOCK assays, offer sensitive and specific detection of CLRDV, with potential for on-site applications. Efficient screening techniques, supported by next-generation sequencing and transcriptomics, are essential for identifying novel resistance sources and elucidating virus-host interactions. CRISPR-based genome editing holds significant promise, as demonstrated in other crops. Targeted disruption of host susceptibility genes using CRISPR-Cas9, or direct degradation of viral genomes with RNA-targeting systems such as Cas12/Cas13, could offer durable, broad-spectrum resistance. By integrating molecular virology, high-throughput genomics, and precision gene editing, this review outlines a roadmap for translating these advances into sustainable, field-level solutions for CLRDV management and long-term cotton productivity.},
}
@article {pmid41176802,
year = {2025},
author = {Kalinina, NO and Spechenkova, NA and Taliansky, ME},
title = {Erratum to: Biotechnological Approaches to Plant Antiviral Resistance: CRISPR-Cas or RNA Interference?.},
journal = {Biochemistry. Biokhimiia},
volume = {90},
number = {10},
pages = {1450},
doi = {10.1134/S0006297925100013},
pmid = {41176802},
issn = {1608-3040},
}
@article {pmid41175344,
year = {2025},
author = {Shamloo, S and Schloßhauer, JL and Tiwari, S and Denise Fischer, K and Almolla, O and Ghebrechristos, Y and Kratzenberg, L and Bejoy, AM and Aifantis, I and Boccalatte, F and Wang, E and Imig, J},
title = {RNA binding of GAPDH controls transcript stability and protein translation in acute myeloid leukemia.},
journal = {RNA biology},
volume = {22},
number = {1},
pages = {1-23},
doi = {10.1080/15476286.2025.2580180},
pmid = {41175344},
issn = {1555-8584},
mesh = {Humans ; *Leukemia, Myeloid, Acute/genetics/metabolism/pathology ; *Glyceraldehyde-3-Phosphate Dehydrogenases/metabolism/genetics ; *Protein Biosynthesis ; *RNA, Messenger/genetics/metabolism ; RNA-Binding Proteins/metabolism/genetics ; 5' Untranslated Regions ; Cell Line, Tumor ; *RNA Stability ; Protein Binding ; Gene Expression Regulation, Leukemic ; Cell Proliferation ; Glyceraldehyde-3-Phosphate Dehydrogenase (Phosphorylating) ; },
abstract = {Dysregulation of RNA binding proteins (RBPs) is a hallmark in cancerous cells. In acute myeloid leukaemia (AML) RBPs are key regulators of tumour proliferation. While classical RBPs have defined RNA binding domains, RNA recognition and function in AML by non-canonical RBPs (ncRBPs) remain unclear. Given the inherent complexity of targeting AML broadly, our goal was to uncover potential ncRBP candidates critical for AML survival using a CRISPR/Cas-based screening. We identified the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as a pro-proliferative factor in AML cells. Based on cross-linking and immunoprecipitation (CLIP), we are defining the global targetome, detecting novel RNA targets mainly located within 5'UTRs, including GAPDH, RPL13a, and PKM. The knockdown of GAPDH unveiled genetic pathways related to ribosome biogenesis, translation initiation, and regulation. Moreover, we demonstrated a stabilizing effect through GAPDH binding to target transcripts including its own mRNA. The present findings provide new insights on the RNA functions and characteristics of GAPDH in AML.},
}
@article {pmid40958477,
year = {2025},
author = {Tao, XL and Lei, YM and Zhou, XM and Chen, ZP and Ma, Y and Ma, PY and Song, DQ and Zhuo, Y},
title = {Allosteric Activation of Cas12a via PAM Topological Engineering for Direct and Rapid Detection of Nucleases.},
journal = {Angewandte Chemie (International ed. in English)},
volume = {64},
number = {45},
pages = {e202515521},
doi = {10.1002/anie.202515521},
pmid = {40958477},
issn = {1521-3773},
support = {22374123//National Natural Science Foundation of China/ ; SWU-XJLJ202303//The Fundamental Research Funds for the Central Universities/ ; },
mesh = {*CRISPR-Associated Proteins/metabolism/chemistry ; Allosteric Regulation ; CRISPR-Cas Systems ; *DNA/chemistry/metabolism ; *Endodeoxyribonucleases/metabolism/chemistry ; Bacterial Proteins ; },
abstract = {A contemporary question in the intensely active field of CRISPR-Cas12a-based molecular diagnostics is how to simplify the multistep conversion process for detecting nonnucleic acid targets. Herein we describe an allosteric Y-shaped DNA structure for Cas12a activation via protospacer-adjacent motif (PAM) topological engineering (Y-COPE) to achieve straightforward and diverse nuclease monitoring. The newly designed topological structure of the Y-COPE is characterized by a split PAM embedded at the three-way junction and protospacers flanking both sides. This unique spatial configuration of the PAM effectively prevents Cas12a activation. Upon target cleavage, the released truncated fragments can dynamically correct the PAM, which promptly restores the dsDNA conformation for Cas12a activation and accomplishes signal output. Theoretical calculation results revealed that, compared with the canonical dsDNA activator, in the Y-COPE, there was a 1.8 Å increase in the center distance between Lys595 of Cas12a and the PAM, which led to a 24.2 kcal mol[-1] increase in binding free energy. This clearly revealed the underlying inhibition mechanism of the topological configuration of the PAM for Cas12a activation. This study advances the understanding of the dynamic response of Cas12a to topological PAM conformations and introduces the universal concept of CRISPR-based nonnucleic acid detection to benefit the next-generation molecular diagnostics.},
}
@article {pmid40888991,
year = {2025},
author = {Lin, HK and Dai, J and Pusztai, L},
title = {Integrating large-scale in vitro functional genomic screen and multi-omics data to identify novel breast cancer targets.},
journal = {Breast cancer research and treatment},
volume = {214},
number = {3},
pages = {319-327},
pmid = {40888991},
issn = {1573-7217},
support = {BCRF-22-133, SAC220225//Breast Cancer Research Foundation Investigator Award (BCRF-22-133), Susan Komen Leadership Grant (SAC220225)/ ; },
mesh = {Humans ; Female ; *Breast Neoplasms/genetics/drug therapy/pathology ; *Genomics/methods ; DNA Copy Number Variations ; Cell Line, Tumor ; *Biomarkers, Tumor/genetics ; Mutation ; Gene Expression Regulation, Neoplastic ; Gene Expression Profiling ; CRISPR-Cas Systems ; Transcriptome ; Molecular Targeted Therapy ; Multiomics ; },
abstract = {PURPOSE: Our goal is to leverage publicly available whole transcriptome and genome-wide CRISPR-Cas9 screen data to identify and prioritize novel breast cancer therapeutic targets.<br><br>METHODS: We used DepMap dependency scores&#x2009;>&#x2009;0.5 to identify genes that are potential therapeutic targets in 48 breast cancer cell lines. We removed genes that were pan-essential or were not expressed in TCGA breast cancer cohort. Genes were prioritized based on druggability using the Drug-Gene Interaction Database. Targets were defined separately for ER+, HER2+, and TNBC. A broader list of genes with dependency score&#x2009;>&#x2009;0.25 were used to assess the associations between dependency scores and mutations and copy number variations (CNV) to identify potential synthetic lethal relationships and to map survival critical genes into biological pathways.<br><br>RESULTS: 66, 53, and 29 genes were prioritized as targets in ER+, HER2+, and TNBC, respectively. These included known actionable targets and many novel targets. ER+ included FOXA1, GATA3, LDB1, TRPS1, NAMPT, WDR26, and ZNF217; HER2+ cancers included STX4, HECTD1, and TBL1XR1; and TNBC included GFPT1 and GPX4. Synthetic lethal associations revealed 5 and 19 significant associations between potential survival critical genes and mutations in HER2+&#x2009;and TNBC, respectively. For example, PIK3CA mutation increased dependency on NDUFS3 in HER2+&#x2009;cancers, and CNTRL mutation increased dependency on electron transport chain (ETC) genes in TNBC. 329, 747, and 622 CNVs showed synthetic lethal association in ER+, HER2+, and TNBC, respectively.<br><br>CONCLUSION: We provide a genome-wide drug target prioritization list for breast cancer derived from integrated large-scale omics data.},
}
@article {pmid40590508,
year = {2025},
author = {Mohammadzadeh, R and Shahbazi, S and Khodaei, N and Sabzi, S},
title = {Emerging Therapeutic Strategies to Combat Antimicrobial Resistance in the Post-Antibiotic Era.},
journal = {Journal of basic microbiology},
volume = {65},
number = {11},
pages = {e70070},
doi = {10.1002/jobm.70070},
pmid = {40590508},
issn = {1521-4028},
mesh = {Humans ; *Bacterial Infections/therapy/microbiology/drug therapy ; *Anti-Bacterial Agents/pharmacology/therapeutic use ; *Drug Resistance, Bacterial/drug effects ; *Bacteria/drug effects/pathogenicity ; Probiotics/therapeutic use ; Bacteriophages ; Antimicrobial Peptides/therapeutic use/pharmacology ; Bacteriocins/therapeutic use ; Animals ; CRISPR-Cas Systems ; },
abstract = {Antimicrobial resistance (AMR) is a serious global health issue. This review aims to explore alternative therapeutic strategies for combating AMR. The goal is to evaluate emerging treatments that target resistant pathogens through novel mechanisms, bypassing the limitations of traditional antibiotics. Recent researches highlight several promising alternatives, including antibodies, antimicrobial peptides, bacteriocins, bacteriophages, and probiotics (in the clinical trials) and synthetic antimicrobial peptides, anti-virulence strategies, genetically modified phages, antibacterial oligonucleotides, CRISPR-Cas9, and predatory bacteria (in the research stage). These therapies demonstrate potential to overcome AMR by targeting specific bacterial mechanisms, reducing toxicity, and evading resistance. Alternative therapies for AMR present significant promise, offering new avenues for treatment. Despite challenges in optimization and delivery, these therapies could revolutionize the way bacterial infections are treated. Continued research is crucial to address hurdles and ensure these therapies can be safely and effectively implemented in clinical settings, shaping the future of infection management.},
}
@article {pmid41174869,
year = {2025},
author = {Li, S and Zhang, B and Ma, P and Zhang, Y and Hu, Z and Wu, X and Chen, Q and Zhao, Y},
title = {The 13-lipoxygenase GmLOX6 is involved in JA biosynthesis and serves as a positive regulator of salt stress tolerance in soybean.},
journal = {The Plant journal : for cell and molecular biology},
volume = {124},
number = {3},
pages = {e70550},
pmid = {41174869},
issn = {1365-313X},
support = {2023ZD04036//Biological Breeding-National Science and Technology Major Project/ ; 2021YFD1201104-02-02//National Key Research and Development Program of China/ ; 2023YFD2300101//National Key Research and Development Program of China/ ; 31971899//National Natural Science Foundation of China/ ; 32272072//National Natural Science Foundation of China/ ; 32272093//National Natural Science Foundation of China/ ; U23A20192//National Natural Science Foundation of China/ ; },
mesh = {*Glycine max/genetics/physiology/enzymology/metabolism ; *Lipoxygenase/metabolism/genetics ; *Oxylipins/metabolism ; *Cyclopentanes/metabolism ; *Salt Tolerance/genetics/physiology ; *Plant Proteins/metabolism/genetics ; Salt Stress ; Gene Expression Regulation, Plant ; Reactive Oxygen Species/metabolism ; Plant Growth Regulators/metabolism ; Plants, Genetically Modified ; alpha-Linolenic Acid/metabolism ; CRISPR-Cas Systems ; },
abstract = {Salinity represents a major abiotic stressor that significantly impairs soybean growth and yield. Although jasmonic acid (JA) has been firmly established as a key regulator of plant defense against salt stress, the precise functions of lipoxygenase (LOX) genes responsible for initiating JA biosynthesis remain poorly defined. Here, a comprehensive genome-wide analysis of the soybean LOX gene family was performed, and a detailed functional characterization of GmLOX6 was carried out. Subcellular localization confirmed that GmLOX6 is targeted to chloroplasts, while enzymatic assays demonstrated that it acts as a 13-LOX enzyme with a strong preference for α-linolenic acid as substrate. To clarify its role under salt stress, we generated both overexpression and CRISPR/Cas9-mediated knockout lines of soybean. Phenotypic and molecular evaluations revealed that GmLOX6 facilitates JA production under salt stress, thereby contributing to enhanced JA accumulation. This elevation in JA levels was associated with improved salt tolerance through multiple physiological adaptations, including the activation of antioxidant enzymes for the detoxification of reactive oxygen species (ROS), enhanced Na[+] extrusion to preserve ionic balance, and reinforced membrane stability. Moreover, GmRWP-RK11 was identified as a transcriptional repressor of GmLOX6. Functional disruption of GmRWP-RK11 via CRISPR/Cas9 conferred greater salt tolerance, further supporting its negative regulatory role. Collectively, these findings uncover a novel regulatory axis in which GmLOX6-mediated JA biosynthesis enhances soybean resistance to salinity through modulation of ROS homeostasis and Na[+] transport. These insights provide an expanded understanding of the transcriptional and biochemical mechanisms underpinning JA-driven stress adaptation in soybean.},
}
@article {pmid41174319,
year = {2026},
author = {Fatmi, MQ and Nadeem, A and Abbasov, M and Sajjad, M},
title = {Computational Methods to Engineer Cas Proteins for Efficient Genome Editing.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2979},
number = {},
pages = {279-300},
pmid = {41174319},
issn = {1940-6029},
mesh = {*Gene Editing/methods ; Molecular Dynamics Simulation ; *CRISPR-Cas Systems/genetics ; *Protein Engineering/methods ; *Computational Biology/methods ; Mutation ; *CRISPR-Associated Proteins/genetics/chemistry ; },
abstract = {The clustered regularly interspaced short palindromic repeats/CRISPR-associated protein (CRISPR/Cas) system has revolutionized genome editing through programmable, sequence-specific deoxyribonucleic acid (DNA) targeting. Yet, its broader application remains limited by off-target effects and context-dependent efficiency. To address these challenges, we present an integrated computational protocol with easy-to-do steps for researchers to guide the rational design of CRISPR/Cas variants with improved stability and specificity. The integrated workflow begins with coevolutionary coupling analysis to identify conserved and covarying residues critical for function. These residues are then evaluated for energetically favorable substitutions through mutant stability prediction, followed by network centrality analysis to evaluate the impact of mutations on intramolecular communication pathways, preserving key allosteric interactions. Finally, molecular dynamics (MD) simulations validate the structural integrity and dynamic behavior of the selected variants. Network analysis and molecular dynamics (MD) simulations are applied iteratively, allowing insights from MD to refine network-based evaluations and vice versa. This multiscale strategy offers a streamlined and systematic approach for engineering optimized Cas proteins for genome editing applications.},
}
@article {pmid41173880,
year = {2025},
author = {Binenbaum, J and Adamkova, V and Fryer, H and Xu, L and Gorringe, N and Włodzimierz, P and Burns, R and Papikian, A and Jacobsen, SE and Henderson, IR and Harris, CJ},
title = {CRISPR targeting of H3K4me3 activates gene expression and unlocks centromere-proximal crossover recombination in Arabidopsis.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {9587},
pmid = {41173880},
issn = {2041-1723},
support = {ERC Starting Grant (TransPlantMemory)//EC | EU Framework Programme for Research and Innovation H2020 | H2020 Priority Excellent Science | H2020 European Research Council (H2020 Excellent Science - European Research Council)/ ; EP/Z001749/1//EC | EU Framework Programme for Research and Innovation H2020 | H2020 Priority Excellent Science | H2020 European Research Council (H2020 Excellent Science - European Research Council)/ ; ERC Advanced Grant (EvoPanCen)//EC | EU Framework Programme for Research and Innovation H2020 | H2020 Priority Excellent Science | H2020 European Research Council (H2020 Excellent Science - European Research Council)/ ; EP/X025306/1//RCUK | Engineering and Physical Sciences Research Council (EPSRC)/ ; URF\R1\201016//Royal Society/ ; doctoral studentship//RCUK | Biotechnology and Biological Sciences Research Council (BBSRC)/ ; BB/V003984/1//RCUK | Biotechnology and Biological Sciences Research Council (BBSRC)/ ; },
mesh = {*Arabidopsis/genetics/metabolism ; *Arabidopsis Proteins/genetics/metabolism ; *Histones/metabolism/genetics ; *Gene Expression Regulation, Plant ; *Centromere/genetics/metabolism ; *Crossing Over, Genetic ; *CRISPR-Cas Systems/genetics ; Histone-Lysine N-Methyltransferase/genetics/metabolism ; Disease Resistance/genetics ; Meiosis/genetics ; Plants, Genetically Modified ; },
abstract = {H3K4me3 is a fundamental and highly conserved chromatin mark across eukaryotes, playing a central role in many genome-related processes, including transcription, maintenance of cell identity, DNA damage repair, and meiotic recombination. However, identifying the causal function of H3K4me3 in these diverse pathways remains a challenge, and we lack the tools to manipulate it for agricultural benefit. Here we use the CRISPR-based SunTag system to direct H3K4me3 methyltransferases in the model plant, Arabidopsis thaliana. Targeting of SunTag-SDG2 activates the expression of the endogenous reporter gene, FWA. We show that SunTag-SDG2 can be employed to increase pathogen resistance by targeting the H3K4me3-dependent disease resistance gene, SNC1. Meiotic crossover recombination rates impose a limit on the speed with which new traits can be transferred to elite crop varieties. We demonstrate that targeting of SunTag-SDG2 to low recombining centromeric regions can significantly stimulate proximal crossover formation. Finally, we reveal that the effect is not specific to SDG2 and is likely dependent on the H3K4me3 mark itself, as the orthogonal mammalian-derived H3K4me3 methyltransferase, PRDM9, produces a similar effect on gene expression with reduced off-target potential. Overall, our study supports an instructive role for H3K4me3 in transcription and meiotic recombination and opens the door to precise modulation of important agricultural traits.},
}
@article {pmid41173850,
year = {2025},
author = {Brown, BC and Tokolyi, A and Morris, JA and Lappalainen, T and Knowles, DA},
title = {Large-scale causal discovery using interventional data sheds light on gene network structure in k562 cells.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {9628},
pmid = {41173850},
issn = {2041-1723},
support = {K99HG012373//U.S. Department of Health &amp; Human Services | NIH | National Human Genome Research Institute (NHGRI)/ ; K99HG012792//U.S. Department of Health &amp; Human Services | NIH | National Human Genome Research Institute (NHGRI)/ ; R01AG057422//U.S. Department of Health &amp; Human Services | NIH | National Institute on Aging (U.S. National Institute on Aging)/ ; U01AG068880//U.S. Department of Health &amp; Human Services | NIH | National Institute on Aging (U.S. National Institute on Aging)/ ; R01MH106842//U.S. Department of Health &amp; Human Services | NIH | National Institute of Mental Health (NIMH)/ ; },
mesh = {Humans ; *Gene Regulatory Networks ; K562 Cells ; CRISPR-Cas Systems ; },
abstract = {Inference of directed biological networks is an important but notoriously challenging problem. The recent proliferation of large-scale CRISPR perturbation data provides a new opportunity to tackle this problem by leveraging the transcriptional response to the presence of a gene-targeting guide. Here, we introduce inverse sparse regression (inspre), an approach to learning causal networks that leverages large-scale intervention-response data. Applied to 788 genes from the genome-wide perturb-seq dataset, inspre discovers a network with small-world and scale-free properties. We integrate our network estimate with external data, finding relationships between gene eigencentrality and both measures of gene essentiality and gene expression heritability. Our analysis helps to elucidate the structure of networks that may underlie complex traits.},
}
@article {pmid39832721,
year = {2025},
author = {Mao, X and Xiong, J and Cai, M and Wang, C and He, Q and Wang, B and Chen, J and Xiao, Z and Wang, B and Han, S and Zhang, Y},
title = {SCARB1 links cholesterol metabolism-mediated ferroptosis inhibition to radioresistance in tumor cells.},
journal = {Journal of advanced research},
volume = {77},
number = {},
pages = {207-219},
doi = {10.1016/j.jare.2025.01.026},
pmid = {39832721},
issn = {2090-1224},
mesh = {*Ferroptosis/genetics ; Humans ; *Radiation Tolerance/genetics ; *Cholesterol/metabolism ; *Scavenger Receptors, Class B/metabolism/genetics ; Lipid Peroxidation ; Cell Line, Tumor ; Animals ; *Neoplasms/metabolism/radiotherapy/pathology/genetics ; Mice ; CRISPR-Cas Systems ; Gene Expression Regulation, Neoplastic ; },
abstract = {INTRODUCTION: Ferroptosis is an iron-dependent form of cell death triggered by the excessive accumulation of lipid peroxides. Understanding the regulatory mechanisms of ferroptosis and developing strategies to target this process hold significant clinical applications in tumor therapy.<br><br>OBJECTIVE: Our study aims to search for novel candidate genes involved in the regulation of ferroptosis and to investigate their mechanism of action in ferroptosis and tumor therapy.<br><br>METHODS: We employed a CRISPR-Cas9 library to perform a genome-wide screen under ferroptosis inducer treatment conditions, revealing Scavenger Receptor Class B Member 1(SCARB1) as a novel candidate gene involved in ferroptosis regulation. Subsequently, lipidomic analyses, metabolic interventions, and relevant cellular experimental analyses were performed to elucidate the role of SCARB1 in ferroptosis, lipid peroxidation, and tumor therapy.<br><br>RESULTS: Our study confirmed that SCARB1 significantly inhibits ferroptosis and lipid peroxidation induced by ferroptosis inducers. Mechanistically, SCARB1 inhibits ferroptosis through the regulation of cholesterol metabolism, and the upregulation of CoQ10 level is demonstrated to mediate the suppression of ferroptosis by SCARB1 after lipidomic analysis and metabolic intervention. Interestingly, SCARB1 exerts a tumor suppressive effect regarding tumor growth, migration and invasion, which is possibly independent of ferroptosis regulation. However, SCARB1 promotes radioresistance through the upregulation of cholesterol metabolism and inhibition of ferroptosis, while the combination of ferroptosis inducers can overcome radioresistance in tumor cells with high SCARB1 expression.<br><br>CONCLUSION: This study establishes a theoretical foundation for the regulation of ferroptosis by SCARB1 and highlights the potential of targeting lipid metabolism to overcome radioresistance in cancer therapy. The identification of SCARB1 as a key player in ferroptosis and its dual role in tumor suppression and radioresistance provides new avenues for therapeutic intervention in cancer treatment.},
}
@article {pmid41172978,
year = {2025},
author = {Sancho-Shimizu, V},
title = {VUS no more: Decoding inborn errors of immunity for clinical action.},
journal = {Cell},
volume = {188},
number = {22},
pages = {6105-6106},
doi = {10.1016/j.cell.2025.09.009},
pmid = {41172978},
issn = {1097-4172},
mesh = {Humans ; *Gene Editing/methods ; CRISPR-Cas Systems ; Phenotype ; },
abstract = {Saturation genome editing meets functional phenotyping to turn sequencing ambiguity into actionable diagnoses.},
}
@article {pmid41172903,
year = {2025},
author = {Zhang, J and Zhu, M and Yan, H and Qiao, J and Liu, Y},
title = {Split CRISPR/Cas systems: Pioneering solutions for molecular diagnostics challenges.},
journal = {Biosensors &amp; bioelectronics},
volume = {293},
number = {},
pages = {118177},
doi = {10.1016/j.bios.2025.118177},
pmid = {41172903},
issn = {1873-4235},
abstract = {Split CRISPR/Cas systems have recently emerged as revolutionary tools in molecular diagnostics, addressing the limitations of conventional CRISPR-based detection methods, such as low sensitivity and specificity for low-abundance targets and limited regulatory flexibility. This review highlights "split-activation" strategies that enhance analytical performance without requiring pre-amplification. It elaborates on two key approaches: split activator-mediated Cas systems for ultrasensitive RNA detection and split crRNA architectures for multiplex nucleic acid analysis. These innovations achieve significant improvements in sensitivity for femtomolar-level biomarkers while maintaining superior single-base discrimination. We examine their applications in clinical and on-site monitoring, analyze current challenges including background noise, multiplex capacity, and cost considerations. Future directions include developing customized Cas variants, nanomaterial-based amplification-free workflows, and integrated microfluidic platforms. This technology holds great promise for precision diagnostics, particularly in resource-limited settings.},
}
@article {pmid41171933,
year = {2025},
author = {Li, S and Vonesch, SC and Roy, KR and Tu, CS and Steudle, F and Nguyen, M and Jann, C and Steinmetz, LM},
title = {The editable landscape of the yeast genome reveals hotspots of structural variant formation.},
journal = {Science advances},
volume = {11},
number = {44},
pages = {eady9875},
pmid = {41171933},
issn = {2375-2548},
mesh = {*Genome, Fungal ; *Gene Editing/methods ; CRISPR-Cas Systems/genetics ; *Saccharomyces cerevisiae/genetics ; *Genomic Structural Variation ; Whole Genome Sequencing ; INDEL Mutation ; },
abstract = {It is unclear how CRISPR editing outcomes vary across the genome and whether undesirable events such as structural variants (SVs) are predictable or preventable. To define a genome-wide map of editability, we performed whole-genome sequencing on 1875 budding yeast clones edited across 16 chromosomes by CRISPR-Cas9 and donor-templated repair. We found that unintended edits, including short indels and SVs, were enriched in specific genomic and sequence contexts. We developed a predictive model, SCORE (System for CRISPR Outcome and Risk Evaluation), which revealed 4.8% of the genome as SV prone, consisting of 562 SV hotspots. Donor repair-enhancing strategies suppressed SV formation in regions with moderate, but not high, predicted risk. Applying SCORE to the Sc2.0 synthetic yeast genome revealed a markedly altered SV landscape due to the removal of endogenous repetitive elements and the insertion of loxP sites. Our study provides the genome-scale map of SV hotspots after CRISPR editing and predictive and experimental tools to mitigate their formation.},
}
@article {pmid41171921,
year = {2025},
author = {Zhang, H and You, J and Zhou, H and Zhang, Z and Wu, H and Zhang, D and Pan, X and Zhang, W and Zhang, X and Rao, Z},
title = {The two-component nuclease-active KELShedu system confers broad antiphage activity via abortive infection.},
journal = {Science advances},
volume = {11},
number = {44},
pages = {eadv4747},
pmid = {41171921},
issn = {2375-2548},
mesh = {*Escherichia coli/virology/genetics/metabolism ; *Bacteriophages/physiology ; *Escherichia coli Proteins/metabolism/genetics ; *DNA-Binding Proteins/metabolism/genetics ; CRISPR-Cas Systems ; Nucleotides/metabolism ; },
abstract = {Bacteriophages and bacteria engage in a continuous evolutionary arms race, driving the development of intricate bacterial defense systems such as CRISPR-Cas, BREX (Bacteriophage Exclusion), Gabija, and Shedu. Here, we characterize a two-component KELShedu system in Escherichia coli that confers resistance to phages via abortive infection. The KELShedu system comprises KELA, a double-stranded DNA-binding protein, and KELB, a metal ion-dependent nuclease harboring the DUF4263 domain. In addition, we find that physiological levels of nucleotide triphosphates (NTPs) inhibit the DNA cleavage activity of the KELShedu system, suggesting that KELShedu's activation depends on reduced intracellular NTP levels during phage invasion. Our research demonstrates that the KELShedu system responds to nucleotide depletion triggered by phage replication, leading to nonspecific degradation of cellular DNA and ultimately inducing abortive infection. These insights into the KELShedu system expand the repertoire of bacterial antiphage mechanisms and lay the groundwork for applications in microbial engineering and therapeutic development.},
}
@article {pmid41171707,
year = {2025},
author = {Vadakkethil, AA and Panda, S and Mitra, A and Dash, M and Baig, MJ and Angadi, UB and Kumar, D and Jaiswal, S and Asif Iquebal, M and Molla, KA},
title = {CRISPR-GATE: a one-stop repository and guide to computational resources for genome editing experimentation.},
journal = {Briefings in bioinformatics},
volume = {26},
number = {5},
pages = {},
pmid = {41171707},
issn = {1477-4054},
support = {//Indian Council of Agricultural Research/ ; },
mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems ; RNA, Guide, CRISPR-Cas Systems/genetics ; *Computational Biology/methods ; *Software ; *Databases, Genetic ; Humans ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Internet ; },
abstract = {Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-CRISPR associated protein (CRISPR-Cas) has emerged and evolved as a revolutionary genome editing technology, transforming research across diverse biological disciplines. Over the past decade, this technology has unveiled numerous opportunities for precise genome manipulation. However, the processes of discovering Cas proteins, repurposing them as editing tools, selecting appropriate candidate tool from the CRISPR-toolbox, designing experiments, and analyzing data are often complex and require careful consideration. To support researchers at every stage of CRISPR experimentation, a wide array of web resources has been developed. In this article, we provide a comprehensive overview of standalone and web-based tools that assist in the identification of CRISPR-Cas systems and the design of guide RNAs (gRNAs). We also highlight tools for evaluating gRNA efficiency, predicting CRISPR-Cas9 mutation profiles, as well as tools for base editing and prime editing, and the analysis and visualization of experimental results. Additionally, we introduce CRISPR-Gateway for Accessing Tools and Resources (CRISPR-GATE), an all-inclusive web repository that consolidates publicly available tools for genome editing research. This repository offers a categorized and user-friendly interface, allowing researchers to quickly access relevant tools based on their specific needs. CRISPR-GATE aims to streamline the search for CRISPR resources, facilitating both education and accelerating innovation. The web repository can be accessed from https://crispr-gate.daasbioinfromaticsteam.in/.},
}
@article {pmid41171314,
year = {2025},
author = {Zaheer, A and AboQuella, NM and Wadan, AS and Saad, HA and Kumar, D and Panjwani, S and Rath, S and Ahmed, SI},
title = {CRISPR-based gene therapy for huntington's disease: current advances and future prospects.},
journal = {Neurogenetics},
volume = {26},
number = {1},
pages = {76},
pmid = {41171314},
issn = {1364-6753},
mesh = {*Huntington Disease/therapy/genetics ; Humans ; *Genetic Therapy/methods/trends ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; Animals ; Huntingtin Protein/genetics ; Disease Models, Animal ; },
abstract = {CRISPR-Cas9 technology offers transformative potential in treating Huntington's Disease (HD) by directly addressing its genetic root causes. This manuscript explores the pathophysiological mechanisms of HD, characterized by toxic mutant huntingtin (mHTT) protein resulting from expanded CAG repeats in the HTT gene, and the challenges posed by current therapeutic limitations. We comprehensively review the mechanisms of CRISPR-based therapeutic strategies, including excision of expanded repeats, allele-specific targeting, and epigenome editing, highlighting their efficacy in preclinical studies using animal models and human iPSCs. Delivery methods, such as viral and non-viral vectors, are analysed for their role in optimizing therapeutic outcomes while minimizing off-target effects and immune responses. Ethical and safety considerations, especially regarding precision and long-term impacts, are critically examined alongside emerging strategies to enhance specificity. With ongoing clinical trials and advancements in delivery systems, CRISPR technology represents a paradigm shift in addressing HD and broader neurodegenerative conditions. This review underscores the promise of gene editing in overcoming existing barriers and paving the way for transformative therapeutic approaches.},
}
@article {pmid41170849,
year = {2025},
author = {Yu, T and Xie, J and Huang, X and Huang, J and Bao, G and Yuan, W and Gao, C and Liu, C and Hu, J and Yang, W and Li, G},
title = {BaeR and H-NS control CRISPR-Cas-mediated immunity and virulence in Acinetobacter baumannii.},
journal = {mSystems},
volume = {},
number = {},
pages = {e0106725},
doi = {10.1128/msystems.01067-25},
pmid = {41170849},
issn = {2379-5077},
abstract = {Acinetobacter baumannii balances its remarkable ability to acquire antibiotic resistance genes via horizontal gene transfer (HGT) with the immune defense functions of its CRISPR-Cas system, forming a dynamic equilibrium governed by intricate transcriptional regulation. However, the regulatory mechanisms underlying the I-Fb CRISPR-Cas system in A. baumannii remain poorly understood. This study elucidated a multitiered regulatory axis mediated by BaeR and H-NS that coordinates immune defense and virulence expression in the I-Fb CRISPR-Cas system. Using DNA pull-down and electrophoretic mobility shift assay (EMSA), we demonstrated that H-NS directly binds AT-rich regions within the cas3 promoter, suppressing both interference activity and adaptive immunity of the I-Fb CRISPR-Cas system. Intriguingly, the two-component regulator BaeR controlled this suppression by positively regulating H-NS expression. The results revealed that Δcas3 mutants exhibited increased biofilm thickness, elevated the extracellular matrix component poly N-acetyl glucosamine (PNAG) production, upregulated pilus expression, and significantly enhanced epithelial cell adhesion. Strikingly, Δh-ns-cas3 and ΔbaeR-cas3 double-knockout strains showed no statistically significant differences in virulence phenotypes compared to the Δcas3 single mutants. These findings indicate CRISPR-Cas-mediated inhibition of biofilm formation is abolished upon cas3 deletion, thereby releasing the regulatory constraints imposed by BaeR and H-NS. This dysregulation leads to excessive biofilm and extracellular matrix component accumulation, ultimately amplifying bacterial colonization capacity and pathogenicity in host environments. This discovery reveals the dual regulatory roles of BaeR and H-NS in the A. baumannii I-Fb CRISPR-Cas system, mediating both immune defense and virulence modulation. These insights establish a theoretical foundation for novel antimicrobial strategies targeting CRISPR-Cas regulatory networks.IMPORTANCEA. baumannii, a leading cause of drug-resistant nosocomial infections, evolves antibiotic resistance through horizontal gene transfer (HGT) while employing CRISPR-Cas systems to limit foreign DNA invasion. This study reveals that the I-Fb CRISPR-Cas system, typically a defense mechanism, functions as a repressor of virulence traits in A. baumannii. We demonstrate that the transcriptional regulators H-NS and BaeR form a hierarchical axis suppressing Cas3 expression, thereby constraining biofilm formation and host adhesion. Strikingly, CRISPR-Cas deficiency enhances virulence, thickens biofilms, elevates PNAG production, and enhances epithelial colonization through escape from BaeR-/H-NS-mediated control. This work redefines CRISPR-Cas as a dual-function module balancing immune defense and pathogenicity, exposing the BaeR-H-NS-Cas3 axis as a druggable target for novel anti-infectives aimed at disrupting bacterial adaptive evolution.},
}
@article {pmid41072237,
year = {2026},
author = {Xin, M and Liu, J and Zhou, H and Bu, S and Hao, Z and Sun, H and Lu, J and Feng, X and Jiang, X and Wang, Q and Wan, J},
title = {An H1N1 virus biosensor based on enzyme activity-gated PER-CRISPR/Cas12a cascade signal amplification.},
journal = {Enzyme and microbial technology},
volume = {192},
number = {},
pages = {110759},
doi = {10.1016/j.enzmictec.2025.110759},
pmid = {41072237},
issn = {1879-0909},
mesh = {*Influenza A Virus, H1N1 Subtype/genetics/isolation &amp; purification ; *Biosensing Techniques/methods ; *CRISPR-Cas Systems ; Humans ; *CRISPR-Associated Proteins/metabolism/genetics ; *Endodeoxyribonucleases/metabolism/genetics ; Limit of Detection ; Influenza, Human/diagnosis/virology ; SARS-CoV-2/isolation &amp; purification/genetics ; RNA, Viral/analysis/genetics ; Nucleic Acid Amplification Techniques/methods ; DNA, Viral/analysis/genetics ; DNA, Single-Stranded ; Bacterial Proteins ; },
abstract = {The rapid and accurate detection of the H1N1 influenza virus is a key link in epidemic prevention and control. This study innovatively constructed a cascade signal amplification biosensor based on DNA polymerase activity regulation, aiming to achieve ultra-sensitive and highly specific detection of viral nucleic acids. This biosensor has the following significant advantages: (i) Molecular lock-key regulation mechanism: A functional DNA inhibitor is designed to form a complex with Taq DNA polymerase, and the target H1N1 RNA is specifically recognized to release enzyme activity inhibition, converting the target presence information into a PER reaction initiation signal. (ii) Cascade signal amplification system: The single-stranded DNA generated by PER activates Cas12a trans-cleavage activity, achieving a three-level signal amplification of enzyme activity activation → nucleic acid synthesis → CRISPR cleavage. The biosensor exhibits a linear detection range between 1 pM and 1 μM, with a detection limit of 25 fM. Moreover, the platform showed high versatility and could be readily adapted for the detection of other pathogens such as SARS-CoV-2 by simply modifying the nucleic acid sequences of the inhibitor and activator. This study not only provides a new tool for the screening of H1N1 influenza virus, but also offers a novel strategy for the development of next-generation molecular detection technologies suitable for point-of-care diagnostics, indicating considerable application potential.},
}
@article {pmid40785075,
year = {2025},
author = {Zhu, L and Lin, Y and Yang, G and He, G and Pan, Z and Yu, G and Yao, J and Li, M and Zhou, L and Jiang, D},
title = {A Rapid On-Site Visualization Detection System for Fusarium oxysporum f. sp. cubense Tropical Race 4 Utilizing RPA-CRISPR/Cas12a.},
journal = {Plant disease},
volume = {109},
number = {10},
pages = {2062-2069},
doi = {10.1094/PDIS-11-24-2512-SR},
pmid = {40785075},
issn = {0191-2917},
mesh = {*Fusarium/genetics/isolation &amp; purification ; *Plant Diseases/microbiology ; *Musa/microbiology ; *CRISPR-Cas Systems/genetics ; *Nucleic Acid Amplification Techniques/methods ; Recombinases/metabolism ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {Fusarium wilt of banana, a disease with devastating impacts on banana plants, is primarily caused by a pathogenic fungus called Fusarium oxysporum f. sp. cubense tropical race 4 (Foc TR4). To facilitate control and interrupt the spread of Fusarium wilt of banana, we developed a rapid on-site visualization system for detecting Foc TR4 based on recombinase polymerase amplification (RPA) combined with CRISPR/Cas12a. Based on Foc TR4-specific sequences, primers, crRNA, and ssDNAs were designed. The detection system exhibited high specificity, with amplification signals observed exclusively in samples containing Foc TR4. Additionally, the system showed high sensitivity, with a limit of detection (LOD) of approximately 20 copies, and high efficiency, with detection results generated within 1 h from time of amplification. Notably, this method does not require large-scale instruments, making it a convenient and rapid approach. The detection system represents the first instance of on-site visualization of Foc TR4. This system can be employed for early detection of Foc TR4 and can provide a technical reference for the rapid detection and applications in the field of other pathogens.},
}
@article {pmid40715781,
year = {2025},
author = {Zhang, H and Li, YC and Pang, D and Xie, C and Zhang, T and Li, Y and Li, Y and Jiang, ZY and Bu, GL and Liu, MM and Chen, YR and Fei, HX and Lin, RB and Wu, PH and Du, WT and Zhao, GX and Luo, YL and Han, P and Zhong, Q and Sun, C and Zeng, MS},
title = {Desmocollin 2 is a dominant entry receptor for Epstein-Barr virus infection of epithelial cells.},
journal = {Nature microbiology},
volume = {10},
number = {11},
pages = {2768-2780},
pmid = {40715781},
issn = {2058-5276},
mesh = {Animals ; Humans ; *Epithelial Cells/virology/metabolism ; *Virus Internalization ; *Desmocollins/metabolism/genetics ; *Herpesvirus 4, Human/physiology ; *Epstein-Barr Virus Infections/virology/metabolism ; Cell Line ; *Receptors, Virus/metabolism/genetics ; Cricetinae ; CRISPR-Cas Systems ; Viral Envelope Proteins/metabolism ; Membrane Glycoproteins/metabolism ; Viral Proteins ; Molecular Chaperones ; },
abstract = {Epstein-Barr virus (EBV) can infect B cells and epithelial cells, and cause lymphomas and various epithelial malignancies. During epithelial cell infection, EBV employs a complex combination of viral glycoproteins and host receptors. However, the exact mechanism and whether a dominant receptor exists remain unclear. Here we identify desmocollin 2 (DSC2) as a dominant EBV entry receptor for epithelial cell infection using CRISPR-Cas9 screening. Knockout of DSC2 reduced EBV infection in both nasopharyngeal and gastric epithelial cell lines, and infection was rescued when DSC2 expression was restored. Expression of human DSC2 in non-EBV-susceptible hamster cell lines enabled susceptibility to EBV. Furthermore, we found that DSC2 directly binds to EBV glycoprotein H/glycoprotein L through its extracellular domain, particularly the preEC-EC2 regions, which could be targeted by polyclonal antibodies, therefore blocking EBV infection in primary epithelial cells. DSC2 enabled virus entry independent of Ephrin receptor A2. These findings could aid development of currently unavailable animal models and support development of targeted therapies.},
}
@article {pmid41170434,
year = {2025},
author = {Araújo, MRB and Dos Santos, LS and Prates, FD and Perini, HF and Silva, JS and Ramos, JN and Bokermann, S and Sacchi, CT and de Mattos Guaraldi, AL and Campos, KR and Cardoso, TDCS and Castro, DLC and Silva, MA and Sousa, M&#xc2;B and Vieira, VV and Santos, MBN and Camargo, CH and Andrade, BS and da Silva, MV and Sant'Anna, LO and Viana, MVC and Azevedo, V},
title = {Virulence and mutations analysis based on the whole genome of a Brazilian Corynebacterium diphtheriae strain isolated from a cutaneous infection.},
journal = {Frontiers in microbiology},
volume = {16},
number = {},
pages = {1579154},
pmid = {41170434},
issn = {1664-302X},
abstract = {Corynebacterium diphtheriae is the main etiological agent of diphtheria, a potentially fatal disease whose most severe signs and symptoms result from the action of an exotoxin, the diphtheria toxin (DT). Although non-toxigenic C. diphtheriae strains have been associated with several diseases, including cutaneous infections and endocarditis, they are not monitored in many countries, and their mechanisms of virulence and antimicrobial resistance remain underexplored. Therefore, this study aimed to provide a comprehensive characterization -through genomic, in vitro, and in vivo analyses - of a non-toxigenic C. diphtheriae strain (46855) isolated from a leg lesion, highlighting its pathogenic potential and resistance profile. The isolate was assigned to a novel sequence type (ST-925) and was found to be resistant to tetracycline and rifampin. Multiple antimicrobial resistance genes were predicted in the genome, such as tet(33), rbpA, and rpoB2, in addition to mutations in the rpoB gene. A diverse set of virulence-associated genes related to adhesion, iron uptake systems, gene regulation, and post-translational modification was also identified. The isolate was able to form biofilm in vitro and exhibited strong virulence in Galleria mellonella larvae and A549 human pneumocyte cells. Finally, the structural analysis of the rpoB gene, carried out for the first time in this study, linked the observed mutations to rifampin resistance in C. diphtheriae. In summary, the data revealed that C. diphtheriae 46855, although non-toxigenic, harbors multiple genes associated with antimicrobial resistance and virulence, emphasizing the need for greater surveillance and functional studies on non-toxigenic strains.},
}
@article {pmid41170433,
year = {2025},
author = {Zhang, S and Chu, M and Sun, X},
title = {The arms race in bacteria-phage interaction: deciphering bacteria defense and phage anti-defense mechanisms through metagenomics.},
journal = {Frontiers in microbiology},
volume = {16},
number = {},
pages = {1687307},
pmid = {41170433},
issn = {1664-302X},
abstract = {Bacteriophages are viruses that specifically infect bacteria and co-evolve with their hosts through mutual interactions. They represent one of the most significant drivers of microbial diversity, influencing its evolution, generation, and maintenance. To counter bacteriophage infection, bacteria have developed sophisticated immune systems, including both passive adaptations, such as inhibiting phage adsorption and preventing DNA entry, and active defense systems such as restriction-modification systems and CRISPR-Cas systems. The ongoing arms race between bacteriophages and bacteria has left distinct evolutionary signatures in their genomic sequences. Advances in large-scale genomic and metagenomic sequencing technologies, coupled with bioinformatics approaches, have greatly enhanced our understanding of bacteria-phage interaction mechanisms, driving progress in bacteriophage biology. This review systematically analyses the diverse immune strategies bacteria employ against phage infection, elucidates the coordination and interrelationships among different anti-phage mechanisms, and highlights potential directions for future research.},
}
@article {pmid41168338,
year = {2025},
author = {Elsayed, EM and Stukenberg, D and Meier, D and Schmeck, B and Becker, A},
title = {RECKLEEN is a lambda Red/CRISPR-Cas9 based single plasmid platform for enhanced genome editing in Klebsiella pneumoniae.},
journal = {Communications biology},
volume = {8},
number = {1},
pages = {1509},
pmid = {41168338},
issn = {2399-3642},
support = {LOEWE/2/13/519/03/06.001(0002)/74//Hessisches Ministerium f&#xfc;r Wissenschaft und Kunst (Hessen State Ministry of Higher Education, Research and the Arts)/ ; },
mesh = {*Klebsiella pneumoniae/genetics ; *Gene Editing/methods ; *CRISPR-Cas Systems ; *Plasmids/genetics ; *Bacteriophage lambda/genetics ; *Genome, Bacterial ; },
abstract = {Klebsiella pneumoniae (Kp) has evolved as a major public health threat due to its multidrug-resistance (MDR) and hypervirulence. Current Kp genome-editing tools are constrained by cumbersome workflows, low flexibility, and limited scalability. Here, we present the RECKLEEN system -Recombineering/CRISPR-based KLebsiella Engineering for Efficient Nucleotide editing - as a single plasmid platform designed for precise genetic manipulation of Kp. RECKLEEN combines lambda Red recombineering with powerful CRISPR-Cas9-based targeted counterselection, achieving up to 99.998% killing efficiency. By implementing the near PAM-less SpG Cas9 variant in RECKLEEN, the compatible target sequence spectrum was significantly broadened. This approach enables deletions, point mutations, and DNA integrations, with efficiencies reaching 100% of the counter-selected clones. Simultaneous multi-target deletions were accomplished with up to 72% efficiency. To streamline the process, we developed a toolbox of eleven plasmids based on a modular cloning standard, enabling time- and resource-efficient assembly of editing constructs. This allows a 5-days workflow, from plasmid construction to the generation of strains with the desired genetic modification(s). The efficacy of RECKLEEN extends to various MDR Kp strains, such as ATCC 700721, ATCC BAA-1705, and ATCC 700603, demonstrating its broad applicability. RECKLEEN significantly enhances genome-editing capabilities for Kp, advancing research into its pathology and MDR mechanisms.},
}
@article {pmid41167881,
year = {2025},
author = {Meng, X and Yue, Y and Huang, M and Duan, Z and Liu, K and Wu, L},
title = {DNAzyme-CRISPR driven dual-mode biosensor with nanozyme signal amplification for on-site Pb[2+] detection.},
journal = {Analytica chimica acta},
volume = {1379},
number = {},
pages = {344711},
doi = {10.1016/j.aca.2025.344711},
pmid = {41167881},
issn = {1873-4324},
mesh = {*Lead/analysis ; *DNA, Catalytic/chemistry/metabolism ; *Biosensing Techniques/methods ; Electrochemical Techniques ; *CRISPR-Cas Systems ; Food Contamination/analysis ; Limit of Detection ; Colorimetry ; Cerium/chemistry ; },
abstract = {BACKGROUND: Lead ion (Pb[2+]) is a toxic heavy metal that poses severe threats to food safety. Traditional methods like inductively coupled plasma mass spectrometry (ICP-MS) and atomic absorption spectrometry (AAS) rely on bulky instrumentation, which are limited by high costs, complex sample preparation requirements, and inability to meet the demands for rapid on-site testing. Nanozyme-based biosensors have emerged as promising alternatives, yet single-mode sensors often suffer from matrix interference in complex food samples. Therefore, developing a rapid and reliable on-site method is critical for Pb[2+] detection.<br><br>RESULTS: To address this challenge, a CRISPR/Cas12a-driven dual-mode biosensor integrating Pt/CeO2 nanozyme-mediated peroxidase activity with GR-5 DNAzyme recognition was developed. The biosensor's design capitalizes on GR-5 DNAzyme for Pb[2+]-specific recognition, triggering CRISPR/Cas12a-mediated cleavage of electrochemical/colorimetric signal probe SH-ssDNA-Pt/CeO2 to generate dual signals. This innovative platform synergizes electrochemical precision and colorimetric simplicity for Pb[2+] detection in complex food matrices. The biosensor achieved an ultra-sensitive electrochemical response (linear range: 0.002-200&#xa0;nM; limit of detection: 0.14 pM) alongside a robust colorimetric readout (linear range: 0.5-2000&#xa0;nM; limit of detection: 0.47&#xa0;nM), representing significant sensitivity improvements over conventional single-mode sensors. Crucially, the intrinsic cross-verification mechanism between orthogonal signal modalities minimized false positives while ensuring >90.5&#xa0;% recovery in spiked corn, edible oil, beef and red wine samples with RSD <5&#xa0;%. These results highlight the potential of the dual-mode sensor as a practical, field deployable sensing platform for the detection of Pb[2+].<br><br>SIGNIFICANCE: This work innovatively integrates the specificity of CRISPR with the catalytic properties of nanozymes into a self-validating electrochemical/colorimetric dual-mode system. The detection method not only establishes a robust platform for highly sensitive, reliable and visualized detection of Pb[2+] in complex food matrices, but also pioneers a new paradigm for multi-modal biosensor design.},
}
@article {pmid41165985,
year = {2026},
author = {Bicknell, R and Koltunow, AMG},
title = {Pilosella: A Dicotyledonous Model for Studying Aposporous, Autonomous Apomixis.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2987},
number = {},
pages = {209-220},
pmid = {41165985},
issn = {1940-6029},
mesh = {*Apomixis/genetics ; Seeds/genetics/growth &amp; development ; *Asteraceae/genetics/physiology/growth &amp; development ; Ovule/genetics ; CRISPR-Cas Systems ; },
abstract = {Pilosella, a member of the Compositae, is a model system used to study the molecular genetics of aposporous apomixis. These plants are small, rapidly growing perennials that are easy to cultivate both in the greenhouse and in tissue culture. Apomixis in Pilosella occurs by apospory where mitotically derived embryo sacs arise adjacent to cells undergoing female gamete meiosis in the ovule. Seed initiation is autonomous, where both embryo and endosperm form without fertilization in the aposporous embryo sac. Apomixis is not fully penetrant in Pilosella. Instead, plants are facultatively apomictic, and apomixis can be easily scored through the simple decapitation of the immature capitulum bud. Natural sexual and facultatively apomictic forms are readily cross-compatible, facilitating comparative studies of inheritance and allele function. A wide range of experimental methods have been described for these plants, including histological techniques for studying the cytological aspects of apomixis, an efficient Agrobacterium-mediated transformation system, CRISPR/Cas9 mutagenesis, and mapping approaches that use deletion mutation and segregation in polyhaploid populations. Freely available online resources include a genome assembly, a molecular map based on cDNA markers and a transcriptome database. Collectively, these resources make Pilosella a highly tractable experimental system for studying the genetic control of native apomixis.},
}
@article {pmid41165983,
year = {2026},
author = {Radoeva, T and Rigola, D and Op den Camp, RHM and van Dijk, PJ and Underwood, CJ},
title = {Targeted Mutagenesis in Natural Apomicts.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2987},
number = {},
pages = {181-191},
pmid = {41165983},
issn = {1940-6029},
mesh = {*Apomixis/genetics ; CRISPR-Cas Systems ; *Mutagenesis ; *Taraxacum/genetics ; Parthenogenesis/genetics ; Chromosome Mapping ; Genes, Plant ; },
abstract = {Apomixis-clonal reproduction through seeds-is an alternative reproductive strategy that takes place in less than 0.1% of plant species and has evolved independently in diverse plant lineages. To date, the genetic basis of apomixis has been unraveled genetically in only a few genera. The identification of causal apomixis genes is technically challenging, as apomictic species are typically polyploid and the genetic loci associated with apomixis are often in low-recombination regions limiting conventional fine-mapping. In triploid apomictic dandelion (Taraxacum officinale), after conventional genetic mapping, deletion mapping, and complete apomixis loci haplotype assembly, we made use of targeted mutagenesis using CRISPR/Cas9 technology to identify the Taraxacum officinale PARTHENOGENESIS (ToPAR) gene that is responsible for embryogenesis in the absence of fertilization. Here, we report the methods used to clone the ToPAR gene by targeted mutagenesis and we expect that the general principles could be applied in other systems to identify novel apomixis genes.},
}
@article {pmid41165418,
year = {2025},
author = {Kumar, U and Dwivedi, D and Das, U},
title = {Advancements in CRISPR-Mediated Multiplex Genome Editing: Transforming Plant Breeding for Crop Improvement and Polygenic Trait Engineering.},
journal = {Biotechnology journal},
volume = {20},
number = {11},
pages = {e70148},
doi = {10.1002/biot.70148},
pmid = {41165418},
issn = {1860-7314},
support = {DBTHRDPMU/JRF/BET-24/I/2024-25/376//Department of Biotechnology/ ; 24J/01/00130//Council of Scientific and Industrial Research/ ; 3/1/3/BRET-2024/HRD (L1)//Indian Council of Medical Research/ ; AICE-JRF/SRF-KK04002406//Indian Council of Agricultural Research/ ; },
mesh = {*Gene Editing/methods ; *Plant Breeding/methods ; *Crops, Agricultural/genetics ; *CRISPR-Cas Systems/genetics ; Plants, Genetically Modified/genetics ; Multifactorial Inheritance/genetics ; Genome, Plant ; },
abstract = {With accelerating climate change and the urgent need to stack polygenic traits, multiplex CRISPR/Cas offers a scalable route to resilient crops-yet low editing efficiency and regeneration bottlenecks remain critical constraints. This review centers on multiplex strategies for polygenic trait engineering in plants, surveying compact nucleases (Cas9, Cas12, Cas13 and emerging ultra-compact variants), polycistronic gRNA platforms (tRNA-gRNA arrays, self-cleaving ribozymes, Csy4 processing), and delivery routes (Agrobacterium, biolistics, protoplast transfection, viral vectors). We highlight concrete outcomes-for example, targeted edits in PYL ABA-receptors increased rice grain yield by up to 31% in field tests-and applications from yield and disease resistance to abiotic-stress tolerance, nutrient biofortification and de novo domestication. Technical risks (off-targets, mosaicism, chromosomal rearrangements, transformability) are appraised alongside emerging fixes: compact/engineered nucleases, RNA-processing arrays, morphogenic regulators, and AI-driven sgRNA design integrated with multi-omics. By prioritizing multiplex approaches for polygenic trait stacking, the review argues that these tools are essential to accelerate precision breeding for climate-adapted agriculture.},
}
@article {pmid41163074,
year = {2025},
author = {Dashti, M and Mohammaddust Sarab, M and Shad, F and Dehnavi, S},
title = {CRISPR-mediated engineering of mesenchymal stromal/stem cells: a summary of recent progress in immunological applications for regenerative medicine and cancer therapy.},
journal = {Stem cell research &amp; therapy},
volume = {16},
number = {1},
pages = {592},
pmid = {41163074},
issn = {1757-6512},
mesh = {Humans ; *Mesenchymal Stem Cells/immunology/metabolism/cytology ; *CRISPR-Cas Systems/genetics ; *Neoplasms/therapy/immunology ; *Regenerative Medicine/methods ; Gene Editing ; Animals ; *Mesenchymal Stem Cell Transplantation ; },
abstract = {Mesenchymal stromal/stem cells (MSCs) have introduced as a cornerstone of regenerative medicine, owing to their immunomodulatory properties and therapeutic potential in autoimmune and inflammatory disorders. Although, their clinical application is often restricted due to immune rejection and heterogeneity in immunoregulatory responses. The advent of Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated protein 9 (CRISPR/Cas9) technology has revolutionized MSC engineering, enabling precise genetic modifications to enhance their immunological efficacy. This review explores how CRISPR-mediated editing of MSCs can mitigate immunogenicity, amplify anti-inflammatory functions, and repurpose MSCs for targeted immunotherapy. Key strategies include knockout of β2-microglobulin to evade T-cell recognition, augmentation of anti-inflammatory mediators like interleukin (IL)-10 and TNF-alpha stimulated gene/protein 6 (TSG-6), and disruption of pro-inflammatory pathways such as toll-like receptor 4 (TLR4)/NF-κB. In addition, CRISPR-engineered MSCs demonstrate promise in reshaping tumor microenvironments and combating bacterial infections through enhanced innate immunity. Despite challenges including off-target effects and delivery optimization, CRISPR-tailored MSCs represent a transformative approach to overcoming immunological barriers, paving the way for universal, off-the-shelf therapies in rheumatoid arthritis, cancer, and beyond.},
}
@article {pmid41147512,
year = {2025},
author = {Mirzaei, F and Mosaffa Jahromi, A and Molavi, H and Kabelitz, D and Kalantar, K and Meri, S},
title = {Targeting RNA-Binding proteins Roquin-1 and Regnase-1 could enhance CAR-iPSC-derived macrophage immunotherapy for solid tumors: a perspective and challenges.},
journal = {RNA biology},
volume = {22},
number = {1},
pages = {1-7},
doi = {10.1080/15476286.2025.2581385},
pmid = {41147512},
issn = {1555-8584},
mesh = {Humans ; *Ribonucleases/genetics/metabolism ; *Neoplasms/therapy/immunology/genetics ; *Macrophages/immunology/metabolism ; Tumor Microenvironment/immunology ; *RNA-Binding Proteins/genetics ; *Induced Pluripotent Stem Cells/cytology/metabolism/immunology ; Animals ; *Immunotherapy/methods ; Receptors, Chimeric Antigen/genetics/metabolism/immunology ; *Ubiquitin-Protein Ligases/genetics ; CRISPR-Cas Systems ; Gene Editing ; *Immunotherapy, Adoptive/methods ; Transcription Factors ; },
abstract = {Solid tumours present major treatment obstacles because of their immunosuppressive microenvironment and poor response to traditional chimeric antigen receptor (CAR)-based immunotherapies. Recent advances in cellular engineering have introduced CAR-macrophages derived from induced pluripotent stem cells (CAR-iMacs) as a promising approach to get around these obstacles. CAR-iMacs are designed to attack tumours, but their phenotypic plasticity can cause them to transform into M2-like macrophages in the tumour environment (TME), where they may instead suppress immune responses and promote tumour progression and metastasis. Roquin-1 and Regnase-1 are RNA-binding proteins that act as negative regulators of inflammatory genes that contribute to the phenotypic plasticity of macrophages. This perspective highlights a novel approach to augmenting anti-tumour responses of CAR-iMacs by simultaneously knocking out Roquin-1 and Regnase-1 via CRISPR-Cas9 gene editing. This approach drives a shift from an immunosuppressive M2-like state to an M1 state, promoting sustained pro-inflammatory signalling, boosting phagocytic and cytotoxic capabilities within the tumour microenvironment. Addressing a serious constraint in conventional adoptive cell therapies, this dual-targeting platform could provide a potent and scalable immunotherapeutic treatment for solid malignancies.},
}
@article {pmid41037400,
year = {2025},
author = {Combredet, C and Ansel, M and Brunet, T},
title = {A selection-based knockout approach for a choanoflagellate reveals regulation of multicellular development by Hippo signaling.},
journal = {Cell reports},
volume = {44},
number = {10},
pages = {116345},
doi = {10.1016/j.celrep.2025.116345},
pmid = {41037400},
issn = {2211-1247},
mesh = {*Choanoflagellata/genetics/growth &amp; development/metabolism ; *Signal Transduction ; *Gene Knockout Techniques/methods ; *Protein Serine-Threonine Kinases/metabolism/genetics ; Animals ; CRISPR-Cas Systems/genetics ; },
abstract = {Choanoflagellates, the closest living relatives of animals, provide crucial insights into animal origins. The multicellular choanoflagellate Salpingoeca rosetta can be genetically modified, but existing knockout (KO) pipelines are time consuming and have variable efficiency. Here, we present a fast and robust KO method for S. rosetta. We use CRISPR-Cas9 to inactivate target genes by interrupting, or fully replacing, their coding sequence with a selectable antibiotic resistance cassette. We inactivated three known S. rosetta multicellular developmental regulators (rosetteless, couscous, and jumble) and two homologs of Hippo pathway genes that control multicellular size in animals (warts and yorkie). Interestingly, warts-KO rosettes were consistently larger than their wild-type counterparts. RNA sequencing revealed that Warts and Yorkie regulated several extracellular matrix genes involved in multicellularity (including couscous), suggesting that Hippo signaling regulates multicellular size in choanoflagellates by modulating matrix secretion. We discuss the potential of our method to accelerate choanoflagellate functional genetics.},
}
@article {pmid41026602,
year = {2025},
author = {Kalchschmidt, J and Kanno, T and Park, S and Dubois, WD and Zhao, Y and Trzaskoma, P and Thomas, CJ and Staudt, LM and O'Shea, JJ and Jung, S and Casellas, R},
title = {Biphasic control of the B cell transcriptome by mTORC1 and GSK3.},
journal = {Cell reports},
volume = {44},
number = {10},
pages = {116361},
doi = {10.1016/j.celrep.2025.116361},
pmid = {41026602},
issn = {2211-1247},
mesh = {Humans ; *Mechanistic Target of Rapamycin Complex 1/metabolism/genetics ; *B-Lymphocytes/metabolism ; *Glycogen Synthase Kinase 3/metabolism/genetics ; *Transcriptome/genetics ; Lymphoma, B-Cell/genetics ; Cell Line, Tumor ; CRISPR-Cas Systems/genetics ; },
abstract = {A central question in immune regulation is how cells coordinate transcriptional responses to environmental cues. It remains unclear whether transcriptional regulation is controlled by isolated mechanism or integrated regulatory programs. Here, we develop a high-sensitivity, genome-wide CRISPR-Cas9 screening platform with 47 transcriptional reporters in human B cell lymphoma, identifying 4,440 regulators and 17,638 regulatory interactions. To enable the exploration of these networks, we establish B-LEARN, an interactive portal for data visualization and discovery. Our results reveal a large number of shared regulators across our 47 screens that act as context-dependent activators or repressors. Globally, we uncover a biphasic regulatory architecture in which mTORC1 and GSK3 exert opposing control over the B cell transcriptome. Notably, mTOR inhibition broadly activates key B cell genes, an effect antagonized by GSK3. Thus, B cell transcription is governed by an integrated, pathway-driven circuit, offering new targets to modulate gene expression in lymphoma and autoimmune disease.},
}
@article {pmid40693307,
year = {2025},
author = {Xia, Y and Guo, R and Lu, T and Jiang, S and You, K and Xia, X and Du, K and Kang, X},
title = {PagHB7/PagABF4-PagEPFL9 Module Regulates Stomatal Density and Drought Tolerance in Poplar.},
journal = {Plant biotechnology journal},
volume = {23},
number = {11},
pages = {4857-4871},
doi = {10.1111/pbi.70273},
pmid = {40693307},
issn = {1467-7652},
support = {2024M760220//the China Postdoctoral Science Foundation/ ; 2021YFD2200105//the National Key R&amp;D Program of China during the 14th Five-year Plan Period/ ; },
mesh = {*Populus/genetics/physiology/metabolism ; *Plant Stomata/physiology/genetics ; Droughts ; *Plant Proteins/metabolism/genetics ; Gene Expression Regulation, Plant ; *Transcription Factors/metabolism/genetics ; Plants, Genetically Modified ; Stress, Physiological ; CRISPR-Cas Systems ; Drought Resistance ; },
abstract = {Epidermal patterning factor-like 9 (EPFL9) influences stomatal density and growth in poplar. There have been no reports on homeobox 7 (HB7) and ABRE binding factor 4 (ABF4) regulating stomatal density or drought tolerance by targeting EPFL9 in poplar. This study revealed that EPFL9 was specifically localised in guard cells in leaves and responded to drought stress. By constructing CRISPR/Cas9-mediated PagEPFL9 gene-edited lines, we found that epfl9 mutant plants showed significantly reduced stomatal density, inhibited growth and enhanced drought resistance. However, PagEPFL9 overexpression increased its drought stress sensitivity by increasing the stomatal density. PagHB7 was demonstrated to be an upstream regulator of PagEPFL9 by yeast one-hybrid screening library experiments, yeast one-hybrid experiments, electrophoretic mobility shift assay and dual luciferase reporter gene assay experiments. Yeast two-hybrid, bimolecular fluorescence complementation, split luciferase complementation assays, GST pull-down, electrophoretic mobility shift assay and dual luciferase reporter gene assay experiments further demonstrated that PagHB7 interacted with PagABF4 and that PagABF4 enhanced the inhibitory effect of PagHB7 on PagEPFL9. Knockout plants of PagHB7, a negative regulator of PagEPFL9, had a significantly increased stomatal density and reduced drought tolerance. Poplars overexpressing PagABF4 showed similar phenotypes to poplars knocking out PagEPFL9, with stomatal density significantly lower than that of WT, which may result in greater drought tolerance. Our study demonstrates that PagHB7 and PagABF4 interact with each other and regulate stomatal density by targeting PagEPFL9, thereby affecting drought resistance in poplar. This study provides new genetic resources for molecular design breeding of plant growth and drought tolerance.},
}
@article {pmid41162659,
year = {2025},
author = {Campbell, IW and Basta, DW and Zingl, FG and Sullivan, EJ and Doranga, S and Waldor, MK},
title = {Anoxia activates CRISPR-Cas immunity in the mouse intestine.},
journal = {Nature microbiology},
volume = {},
number = {},
pages = {},
pmid = {41162659},
issn = {2058-5276},
support = {P30 DK034854/DK/NIDDK NIH HHS/United States ; R01 AI042347/AI/NIAID NIH HHS/United States ; Investigator program//Howard Hughes Medical Institute (HHMI)/ ; },
abstract = {The natural context in which CRISPR-Cas systems are active in Enterobacteriaceae has remained enigmatic. Here we find that the Citrobacter rodentium type I-E CRISPR-Cas system is activated by the oxygen-responsive transcriptional regulator Fnr in the anoxic environment of the mouse intestine. Since Fnr-dependent regulation is predicted in ~41% of Enterobacteriaceae cas3 orthologues, we propose that anoxic regulation of CRISPR-Cas immunity is an adaptation that protects Enterobacteriaceae against threats from foreign DNA within the intestinal microbiome.},
}
@article {pmid41162404,
year = {2025},
author = {Yan, X and Liu, B and Zhou, S and Fan, Y and Wei, S and Qiu, D and Xiang, H and Zhou, J and Mergny, JL and Monchaud, D and Ju, H and Zhou, J},
title = {A Chimeric Photo-Controllable CRISPR/Cas12a System for Universal and Fast Diagnostics.},
journal = {Analytical chemistry},
volume = {},
number = {},
pages = {},
doi = {10.1021/acs.analchem.5c04782},
pmid = {41162404},
issn = {1520-6882},
abstract = {The potential of clustered regularly interspaced short palindromic repeats (CRISPR) and corresponding CRISPR-associated (Cas) protein systems (CRISPR/Cas) systems for biomedical applications is tremendous; however, precise control of their activity is essential to better harness this potential and, beyond this, to develop reliable diagnostic reagents. Herein, we report on such a strategy by controlling the CRISPR/Cas12a activity using a photo-controllable CRISPR RNA (crRNA). To this end, the 3' end of crRNA was conjugated to a G-quadruplex (G4) block through a photocleavable linker: upon photo irradiation, the G4 trigger is removed, thus allowing for the DNA target to access and hybridize with the crRNA, and thus be processed by the CRISPR/Cas12a system. The efficiency of this approach was demonstrated by the detection of human papillomavirus 16 DNA in 50 clinical samples: our one-pot strategy was found to be as efficient as the routinely implemented method (qPCR), with 95.7% sensitivity and 100% specificity, in addition to be faster (25 versus 60 min) and both simpler and less expensive (being implementable as lateral flow test strips). Collectively, this new and fully controllable CRISPR/Cas system holds great potential for next-generation clinical diagnostics.},
}
@article {pmid41162070,
year = {2025},
author = {Gautam, V and Jambagi, SR and Muthugounder, M},
title = {Genome editing of detoxification gene repertoires in insects using clustered regularly interspaced short palindromic repeats (CRISPR): A systematic review and meta-analysis.},
journal = {Pesticide biochemistry and physiology},
volume = {215},
number = {},
pages = {106687},
doi = {10.1016/j.pestbp.2025.106687},
pmid = {41162070},
issn = {1095-9939},
mesh = {Animals ; *Gene Editing ; *Insecta/genetics ; *Insecticide Resistance/genetics ; Insecticides/pharmacology ; CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Inactivation, Metabolic/genetics ; },
abstract = {Decoding the molecular mechanisms underlying insect resistance to insecticides and host plant adaptation is essential for effective and sustainable Insecticide Resistance Management (IRM). Reverse genetic approaches targeting "target site resistance" can help control pests without harming pollinators and beneficial biocontrol agents. Understanding the role of mutations involved in xenobiotic resistance enables the judicious use of pesticides. CRISPR-based genome editing allows precise manipulation of detoxification genes, helping to decipher their roles in resistance development. This review provides a comprehensive overview of CRISPR-mediated genome editing in insect detoxification genes and their involvement in resistance mechanisms. In addition to synthesizing overall data trajectories, we present study-level effect sizes that highlight context-specific responses to gene editing, offering insights that can inform future experimental designs and functional validation studies.},
}
@article {pmid41161594,
year = {2025},
author = {Fu, H and Xu, W and Huang, M and Cong, Y},
title = {Molecular detection of Salmonella.},
journal = {Journal of food protection},
volume = {},
number = {},
pages = {100659},
doi = {10.1016/j.jfp.2025.100659},
pmid = {41161594},
issn = {1944-9097},
abstract = {The genus Salmonella consists of a group of globally significant foodborne pathogens that pose substantial public health risks. Traditional detection methods are inadequate for rapid diagnosis and effective epidemic surveillance due to limitations such as time-consuming procedures and insufficient sensitivity. In recent years, development of molecular techniques has driven innovations in Salmonella detection. Nucleic acid-based detection methods including polymerase chain reaction (PCR), real-time fluorescent quantitative PCR (qPCR), whole genome sequencing (WGS) and more emerge as crucial approaches for Salmonella detection due to their high sensitivity, specificity, and rapidity. Our review systematically summarized technological advancements in molecular detection of Salmonella, including specific genetic targets and drug resistance genes used for molecular detection, typing technologies, and emerging techniques such as CRISPR-Cas systems and microfluidic chips. This review comprehensively covers a wide array of molecular detection and characterization technologies, including conventional PCR, qPCR, multiplex PCR, digital PCR (dPCR), isothermal amplification techniques (such as loop-mediated isothermal amplification, recombinase polymerase amplification), genotyping methods (including pulsed-field gel electrophoresis, multilocus sequence typing etc.), WGS, melting curve analysis (MCA), and other emerging technologies. The review also discusses the balance between sensitivity and specificity in complex samples, challenges regarding the cost and accessibility of advanced technologies, as well as prospects for future development directions including portable point-of-care testing devices, automated detection equipment. Ongoing optimization of molecular detection technologies will provide critical support for the prevention and control of Salmonella infections.},
}
@article {pmid41161575,
year = {2025},
author = {Verma, R and Das, G and Manjunathachar, H and Muwel, N and Choudhary, R and Kumar, S and Nath, S and Gattani, A and Gupta, V and Sharma, RK and Ajith, Y},
title = {CRISPR-Cas systems: Pioneering next-generation diagnostic tools for parasitic diseases.},
journal = {Molecular and biochemical parasitology},
volume = {},
number = {},
pages = {111708},
doi = {10.1016/j.molbiopara.2025.111708},
pmid = {41161575},
issn = {1872-9428},
abstract = {Parasitic diseases pose significant threats to both human and veterinary health, causing morbidity, mortality, and economic losses. Effective diagnostics are critical, yet conventional methods such as microscopy, serology, and polymerase chain reaction (PCR) are limited by low sensitivity, cross-reactivity, or dependence on costly equipment and skilled personnel. Isothermal amplification techniques, such as loop-mediated isothermal amplification (LAMP) and recombinase polymerase amplification (RPA), have improved point-of-care (POC) applications but remain limited by nonspecific amplification and reduced sensitivity for low-copy targets. Clustered regularly interspaced short palindromic repeats (CRISPR)-associated (Cas) systems have emerged as transformative tools in molecular diagnostics, offering high sensitivity, specificity, rapidity, and cost-effectiveness. This review presents an overview of CRISPR-Cas systems, their historical development, classification (Class 1 and Class 2, Types I-VI), molecular mechanisms, and diagnostic potential in parasitic diseases, with illustrative examples from studies published between 2017 and May 2025. Despite significant progress, CRISPR-based diagnostics face challenges such as off-target activity, dependence on nucleic acid amplification, and complex sample preparation. Future directions focus on amplification-free detection, multiplexed assay development, and integration with nanotechnology, microfluidics, smartphone-based devices, and artificial intelligence. CRISPR-Cas technologies thus represent a promising frontier in next-generation diagnostics for parasitic disease surveillance, control, and personalized healthcare in both human and veterinary health.},
}
@article {pmid41161091,
year = {2025},
author = {Hong, SH and Kim, I and Lee, G and Kim, EH and Bae, E and Suh, JY},
title = {Structural and mechanistic investigation of the anti-CRISPR protein AcrIE5 using NMR spectroscopy and AlphaFold modeling.},
journal = {Biochemical and biophysical research communications},
volume = {789},
number = {},
pages = {152854},
doi = {10.1016/j.bbrc.2025.152854},
pmid = {41161091},
issn = {1090-2104},
abstract = {The CRISPR-Cas system employs RNA-guided endonucleases to protect bacteria and archaea from invading bacteriophages and plasmids. In response, bacteriophages have evolved anti-CRISPR proteins that inhibit diverse types of the CRISPR-Cas system. AcrIE5 was discovered from the mobile genetic elements of Pseudomonas aeruginosa, and potently inhibits the type I-E CRISPR-Cas system of P. aeruginosa. Here, we determined the solution structure of AcrIE5 using NMR spectroscopy, which adopts a novel αβ fold comprising three α-helices and two β-strands. AcrIE5 harbors a mobile loop between the β-strands that is conserved among homologs encoded by MGEs infecting Pseudomonas species, but truncated in homologs from MGEs of other bacteria. AlphaFold correctly reproduced the experimental structure of AcrIE5 and predicted its binding at a cleft formed by Cas8e, Cas7e, and Cas5e within the P. aeruginosa type I-E Cascade. The mobile loop and α-helices of AcrIE5 mediated key interactions with Cas8e at the PAM recognition site, as well as with adjacent Cas7e and Cas5e. AcrIE5 did not bind individual subunits of Cascade with high affinity, suggesting that it recognizes a composite interface of the functional Cascade assembly. Taken together, our findings suggest that AcrIE5 may compete with DNA binding to the PAM recognition site of type I-E Cascade, similar to AcrIE3 and AcrIE4, and also highlight a potential functional role of the conserved mobile loop in host-specific anti-CRISPR activity.},
}
@article {pmid41160700,
year = {2025},
author = {Elliott, SD and Ready, PJ and Wrinn, CM and Ma, Q and Edward, M and Niescier, RF and Escobar, I and Sun, J and Ganga, AK and McAtee, CK and Atiş, &#x130;S and Koleske, AJ and Bordey, A and Breslow, DK},
title = {A CRISPR activation screen reveals a cilia disassembly pathway mutated in focal cortical dysplasia.},
journal = {Science advances},
volume = {11},
number = {44},
pages = {eaeb7238},
pmid = {41160700},
issn = {2375-2548},
mesh = {*Cilia/metabolism/genetics/pathology ; Humans ; *Mutation ; *Malformations of Cortical Development/genetics/metabolism/pathology ; Cytoskeletal Proteins/genetics/metabolism ; *CRISPR-Cas Systems ; rhoA GTP-Binding Protein/genetics/metabolism ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Armadillo Domain Proteins/genetics/metabolism ; Animals ; Signal Transduction ; Focal Cortical Dysplasia ; },
abstract = {Defective assembly of primary cilia causes ciliopathies, but cilia disassembly and its role in disease remain poorly understood. From a genome-wide CRISPR activation (CRISPRa) screen for negative regulators of ciliary function, we find here that the F2R G protein-coupled receptor, sterile alpha and TIR motif-containing 1 (SARM1) hydrolase, ryanodine receptors, peri-centrosomal calcium signaling, and RhoA form a functional pathway that is necessary and sufficient for cilia disassembly. Highlighting the significance of this pathway, several components are somatically mutated in focal cortical dysplasia (FCD), a neurological disorder characterized by intractable epilepsy. Supporting the functional impact of these variants, patient-derived SARM1 and RhoA mutations potentiate cilia loss, and a RhoA variant impairs cortical development. Conversely, SARM1 inhibition restores cilia in cells with FCD-associated alterations. Together, our work identifies a pathway for cilia disassembly, implicates aberrant pathway activation as a feature of FCD-associated mutations, and illustrates the potential of CRISPRa screening to provide insight into diseases caused by somatic mutations.},
}
@article {pmid40991409,
year = {2025},
author = {Smith, SL and Iwamoto, Y and Manimaran, A and Drubin, DG},
title = {Harnessing fusion of genome-edited human stem cells to rapidly screen for novel protein functions in vivo.},
journal = {Molecular biology of the cell},
volume = {36},
number = {11},
pages = {ar141},
doi = {10.1091/mbc.E25-06-0301},
pmid = {40991409},
issn = {1939-4586},
mesh = {Humans ; *Gene Editing/methods ; *Induced Pluripotent Stem Cells/metabolism/cytology ; Endocytosis/genetics ; Cell Fusion/methods ; Actin Cytoskeleton/metabolism ; CRISPR-Cas Systems/genetics ; Clathrin/metabolism ; Lysosomes/metabolism ; },
abstract = {Genome editing has enabled the integration of fluorescent protein coding sequences into genomes, resulting in expression of in-frame fusion proteins under the control of their natural gene regulatory sequences. While this technique overcomes the well-documented artifacts associated with gene overexpression for biological processes sensitive to altered protein stoichiometry, such as clathrin-mediated endocytosis (CME), editing genomes of metazoan cells incurs a significant time cost compared with simpler organisms, such as yeast. Editing two or more genes to express multiple fluorescent fusion proteins in a single cell line has proven to be a powerful strategy for uncovering spatial dynamic, and therefore functional, relationships among different proteins, but it can take many months to edit each gene within the same cell line. Here, by utilizing cell fusions, we quickly generated cells expressing pairwise permutations of fluorescent fusion proteins in genome-edited human cells to reveal previously undetected protein-organelle interactions. We fused human induced pluripotent stem cells (hiPSCs) that express in-frame fusions of CME and actin cytoskeleton proteins with hiPSCs that express fluorescently tagged organelle markers, uncovering novel interactions between CME proteins, branched actin filament networks, and lysosomes.},
}
@article {pmid40963020,
year = {2025},
author = {Chauhan, VP and Sharp, PA and Langer, R},
title = {Engineered prime editors with minimal genomic errors.},
journal = {Nature},
volume = {646},
number = {8087},
pages = {1254-1260},
pmid = {40963020},
issn = {1476-4687},
mesh = {*Gene Editing/methods ; *INDEL Mutation/genetics ; CRISPR-Associated Protein 9/metabolism/genetics ; *CRISPR-Cas Systems/genetics ; Deoxyribonuclease I/metabolism/genetics ; Humans ; *Genome/genetics ; DNA/genetics/metabolism/chemistry ; *Genomics ; },
abstract = {Prime editors make programmed genome modifications by writing new sequences into extensions of nicked DNA 3' ends[1]. These edited 3' new strands must displace competing 5' strands to install edits, yet a bias towards retaining the competing 5' strands hinders efficiency and can cause indel errors[2]. Here we discover that nicked end degradation, consistent with competing 5' strand destabilization, can be promoted by Cas9-nickase mutations that relax nick positioning. We exploit this mechanism to engineer efficient prime editors with strikingly low indel errors. Combining this error-suppressing strategy with the latest efficiency-boosting architecture, we design a next-generation prime editor (vPE). Compared with previous editors, vPE features comparable efficiency yet up to 60-fold lower indel errors, enabling edit:indel ratios as high as 543:1.},
}
@article {pmid41160667,
year = {2025},
author = {Du, X and Goh, PK and Ma, C and Coughlan, E and Greatorex, S and Porter, LH and Russ, B and Cummins, KD and Sek, K and Slaney, CY and Scott, AM and Oliaro, J and Neeson, PJ and Risbridger, GP and Taylor, RA and Trapani, JA and Turner, SJ and Darcy, PK and Wiede, F and Tiganis, T},
title = {Targeting PTPN2 enhances human CAR T cell efficacy and the development of long-term memory in mouse xenograft models.},
journal = {Science translational medicine},
volume = {17},
number = {822},
pages = {eadk0627},
doi = {10.1126/scitranslmed.adk0627},
pmid = {41160667},
issn = {1946-6242},
mesh = {Animals ; Humans ; *Protein Tyrosine Phosphatase, Non-Receptor Type 2/metabolism/antagonists &amp; inhibitors ; *Xenograft Model Antitumor Assays ; Mice ; *Immunologic Memory ; *Receptors, Chimeric Antigen/metabolism/immunology ; Immunotherapy, Adoptive ; CRISPR-Cas Systems/genetics ; Cell Line, Tumor ; *T-Lymphocytes/immunology ; CD8-Positive T-Lymphocytes/immunology ; },
abstract = {Chimeric antigen receptor (CAR) T cells have been ineffective against solid tumors, where the hostile tumor microenvironment limits CAR T cell function and persistence. Protein tyrosine phosphatase N2 (PTPN2) attenuates T cell receptor and cytokine signaling to maintain T cell tolerance. Here, we used CRISPR-Cas9 gene editing or an inhibitor to target PTPN2 in human CAR T cells specific for the Lewis Y (LeY) neoantigen, which is expressed in most epithelial tumors. Targeting PTPN2 increased CAR and cytokine signaling, including interferon signaling, and enhanced the antigen-induced expansion, activation, and cytotoxicity of anti-LeY CAR T cells in vitro and in vivo. The deletion of PTPN2 in CAR T cells repressed the growth of human tumor and patient-derived xenografts in mice, when compared with unedited CAR T cells, and prolonged mouse survival. The administration of inhibitor also enhanced the ability of α-LeY CAR T cells to repress tumor growth. Cellular indexing of transcriptomes and epitopes by sequencing analysis of splenic PTPN2-deficient CD8[+] CAR T cells in tumor-bearing mice revealed that PTPN2 deficiency favored the generation of CD45RA[+] CAR T cells expressing markers of long-lived stem cell memory (SCM) CAR T cells. Flow cytometric analysis reaffirmed that the deletion or inhibition of PTPN2 promoted the intratumoral accumulation of SCM CD8[+] CAR T cells and the overall persistence of CD8[+] CAR T cells. These data support the use of gene editing or small-molecule inhibitors targeting PTPN2 in human CAR T cells to treat solid tumors.},
}
@article {pmid41160625,
year = {2025},
author = {Bish, LM and Fuss, JL and Panaccione, DG},
title = {Gene editing of the thioester reductase step in the biosynthesis of lysergic acid amides.},
journal = {PloS one},
volume = {20},
number = {10},
pages = {e0334651},
doi = {10.1371/journal.pone.0334651},
pmid = {41160625},
issn = {1932-6203},
mesh = {*Gene Editing/methods ; CRISPR-Cas Systems ; *Lysergic Acid/analogs &amp; derivatives/metabolism ; *Amides/metabolism ; *Oxidoreductases/genetics/metabolism ; Peptide Synthases/genetics/metabolism ; Fungal Proteins/genetics/metabolism ; },
abstract = {Ergot alkaloids derived from lysergic acid are important in agriculture, as food and feed contaminants, and in medicine, as the foundation of several pharmaceuticals. The fungus Metarhizium brunneum makes several lysergic acid amides, with lysergic acid α-hydroxyethylamide (LAH) being produced in by far the highest concentration. The multifunctional enzyme lysergyl peptide synthetase 3 (Lps3) has multiple domains that play important roles in lysergic acid amide synthesis. We hypothesized a role for the reductase domain of Lps3 in liberating LAH from an enzyme-bound precursor and tested this hypothesis with CRISPR/Cas9-based gene editing experiments. We transformed M. brunneum with a Cas9/single guide RNA complex and a donor DNA that replaced the tyrosine at the active site of the reductase domain of Lps3 with a phenylalanine. Sanger sequencing of edited and wild-type genes demonstrated successful editing of the reductase domain without non-target mutations in Lps3. High performance liquid chromatography of the edited strain showed a significant reduction of LAH and accumulation of the precursor lysergic acid. The phenotype was similar when the edited allele of lpsC was in a wild-type background or in backgrounds with late pathway genes easO or easP knocked out, except no LAH was detectable when the edit was in the easO knockout background. The data demonstrate that the reductase domain plays a key role or roles in formation of LAH. The abundant lysergic acid accumulating in the mutants, as opposed to later pathway intermediates in LAH biosynthesis (such as lysergyl-alanine), indicated severe debilitation of Lps3. The data indicate a requirement for the reductase domain of Lps3 in synthesis of lysergic acid amides and demonstrate the feasibility of the CRISPR/Cas9-based approach for editing genes in Metarhizium species.},
}
@article {pmid41160062,
year = {2025},
author = {Yilmaz Çolak, &#xc7;},
title = {Harnessing CRISPR technology for the diagnosis of Bordetella pertussis: advances and implications.},
journal = {Future microbiology},
volume = {},
number = {},
pages = {1-8},
doi = {10.1080/17460913.2025.2581522},
pmid = {41160062},
issn = {1746-0921},
abstract = {Following the discovery of the prokaryotic adaptive immune system known as clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) proteins, this technology has revolutionized biotechnology as a multifaceted genome-editing tool with a wide range of applications. CRISPR technology has not only provided novel treatment options, especially for genetic diseases, but also transformed molecular diagnostic platforms. The specific, sensitive, and adaptable nature of the CRISPR-Cas systems has led to the development of innovative solutions for the detection of diseases, including viral and bacterial infections. This review provides an overview of the CRISPR-Cas systems and mainly focuses on the application of CRISPR-based assays for the detection of Bordetella pertussis, which is the main causative agent of a highly infectious disease, whooping cough. The review emphasizes the need for novel diagnostic tools for B. pertussis, along with highlighting some future perspectives, since its diagnosis can be challenging due to nonspecific early symptoms and interference from closely related Bordetella species. In this regard, CRISPR-based diagnostic platforms can offer a promising avenue for rapid and accurate detection of B. pertussis, helping the management of whooping cough.},
}
@article {pmid41159723,
year = {2025},
author = {Duan, Z and Yang, R and Lai, T and Jiang, W and Zhang, J and Chen, B and Liao, L},
title = {Development of a CRISPR/Cas9-induced gene editing system for Pseudoalteromonas fuliginea and its applications in functional genomics.},
journal = {Applied and environmental microbiology},
volume = {},
number = {},
pages = {e0177125},
doi = {10.1128/aem.01771-25},
pmid = {41159723},
issn = {1098-5336},
abstract = {Pseudoalteromonas has been used as a model system to study cold adaptation and is of widespread interest in biotechnology and ecology. To explore its physiological responses to extreme cold, uncover functional genes, and clarify their ecological roles, efficient genetic tools are essential. However, existing genetic manipulation methods in Pseudoalteromonas rely on traditional homology-based recombination, which is laborious and time-consuming in this bacterial system. Consequently, improving editing efficiency is crucial for advancing both basic research and applied potential. Here, we established a CRISPR/Cas9 system in Pseudoalteromonas and carried out an extensive investigation of the Type II CRISPR/Cas9 platform for gene editing in Pseudoalteromonas fuliginea, a representative species thriving in the frigid polar oceans. To validate the feasibility of the CRISPR/Cas system in P. fuliginea, multiple genes were selected as targets, and the gene editing effects were confirmed through phenotypic changes or gene expression. We have successfully achieved both gene knockouts and insertions in P. fuliginea, encompassing the deletion of genes such as fliJ, indA, and genes encoding Pf sRNAs, as well as the in vivo insertion of 3×FLAG and the gfp gene. The average CRISPR/Cas9 gene editing efficiency in P. fuliginea exceeded 70%. In summary, we developed an efficient CRISPR/Cas9-based editing system in P. fuliginea, which can be utilized to accelerate the development of Pseudoalteromonas as a model system for addressing fundamental questions related to extreme environmental adaptation and to fulfill its potential biotechnological applications.IMPORTANCEPseudoalteromonas fuliginea is a marine bacterium with great potential for ecological and biotechnological research, yet its genetic manipulation has long been a technical challenge. In this study, we developed a gene editing system based on CRISPR technology that enables efficient and precise genome modification in this organism. Using this system, we successfully deleted, inserted, and tagged multiple genes, including regulatory and non-coding elements, with high success rates. Notably, several of these genes are linked to key traits such as motility and stress response, which contribute to microbial adaptation in polar environments. This tool allows researchers to directly test gene function and study microbial adaptation in cold marine environments. The ability to perform reliable genetic edits in P. fuliginea opens new possibilities for its use as a model organism and will support future advances in microbial ecology, environmental microbiology, and marine biotechnology.},
}
@article {pmid41157107,
year = {2025},
author = {Shpiliukova, K and Kachanov, A and Brezgin, S and Chulanov, V and Ivanov, A and Kostyushev, D and Kostyusheva, A},
title = {m[6]A RNA Modification: Technologies Behind Future Anti-Cancer Therapy.},
journal = {Molecules (Basel, Switzerland)},
volume = {30},
number = {20},
pages = {},
doi = {10.3390/molecules30204091},
pmid = {41157107},
issn = {1420-3049},
support = {075-15-2025-519//Ministry of Science and Higher Education of the Russian Federation (Federal scientific and technical program for the development of genetic technologies for 2019-2030/ ; },
mesh = {Humans ; *Adenosine/analogs &amp; derivatives/metabolism/genetics ; *Neoplasms/genetics/drug therapy/therapy/metabolism ; Methylation ; Animals ; Epigenesis, Genetic ; *Antineoplastic Agents/pharmacology/therapeutic use ; *RNA/genetics/metabolism ; *RNA Processing, Post-Transcriptional ; RNA Methylation ; },
abstract = {N6-methyladenosine (m[6]A) modifications are among the most prevalent epigenetic marks in eukaryotic RNAs, regulating both coding and non-coding RNAs and playing a pivotal role in RNA metabolism. Given their widespread influence, m[6]A modifications are deeply implicated in the pathogenesis of various cancers, including highly aggressive malignancies such as lung cancer, melanoma, and liver cancer. Dysregulation of m[6]A dynamics-marked by an imbalance in methylation and demethylation-can drive tumor progression, enhance metastatic potential, increase aggressiveness, and promote drug resistance, while also exerting context-dependent tumor-suppressive effects. Given this dual role, precise modulation of m[6]A levels and the activity of its regulatory enzymes (writers, erasers, and readers) represent a promising therapeutic avenue. In this review, we highlight recent advances in targeting m[6]A machinery, including small-molecule inhibitors, antisense oligonucleotides, and CRISPR/Cas-based editing tools, capable of both writing and erasing m[6]A marks and altering m[6]A methylation sites per se. By evaluating these strategies, we aim to identify the most effective approaches for restoring physiological m[6]A homeostasis or for strategically manipulating the m[6]A machinery for therapeutic benefit.},
}
@article {pmid41156655,
year = {2025},
author = {Peláez Sánchez, RG and González Restrepo, J and Pineda, S and Cuartas-López, AM and Martínez Garro, JM and Torres-Castro, M and Urrego, R and López-Rojas, LE and Salazar Florez, JE and Monroy, FP},
title = {Bioinformatic Identification of CRISPR-Cas Systems in Leptospira Genus: An Update on Their Distribution Across 77 Species.},
journal = {Pathogens (Basel, Switzerland)},
volume = {14},
number = {10},
pages = {},
doi = {10.3390/pathogens14101044},
pmid = {41156655},
issn = {2076-0817},
mesh = {*CRISPR-Cas Systems/genetics ; *Leptospira/genetics/classification ; *Computational Biology/methods ; Genome, Bacterial ; Bacteriophages/genetics ; Leptospirosis/microbiology ; },
abstract = {UNLABELLED: Leptospirosis is a globally distributed zoonotic disease caused by pathogenic bacteria of the Leptospira genus. Genome editing in Leptospira has been difficult to perform. Currently, the functionality of the CRISPR-Cas system has been demonstrated in species such as Leptospira interrogans. However, the different CRISPR-Cas systems present in most of the 77 species are unknown. Therefore, the objective of this study was to identify these arrays across the genomes of all described Leptospira species using bioinformatics tools.<br><br>METHODS: a bioinformatics workflow was followed: genomes were downloaded from the NCBI database; Cas protein detection was carried out using the CRISPR-CasFinder and RAST web servers; functional analyses of Cas proteins were performed with InterProScan, ProtParam, Swiss Model, Alphafold3, Swiss PDB Viewer, and Pymol; conservation pattern detection was conducted using MEGA12, and Seqlogos; spacer identification was carried out with the Actinobacteriophages database and BLAST version 1.4.0; and bacteriophage detection was performed using PHASTER, and PHASTEST.<br><br>RESULTS: Cas proteins were detected in 36 out of the 77 species of the Leptospira species, including Cas1 to Cas9 and Cas12. These proteins were classified into Class 1 and Class 2 systems, corresponding to types I, II, and V. Direct repeats and spacers were detected in 19 species, with the direct repeats exhibiting two conserved nucleotide motifs. Analysis of spacer sequences revealed 323 distinct bacteriophages. Additionally, three intact bacteriophages were detected in the genomes of four Leptospira species. Notably, two saprophytic species have complete CRISPR-Cas systems.<br><br>CONCLUSIONS: The presence of Cas proteins, direct repeats, and spacer sequences with homology to bacteriophage genomes provides evidence for a functional CRISPR-Cas system in at least 19 species.},
}
@article {pmid41155971,
year = {2025},
author = {Serrano, DR and Juste, F and Anaya, BJ and Ramirez, BI and Sánchez-Guirales, SA and Quispillo, JM and Hernandez, EM and Simon, JA and Trallero, JM and Serrano, C and Rawat, S and Lalatsa, A},
title = {Exosome-Based Drug Delivery: A Next-Generation Platform for Cancer, Infection, Neurological and Immunological Diseases, Gene Therapy and Regenerative Medicine.},
journal = {Pharmaceutics},
volume = {17},
number = {10},
pages = {},
doi = {10.3390/pharmaceutics17101336},
pmid = {41155971},
issn = {1999-4923},
support = {PID2024-156769OB-I00//Ministerio de Ciencia, Innovaci&#xf3;n y Universidades/ ; Innovation in Pharmacology, Nanotechnology, and personalized medicine by 3D printing//Universidad Complutense de Madrid/ ; },
abstract = {Exosomes, naturally derived extracellular vesicles, have emerged as powerful bio-nanocarriers in precision medicine. Their endogenous origin, biocompatibility, and ability to encapsulate and deliver diverse therapeutic payloads position them as transformative tools in drug delivery, gene therapy, and regenerative medicine. This review presents a comprehensive analysis of exosome-based therapeutics across multiple biomedical domains, including cancer, neurological and infectious diseases, immune modulation, and tissue repair. Exosomes derived from stem cells, immune cells, or engineered lines can be loaded with small molecules, RNA, or CRISPR-Cas systems, offering highly specific and low-immunogenic alternatives to viral vectors or synthetic nanoparticles. We explore endogenous and exogenous loading strategies, surface functionalization techniques for targeted delivery, and innovations that allow exosomes to traverse physiological barriers such as the blood-brain barrier. Furthermore, exosomes demonstrate immunomodulatory and regenerative properties in autoimmune and degenerative conditions, with promising roles in skin rejuvenation and cosmeceuticals. Despite their potential, challenges remain in large-scale production, cargo loading efficiency, and regulatory translation. Recent clinical trials and industry efforts underscore the accelerating momentum in this field. Exosomes represent a promising platform in precision medicine, though further standardization and validation are required before widespread clinical use. This review offers critical insights into current technologies, therapeutic mechanisms, and future directions to unlock the full translational potential of exosomes in clinical practice.},
}
@article {pmid41155374,
year = {2025},
author = {Honjo, A and Yako, H and Miyamoto, Y and Yagi, M and Yamamoto, M and Nishi, A and Sakagami, H and Yamauchi, J},
title = {Knocking Down FRMD4A, a Factor Associated with the Brain Development Disorder and a Risk Factor for Alzheimer's Disease, Using RNA-Targeting CRISPR/Cas13 Reveals Its Role in Cell Morphogenesis.},
journal = {International journal of molecular sciences},
volume = {26},
number = {20},
pages = {},
doi = {10.3390/ijms262010083},
pmid = {41155374},
issn = {1422-0067},
mesh = {*Alzheimer Disease/genetics/metabolism/pathology ; Animals ; *CRISPR-Cas Systems ; Neurons/metabolism/cytology ; Mice ; *Morphogenesis/genetics ; *Cytoskeletal Proteins/genetics/metabolism ; *Membrane Proteins/genetics/metabolism ; Gene Knockdown Techniques ; Humans ; *Brain/metabolism ; Cell Line ; },
abstract = {Genetic truncation or mutation of the gene encoding band 4.1, ezrin, radixin, and moesin (FERM) domain protein containing 4A (FRMD4A) is associated with brain developmental diseases, including microcephaly with global developmental delay. It has also been identified as a risk factor for Alzheimer's disease. By analogy with other FERM domain-containing proteins, FRMD4A is believed to regulate cell morphogenesis and/or cell polarization in central nervous system (CNS) cells; however, it remains unclear whether and how dysfunction of FRMD4A and/or its closely homologous protein FRMD4B causes abnormal morphogenesis in neuronal cells. Here, we describe for the first time the roles of FRMD4A and FRMD4B in process elongation in neuronal cells. Knockdown of Frmd4a or Frmd4b using specific RNA-targeting clustered regularly interspaced short palindromic repeat (CRISPR) and Cas13-fitted gRNAs led to decreased process elongation in primary cortical neurons. Similar decreases in neuronal marker expression were observed in the N1E-115 cell line, a model of neuronal differentiation. Furthermore, hesperetin, an aglycone of the citrus flavonoid hesperidin known to promote neuroprotective signaling, recovered the decreased process elongation induced by the knockdown of Frmd4a or Frm4b. Hesperetin also stimulated phosphorylation of mitogen-activated protein kinases/extracellular signal-regulated kinases (MAPKs/ERKs), which could help promote neuronal processes. These results suggest that FRMD4A and FRMD4B regulate process elongation through a possible signaling pathway linked to the sustained phosphorylation of MAPKs/ERKs. Crucially, this study reveals that, at the molecular and cellular levels, hesperetin can restore normal phenotypes when FRMD4A protein or FRMD4B protein is impaired.},
}
@article {pmid41155368,
year = {2025},
author = {Hossain, MM and Sultana, F and Mostafa, M and Khan, I and Tran, LP and Mostofa, MG},
title = {Reinforced Defenses: R-Genes, PTI, and ETI in Modern Wheat Breeding for Blast Resistance.},
journal = {International journal of molecular sciences},
volume = {26},
number = {20},
pages = {},
doi = {10.3390/ijms262010078},
pmid = {41155368},
issn = {1422-0067},
mesh = {*Triticum/genetics/microbiology/immunology ; *Disease Resistance/genetics ; *Plant Diseases/microbiology/genetics/immunology ; *Plant Breeding ; *Plant Immunity/genetics ; *Genes, Plant ; Magnaporthe/pathogenicity ; Host-Pathogen Interactions/genetics/immunology ; },
abstract = {Wheat blast, caused by Magnaporthe oryzae pathotype Triticum (MoT), poses a major threat to wheat (Triticum aestivum) cultivation, particularly in South America and Bangladesh. The rapid evolution and spread of the pathogen necessitate the development of durable and broad-spectrum resistance in wheat cultivars. This review summarizes current insights into the multi-layered defense mechanisms of wheat, encompassing resistance (R) genes, pattern-triggered immunity (PTI), and effector-triggered immunity (ETI) against MoT. The R-genes provide race-specific resistance through ETI, while both ETI and PTI are required to form integral layers of the plant immune system that synergistically reinforce host defense network. Recent advances in genomics, transcriptomics, and molecular breeding have facilitated the discovery and deployment of key R-genes and signaling components involved in PTI and ETI pathways. Integrating these immune strategies through gene pyramiding, marker-assisted selection (MAS), and genome editing offers a promising route towards enhanced and durable resistance in hosts. Harnessing and optimizing these multilayered immune systems will be pivotal to securing wheat productivity amid the growing threat of wheat blast.},
}
@article {pmid41155240,
year = {2025},
author = {Carbone, F},
title = {Special Issue: Latest Research on Plant Genomics and Genome Editing.},
journal = {International journal of molecular sciences},
volume = {26},
number = {20},
pages = {},
doi = {10.3390/ijms26209946},
pmid = {41155240},
issn = {1422-0067},
mesh = {*Gene Editing/methods ; *Genome, Plant ; *Genomics/methods ; *Plants/genetics ; CRISPR-Cas Systems ; High-Throughput Nucleotide Sequencing ; },
abstract = {Over the past ten years, plant science has undergone a remarkable transformation driven by the convergence of next-generation sequencing, increasingly sophisticated bioinformatics tools, and the rise of targeted genome editing platforms [...].},
}
@article {pmid41154642,
year = {2025},
author = {Zhang, H and Yang, Y and Yang, T and Cao, P and Yu, C and Liang, L and Liu, R and Chen, Z},
title = {Engineering a High-Fidelity MAD7 Variant with Enhanced Specificity for Precision Genome Editing via CcdB-Based Bacterial Screening.},
journal = {Biomolecules},
volume = {15},
number = {10},
pages = {},
doi = {10.3390/biom15101413},
pmid = {41154642},
issn = {2218-273X},
support = {2023YFC3402300//National Key R&amp;D Program of China/ ; 22208044//National Natural Science Foundation of China/ ; 22278058//National Natural Science Foundation of China/ ; XLYC2203075//"Xingliao Talent Plan"project/ ; 2024-MSBA-09//Natural Science Foundation of Liaoning Province/ ; 2025JH2/101330156//Natural Science Foundation of Liaoning Province/ ; 2023JJ12SN030//Science and Technology Innovation Foundation of Dalian/ ; DUT24YG131//Fundamental Research Funds for the Central Universities/ ; DUT25LAB105//Fundamental Research Funds for the Central Universities/ ; DUT25RC(3)019//Fundamental Research Funds for the Central Universities/ ; },
mesh = {*Gene Editing/methods ; CRISPR-Cas Systems/genetics ; *Escherichia coli/genetics ; *Bacterial Proteins/genetics/metabolism ; RNA, Guide, CRISPR-Cas Systems/genetics ; *Endonucleases/genetics/metabolism ; Mutation ; },
abstract = {CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-associated protein) nucleases enable precise genome editing, but off-target cleavage remains a critical challenge. Here, we report the development of MAD7_HF, a high-fidelity variant of the MAD7 nuclease engineered through a bacterial screening system leveraging the DNA gyrase-targeting toxic gene ccdB. This system couples survival to efficient on-target cleavage and minimal off-target activity, mimicking the transient action required for high-precision editing. Through iterative selection and sequencing validation, we identified MAD7_HF, harboring three substitutions (R187C, S350T, K1019N) that enhanced discrimination between on- and off-target sites. In Escherichia coli assays, MAD7_HF exhibited a >20-fold reduction in off-target cleavage across multiple mismatch contexts while maintaining on-target efficiency comparable to wild-type MAD7. Structural modeling revealed that these mutations stabilize the guide RNA-DNA hybrid at on-target sites and weaken interactions with mismatched sequences. This work establishes a high-throughput bacterial screening strategy that allows the identification of Cas12a variants with improved specificity at a given target site, providing a useful framework for future efforts to develop precision genome-editing tools.},
}
@article {pmid41153450,
year = {2025},
author = {Nardon, E and Azzalini, E and Paladin, D and Boscarino, D and Bonin, S},
title = {CRISPR/Cas Tools for the Detection of Borrelia sensu lato in Human Samples.},
journal = {Genes},
volume = {16},
number = {10},
pages = {},
doi = {10.3390/genes16101233},
pmid = {41153450},
issn = {2073-4425},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Lyme Disease/diagnosis/microbiology/genetics ; *Borrelia burgdorferi Group/genetics/isolation &amp; purification ; DNA, Bacterial/genetics ; Real-Time Polymerase Chain Reaction/methods ; Sensitivity and Specificity ; Bacterial Outer Membrane Proteins/genetics ; Antigens, Surface ; Bacterial Vaccines ; Lipoproteins ; },
abstract = {BACKGROUND/OBJECTIVES: Lyme disease diagnosis remains challenging due to the limitations of current methods. While PCR-based assays are widely used, their sensitivity can be affected by sample type and the inhibition of host DNA. This study aimed to evaluate the feasibility and sensitivity of a CRISPR/Cas12-based detection system for Borrelia burgdorferi sensu lato, comparing its performance with real-time PCR.<br><br>METHODS: DNA from three Borrelia genospecies (B. burgdorferi, B. garinii, and B. afzelii) was amplified targeting the OspA gene. Detection was performed using a Cas12/crRNA system with a fluorescent ssDNA reporter. Sensitivity assays were conducted on serial dilutions of Borrelia DNA, with and without human genomic DNA, and results were compared with qPCR.<br><br>RESULTS: Direct detection of Borrelia DNA without amplification was not feasible. However, when combined with PCR, the Cas12/crRNA system reliably detected as few as 5 genome copies per reaction. End-point PCR extended to 60 cycles improved detection robustness for B. garinii and B. afzelii, although sensitivity decreased in the presence of human genomic DNA.<br><br>CONCLUSIONS: The Cas12/crRNA-based system offers a sensitive and accessible alternative to qPCR, especially in settings lacking real-time PCR instrumentation. Future developments may include integration with isothermal amplification and microfluidic platforms to enhance direct detection capabilities.},
}
@article {pmid41153439,
year = {2025},
author = {Stunf Pukl, S},
title = {Genetic Therapy of Fuchs Endothelial Corneal Dystrophy: Where Are We? A Review.},
journal = {Genes},
volume = {16},
number = {10},
pages = {},
doi = {10.3390/genes16101222},
pmid = {41153439},
issn = {2073-4425},
mesh = {*Fuchs' Endothelial Dystrophy/therapy/genetics ; Humans ; *Genetic Therapy/methods ; CRISPR-Cas Systems ; Collagen Type VIII/genetics ; Mutation ; Transcription Factor 4/genetics ; Animals ; Gene Editing ; },
abstract = {OBJECTIVES: The incidence of Fuchs endothelial corneal dystrophy (FECD) is growing, and with it, the unmet need for a corneal transplant. Among alternative treatment modalities, only genetic therapy represents a causal therapy.<br><br>METHODS: Following the SNARA protocol, the PubMed and ClinicalTrials databases were searched using the keywords Fuchs endothelial corneal dystrophy, FECD, genetic therapy, and CRISPR-Cas9.<br><br>RESULTS: FECD is polyfactorial disease and mutations or polymorphisms in at least 15 different genes were connected to the disease. For the early-onset form of the disease, exclusive connection to mutations in COL8A2 was confirmed, while for the late-onset form, the most characteristic mutation is the expansion of the CTG18.1 triplet in the TCF4 gene, making these two possible targets. While the CRISPR-Cas9 approach represents the mainstay of genetic therapy development recently, the application of this method to FECD contains several obstacles, studied in preclinical settings. DT-168 and the Ad-Cas9-Col8a2gRNA molecules were developed for FECD treatment and preclinically tested, and phase I and II clinical studies for DT-168 are also already being performed.<br><br>CONCLUSIONS: The review of the literature proved that genetic therapy for FECD is at the level of preclinical research and that there are several specific challenges connected to the target genetic mutation as well as the delivery of possible treatment and duration of the effect. Further studies in the field might bring solutions in the future for alternative treatments for this common corneal disease.},
}
@article {pmid41153385,
year = {2025},
author = {Boti, MA and Diamantopoulos, MA and Scorilas, A},
title = {RNA-Targeting Techniques: A Comparative Analysis of Modern Approaches for RNA Manipulation in Cancer Research and Therapeutics.},
journal = {Genes},
volume = {16},
number = {10},
pages = {},
doi = {10.3390/genes16101168},
pmid = {41153385},
issn = {2073-4425},
mesh = {Humans ; *Neoplasms/genetics/therapy ; RNA Interference ; CRISPR-Cas Systems ; Oligonucleotides, Antisense/therapeutic use/genetics ; Animals ; *RNA/genetics ; },
abstract = {RNA-targeting techniques have emerged as powerful tools in cancer research and therapeutics, offering precise and programmable control over gene expression at the post-transcriptional level. Once viewed as passive intermediates in the central dogma, RNA molecules are now recognized as dynamic regulators of cellular function, capable of influencing transcription, translation, and epigenetic regulation. Advances in high-throughput sequencing technologies, transcriptomics, and structural RNA biology have uncovered a diverse landscape of coding and non-coding RNAs involved in oncogenesis, drug resistance, and tumor progression. In response, several RNA-targeting strategies have been developed to modulate these transcripts, including antisense oligonucleotides (ASOs), RNA interference (RNAi), CRISPR-Cas13 systems, small molecules, and aptamers. This review provides a comparative analysis of these technologies, highlighting their molecular mechanisms, therapeutic potential, and current limitations. Emphasis is placed on the translational progress of RNA-targeting agents, including recent FDA approvals and ongoing clinical trials for cancer indications. Through a critical comparison of these strategies, this review underscores the growing significance of RNA-targeting technologies as a foundation for next-generation cancer therapeutics and precision oncology.},
}
@article {pmid41152294,
year = {2025},
author = {Nemoto, A and Imaizumi, K and Miya, F and Hiroi, Y and Yamada, M and Ideno, H and Saitoh, S and Kosaki, K and Okuno, H and Okano, H},
title = {Rescue of imprinted genes by epigenome editing in human cellular models of Prader-Willi syndrome.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {9442},
pmid = {41152294},
issn = {2041-1723},
mesh = {*Prader-Willi Syndrome/genetics/therapy ; Humans ; *Genomic Imprinting/genetics ; Induced Pluripotent Stem Cells/metabolism ; *Gene Editing/methods ; DNA Methylation/genetics ; CRISPR-Cas Systems ; *Epigenome/genetics ; Hypothalamus/metabolism ; Organoids/metabolism ; Chromosomes, Human, Pair 15/genetics ; Epigenesis, Genetic ; Epigenome Editing ; },
abstract = {Prader-Willi syndrome (PWS) is a genomic imprinting disorder caused by the loss of function of the paternal chromosome 15q11-13, resulting in a spectrum of symptoms associated with hypothalamic dysfunction. PWS patients lack the expression of paternally expressed genes (PEGs) in the 15q11-13 locus but possess an epigenetically silenced set of these genes in the maternal allele. Thus, activation of these silenced genes can serve as a therapeutic target for PWS. Here, we leverage CRISPR-based epigenome editing system to modulate the DNA methylation status of the PWS imprinting control region (PWS-ICR) in induced pluripotent stem cells (iPSCs) derived from PWS patients. Successful demethylation in the PWS-ICR restores the PEG expression from the maternal allele and reorganizes the methylation patterns in other PWS-associated imprinted regions beyond the PWS-ICR. Remarkably, these corrected epigenomic patterns and PEG expression are maintained following the differentiation of these cells into hypothalamic organoids. Finally, the single-cell transcriptomic analysis of epigenome-edited organoids demonstrates a partial restoration of the transcriptomic dysregulation observed in PWS. This study highlights the utility of epigenome editing technology as a therapeutic approach in addressing PWS and potentially other imprinting disorders.},
}
@article {pmid41152284,
year = {2025},
author = {Mishal, R and Meléndez-Zajgla, J and Rueda-Zarazúa, B and Labra-Barrios, ML and Castañón-Sánchez, CA and Uribe Carvajal, S and Padierna-Mota, L and Hernández-Hernández, JM and Leon-Avila, G and Pérez Rangel, A and Hernández-Martínez, E and Angeles-Morales, EB and Albalawi, IK and Luna-Arias, JP},
title = {RNA-seq analysis of wild-type and mutated TBPL1 gene in breast cancer cells lines through CRISPR/Cas9 approach reveals novel molecular signatures.},
journal = {Scientific reports},
volume = {15},
number = {1},
pages = {37578},
pmid = {41152284},
issn = {2045-2322},
support = {Ph.D. fellowship 465228//Consejo Nacional de Humanidades, Ciencia y Tecnologia, Mexico/ ; Ciencia de Frontera 2019_Proyecto No. 116337//Consejo Nacional de Humanidades, Ciencia y Tecnolog&#xed;a, Mexico/ ; Ciencia de Frontera 2019_Proyecto No. 116337//Consejo Nacional de Humanidades, Ciencia y Tecnolog&#xed;a, Mexico/ ; Ciencia de Frontera 2019_Proyecto No. 116337//Consejo Nacional de Humanidades, Ciencia y Tecnolog&#xed;a, Mexico/ ; PhD. fellowship 752614//Consejo Nacional de Humanidades, Ciencia y Tecnolog&#xed;a, Mexico/ ; Ciencia de Frontera 2019_Proyecto No. 116337//Consejo Nacional de Humanidades, Ciencia y Tecnolog&#xed;a, Mexico/ ; Ciencia de Frontera 2019_Proyecto No. 116337//Consejo Nacional de Humanidades, Ciencia y Tecnolog&#xed;a, Mexico/ ; },
mesh = {Humans ; *Breast Neoplasms/genetics/pathology ; *CRISPR-Cas Systems ; Female ; Cell Line, Tumor ; Gene Expression Regulation, Neoplastic ; *TATA-Box Binding Protein/genetics/metabolism ; *Mutation ; RNA-Seq ; Transcriptome ; Cell Proliferation/genetics ; Gene Expression Profiling ; Cell Movement/genetics ; },
abstract = {Breast cancer is the leading cause of death among women globally. Several genes have been found to be transcriptionally dysregulated in cancer, according to recent studies. TATA-box binding protein (TBP) and its two paralogs, TBPL1 and TBPL2, play roles in human transcription. The TBPL1 gene is implicated in colorectal carcinomas by suppressing the expression of miR-18a. However, its function in breast cancer remains undisclosed. TBPL1 is distantly related to TBP and possesses a 40% similarity with TBP's core domain. In the present study, we explored the potential role of the TBPL1 gene in transcriptome regulation by knocking out the TBPL1 gene through the CRISPR/Cas9 method. Following the knockout of the TBPL1 gene, we examined the gene transcription patterns and compared them to wild-type cell lines. We observed disparate signatures of upregulated and downregulated genes in wild-type and mutated conditions. Healthy breast MCF-12F, and T47D, SKBR3, and MDA-MB-231 breast cancer cell lines were assessed, as these cancer cells exhibit overexpression of the TBPL1 gene. Next-generation sequencing data revealed distinct marker genes regulated by the TBPL1 gene and their potential involvement in cell migration, proliferation, anti-apoptosis, and metastasis. Additionally, we also discovered novel lncRNAs implicated in the transcriptome analysis of the TBPL1 knocked-out gene. Our investigation indicated that this gene might affect varied stages of breast cancer cell lines' cellular properties, such as cell duplication, morphology, and growth. It might also contribute to tumor formation in more aggressive cell lines like MDA-MB-231 in vivo.},
}
@article {pmid41152217,
year = {2025},
author = {Song, Z and Guo, J and Fan, Z and Huang, S and Li, G and Zhao, Z and Chen, B and Huang, S and Zheng, W and Wei, Y and Chen, Y and Huang, X and Liu, J and Wu, L and Wang, X},
title = {Noncanonical target-strand cytosine base editing via engineered Un1Cas12f1 platform.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {9499},
pmid = {41152217},
issn = {2041-1723},
mesh = {*Gene Editing/methods ; Animals ; *CRISPR-Cas Systems/genetics ; *Cytosine/metabolism ; Mice ; Male ; Humans ; *CRISPR-Associated Proteins/metabolism/genetics ; Protein Engineering ; },
abstract = {CRISPR/Cas-derived base editors harness various deaminase or glycosylase activities to target bases within non-target strand (NTS) of the R-loop, catalyzing base conversions independent of double-strand break formation. To develop miniature BEs compatible with therapeutic viral vectors, we explore the compact Cas12f system. Through computational modeling and mutagenesis, we establish a highly active enUn1Cas12f1 protein, and subsequently construct the derivative cytosine BE (CBE). Remarkably, the engineered CBE exhibits an unexpected activity to also edit the target strand (TS), indicating its substantially expanded editable space. We refine this activity via a focused alanine scan, establishing a nickase-CBE that preferentially install TS edits (TSminiCBE). Further engineering with a non-specific DNA binding domain yields an optimized TS-editing BE that enables in vivo base edits in mice (male). Overall, through extensive engineering of the Cas12f platform and repurposing its intrinsic dynamics, our work establishes a strand-selectable miniature CBE toolkit with strong potential for diverse applications.},
}
@article {pmid41151616,
year = {2025},
author = {Terhalle, E and Rademacher, J},
title = {[Nontuberculous Mycobacteria: Diagnostic Challenges and Individualized Therapeutic Approaches].},
journal = {Deutsche medizinische Wochenschrift (1946)},
volume = {150},
number = {22},
pages = {1360-1366},
doi = {10.1055/a-2502-6525},
pmid = {41151616},
issn = {1439-4413},
mesh = {Humans ; *Mycobacterium Infections, Nontuberculous/diagnosis/drug therapy/epidemiology/therapy ; *Nontuberculous Mycobacteria/isolation &amp; purification ; Anti-Bacterial Agents/therapeutic use ; Precision Medicine ; },
abstract = {Non-tuberculous mycobacteria (NTM) are increasingly recognized as clinically relevant pathogens, particularly in countries with a low tuberculosis incidence. Recent data from Denmark demonstrate a continuous annual rise in NTM-related pulmonary disease (NTM-PD) of 4.6% over 3 decades, with more than half of the isolates associated with true disease. Structural lung diseases such as bronchiectasis, prior tuberculosis, and chronic pulmonary conditions are major risk factors, alongside immunodeficiencies and immunosuppressive therapies. The diagnosis of NTM-PD requires a combination of clinical symptoms, radiological findings, and the microbiological confirmation. Novel diagnostic tools, such as anti-GPL IgA serology and a CRISPR-Cas-based cfDNA assay, show promise for differentiating colonization from disease and monitoring treatment response, but the sputum culture remains essential for species identification and drug susceptibility testing. Treatment is complex and species-specific, with macrolides forming the backbone of most regimens. Refractory cases, particularly those involving Mycobacterium abscessus, pose therapeutic challenges and often require multidisciplinary management. Inhaled liposomal amikacin (ALIS) has shown benefit in refractory MAC disease. Clinical decision-making must balance efficacy, tolerability, and long-term adherence, highlighting the need for individualized treatment strategies and regular monitoring. This review outlines current evidence and practical recommendations for clinicians managing NTM-PD.},
}
@article {pmid41149352,
year = {2025},
author = {Qin, Y and Xie, J and Zhen, S},
title = {CRISPR-Cas12a and DNA Tetrahedron Assemblies Amplified Fluorescence Anisotropy for the Sensitive Detection of Hepatitis B Virus DNA.},
journal = {Biosensors},
volume = {15},
number = {10},
pages = {},
pmid = {41149352},
issn = {2079-6374},
support = {22322409//National Natural Science Foundation of China/ ; 21974109//National Natural Science Foundation of China/ ; SWU-KF25011//Fundamental Research Funds for the Central Universities/ ; },
mesh = {*Hepatitis B virus/genetics ; *DNA, Viral/analysis ; *Fluorescence Polarization/methods ; Humans ; *CRISPR-Cas Systems ; *Biosensing Techniques ; Limit of Detection ; Hepatitis B ; },
abstract = {Fluorescence anisotropy (FA) has been widely used for analyzing biomolecules due to its high throughput, homogeneous detection, and strong resistance to photobleaching. However, the traditional FA method suffers from low sensitivity when the target molecules are small and rotate rapidly, often producing insignificant changes in the FA value. In this study, by combining double signal amplification through the trans-cleavage of CRISPR-Cas12a and DNA tetrahedron assemblies with a large molecular size, a new, fast, simple and highly sensitive FA method was constructed to achieve the quantitative detection of hepatitis B virus DNA (HBV-DNA). The experimental results showed that the linear range of this method was 0.5-9 nmol/L, and the detection limit (LOD = 3σ/k) was 48 pmol/L. In addition, the method demonstrated excellent selectivity and anti-interference, and it was successfully applied to detect HBV-DNA in human serum, indicating that this method has the potential for clinical diagnosis.},
}
@article {pmid41149312,
year = {2025},
author = {Zhou, Z and Cho, IH and Kadam, US},
title = {CRISPR-Cas-Based Diagnostics in Biomedicine: Principles, Applications, and Future Trajectories.},
journal = {Biosensors},
volume = {15},
number = {10},
pages = {},
pmid = {41149312},
issn = {2079-6374},
mesh = {*CRISPR-Cas Systems ; Humans ; *Biosensing Techniques ; },
abstract = {CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)-Cas (CRISPR-associated) systems, originally identified as prokaryotic adaptive immune mechanisms, have rapidly evolved into powerful tools for molecular diagnostics. Leveraging their precise nucleic acid targeting capabilities, CRISPR diagnostics offer rapid, sensitive, and specific detection solutions for a wide array of targets. This review delves into the fundamental principles of various Cas proteins (e.g., Cas9, Cas12a, Cas13a) and their distinct mechanisms of action (cis- and trans-cleavage). It highlights the diverse applications spanning infectious disease surveillance, cancer biomarker detection, and genetic disorder screening, emphasizing key advantages such as speed, high sensitivity, specificity, portability, and cost-effectiveness, particularly for point-of-care (POC) testing in resource-limited settings. The report also addresses current challenges, including sensitivity limitations without pre-amplification, specificity issues, and complex sample preparation, while exploring promising future trajectories like the integration of artificial intelligence (AI) and the development of universal diagnostic platforms to enhance clinical translation.},
}
@article {pmid41149303,
year = {2025},
author = {Zhu, Z and Ma, H and Yao, H and Yuan, Y and Miao, X and Su, S},
title = {CRISPR-Enhanced Colorimetric Aptasensor for Adenosine Triphosphate Detection Based on MoS2-Based Nanozymes.},
journal = {Biosensors},
volume = {15},
number = {10},
pages = {},
pmid = {41149303},
issn = {2079-6374},
support = {SYW2025037//the Science and Technology Program of Suzhou/ ; 2024GRFX045//the Visiting Scholar Project for Higher Vocational Colleges in Jiangsu Province/ ; TC2024JCYL23//the Science and Technology Program of Taicang/ ; 2023JXKYTD01//the Innovation Team Funds of Suzhou Chien-shiung Institute of Technology/ ; },
mesh = {Colorimetry/methods ; *Adenosine Triphosphate/analysis ; *Biosensing Techniques/methods ; *Aptamers, Nucleotide ; Molybdenum/chemistry ; Gold/chemistry ; Metal Nanoparticles/chemistry ; Disulfides/chemistry ; CRISPR-Cas Systems ; },
abstract = {As the direct energy source in organisms, accurate and simple detection of adenosine triphosphate (ATP) is of great significance. Herein, a colorimetric aptasensor for ATP determination was designed by integrating the CRISPR/Cas12a system with an aptamer, and with Prussian blue nanocube and gold nanoparticle co-functionalized MoS2 (MoS2-PBNCs-AuNPs) nanozymes. As expected, the introduced CRISPR/Cas12a system and aptamer could efficiently amplify the detection signal and improve the specific recognition ability, respectively. Meanwhile, the catalytic activity of the MoS2-PBNCs-AuNPs nanozymes can be regulated with the concentration of ATP. The high-affinity binding of ATP to the aptamer competitively inhibited aptamer-crRNA hybridization, causing fewer Cas12 proteins to be activated. As a result, the uncleaved single-stranded DNA (ssDNA) adsorbed onto the surface of nanozymes to effectively enhance their catalytic oxidation capability toward 3,3',5,5'-tetramethylbenzidine (TMB). According to this phenomenon, this CRISPR-enhanced colorimetric aptasensor can detect down to 0.14 μM ATP with high selectivity, reproducibility, and stability. In addition, acceptable recoveries and low relative standard deviations of the aptasensor for ATP determination suggest that it is promising for application in early detection of clinical-related diseases.},
}
@article {pmid41148810,
year = {2025},
author = {Liu, H and Zhang, P},
title = {Advances in β-Thalassemia Gene Therapy: CRISPR/Cas Systems and Delivery Innovations.},
journal = {Cells},
volume = {14},
number = {20},
pages = {},
pmid = {41148810},
issn = {2073-4409},
mesh = {*beta-Thalassemia/therapy/genetics ; Humans ; *Genetic Therapy/methods ; *CRISPR-Cas Systems/genetics ; Gene Editing/methods ; Animals ; *Gene Transfer Techniques ; beta-Globins/genetics ; },
abstract = {β-thalassemia is an inherited blood disorder caused by mutations in the β-globin (HBB) gene, leading to reduced or absent β-globin production, resulting in chronic anemia. While current therapies, including blood transfusions and hematopoietic stem cell transplantation, offer symptomatic relief, they are limited by complications and their limited accessibility. CRISPR-based gene editing technologies provide new therapeutic avenues by enabling the precise correction of HBB mutations or the reactivation of fetal hemoglobin (HbF) through the targeting of regulatory elements such as BCL11A. These approaches have shown promising preclinical and clinical outcomes. However, efficient and safe delivery remains a major challenge. Viral vectors offer high efficiency but raise concerns about immunogenicity and insertional mutagenesis, whereas non-viral systems such as lipid nanoparticles and engineered exosomes offer lower toxicity and modularity but face targeting limitations. This review highlights recent progress in CRISPR-based therapies for β-thalassemia and emerging delivery strategies to enhance clinical translation.},
}
@article {pmid41134871,
year = {2025},
author = {Charlier, J and Sherkatghanad, Z and Makarenkov, V},
title = {Similarity-based transfer learning with deep learning networks for accurate CRISPR-Cas9 off-target prediction.},
journal = {PLoS computational biology},
volume = {21},
number = {10},
pages = {e1013606},
doi = {10.1371/journal.pcbi.1013606},
pmid = {41134871},
issn = {1553-7358},
mesh = {*Deep Learning ; *CRISPR-Cas Systems/genetics ; Neural Networks, Computer ; Computational Biology/methods ; *Gene Editing/methods ; Humans ; Machine Learning ; },
abstract = {Transfer learning has emerged as a powerful tool for enhancing predictive accuracy in complex tasks, particularly in scenarios where data is limited or imbalanced. This study explores the use of similarity-based pre-evaluation as a methodology to identify optimal source datasets for transfer learning, addressing the dual challenge of efficient source-target dataset pairing and off-target prediction in CRISPR-Cas9, while existing transfer learning applications in the field of gene editing often lack a principled method for source dataset selection. We use cosine, Euclidean, and Manhattan distances to evaluate similarity between the source and target datasets used in our transfer learning experiments. Four deep learning network architectures, i.e. Multilayer Perceptron (MLP), Convolutional Neural Networks (CNNs), Feedforward Neural Networks (FNNs), and Recurrent Neural Networks (RNNs), and two traditional machine learning models, i.e. Logistic Regression (LR) and Random Forest (RF), were tested and compared in our simulations. The results suggest that similarity scores are reliable indicators for pre-selecting source datasets in CRISPR-Cas9 transfer learning experiments, with cosine distance proving to be a more effective dataset comparison metric than either Euclidean or Manhattan distances. An RNN-GRU, a 5-layer FNN, and two MLP variants provided the best overall prediction results in our simulations. By integrating similarity-based source pre-selection with machine learning outcomes, we propose a dual-layered framework that not only streamlines the transfer learning process but also significantly improves off-target prediction accuracy. The code and data used in this study are freely available at: https://github.com/dagrate/transferlearning_offtargets.},
}
@article {pmid41117290,
year = {2025},
author = {Ye, T and Xue, M and Xu, Y and Fan, M and Yuan, M and Yu, J and Cao, H and Hao, L and Wu, X and Yin, F and Xu, F},
title = {Inverted Tetrahedral DNA Reporters Enable Label-Free Ratiometric CRISPR Electrochemical Aptasensing of Kanamycin.},
journal = {Journal of agricultural and food chemistry},
volume = {73},
number = {43},
pages = {27694-27702},
doi = {10.1021/acs.jafc.5c06928},
pmid = {41117290},
issn = {1520-5118},
mesh = {*Electrochemical Techniques/methods/instrumentation ; *Kanamycin/analysis ; *Biosensing Techniques/methods/instrumentation ; *DNA/chemistry/genetics ; Milk/chemistry ; CRISPR-Cas Systems ; *Aptamers, Nucleotide/chemistry/genetics ; Animals ; *Anti-Bacterial Agents/analysis ; Limit of Detection ; Food Contamination/analysis ; },
abstract = {Integrating CRISPR technology with electrochemical sensing has promising potential in point-of-care testing applications. However, inappropriate immobilization of the reporter on the heterogeneous surface leads to a poor trans-cleavage efficiency. Additionally, the accuracy and reliability of electrochemical sensing still face challenges. Herein, an inverted tetrahedral DNA reporter was developed for electrochemical CRISPR aptasensing. Thiol-modified single-strand oligonucleotides were self-assembled on the edge of tetrahedral DNA nanostructures (TDNs) as a scaffold, enabling an inverted immobilization of DNA tetrahedra via the Au-S bond. The trans-cleavage activity of CRISPR/Cas12a on the single-stranded oligonucleotides resulted in the TDNs dissociating from the electrode surface. The recovery of electron transfer of potassium ferricyanide on the electrode enhances the electrochemical response, while the signal of adsorbed methylene blue on the skeleton of TDNs decreases, enabling a ratiometric signal output. As a proof of concept, the proposed inverted tetrahedral DNA reporters were employed to develop a label-free ratiometric electrochemical aptasensing method for kanamycin detection. Under the optimal conditions, as low as 0.35 pM kanamycin was detected in 50 min with a 4 orders of magnitude dynamic range from 1 pM to 10 nM. Furthermore, the practical application ability of the proposed method for kanamycin detection in a spiked milk sample was also demonstrated. This work offers a new perspective for electrochemical CRISPR sensing development.},
}
@article {pmid41052905,
year = {2025},
author = {Sun, Y and Hong, Z and Wang, W and Zhang, H and Ren, X and He, X and Kan, T and Fan, Y and Wang, C and Cao, Y and Zhang, H},
title = {Establishment of an efficient and versatile genome editing platform for L. ruthenicum.},
journal = {Plant physiology},
volume = {199},
number = {2},
pages = {},
doi = {10.1093/plphys/kiaf486},
pmid = {41052905},
issn = {1532-2548},
support = {2022BBF01001-03//Key Research &amp; Development Program of Ningxia Hui Autonomous Region/ ; 32170547//National Natural Science Foundation of China/ ; 2024AAC03123//Natural Science Foundation of Ningxia Hui Autonomous Region/ ; },
mesh = {*Gene Editing/methods ; CRISPR-Cas Systems/genetics ; Plants, Genetically Modified/genetics ; *Genome, Plant ; Promoter Regions, Genetic ; },
abstract = {Black goji berry (Lycium ruthenicum Murr.) is a valuable functional food and traditional medicinal plant owing to its rich content of anthocyanins, trace minerals, vitamins, and polysaccharides. However, limited genetic manipulation tools have hindered functional genomic studies and trait improvement in this species. In this study, we optimized the genetic transformation system for L. ruthenicum, achieving a remarkably high transformation efficiency of 95.4%. Based on this system, we developed a clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9)-mediated gene knockout approach, using the Arabidopsis U6 (AtU6) promoter to drive sgRNA expression and the cauliflower mosaic virus 35S (35S) promoter to drive Cas9 expression, achieving editing efficiencies of 68.8% at the phytoene desaturase (LrPDS) locus and 96.0% at the betaine aldehyde dehydrogenase (LrBADH2) locus. Furthermore, we established an adenine base editing (ABE) system using the ribosomal protein subunit 5A (RPS5A) promoter to drive tRNA adenine deaminase-8e (TadA-8e-nSpCas9) cassette expression, achieving an editing efficiency of 72.2% at the LrPDS locus. To broaden protospacer adjacent motif (PAM) compatibility, we introduced the PAM-relaxed variant SpRY, enabling successful A-to-G editing at an acetolactate synthase (LrALS) target site containing a noncanonical NAN PAM, with an efficiency of 5.3%. Additionally, we developed a multiplex ABE system based on the tRNA-processing strategy, which enabled simultaneous editing at 2 independent loci with an efficiency of 33.3%. Our study establishes a robust genome editing toolkit for L. ruthenicum, offering valuable tools for functional gene analysis and molecular breeding in this economically important species.},
}
@article {pmid39565542,
year = {2025},
author = {Zhou, L and Li, X and Ji, Z and Zhou, C and Yang, L and Li, Y and Fu, C and Gu, L and Zhang, S and Gao, J and Yue, P and Yu, H},
title = {Generation of Ext1 Gene-Edited Mice Model Via Dual sgRNAs/Cas9 System and Phenotypic Analyses.},
journal = {Molecular biotechnology},
volume = {67},
number = {11},
pages = {4233-4246},
pmid = {39565542},
issn = {1559-0305},
support = {32160147//National Natural Science Foundation of China/ ; YCSW2020229//Youth and Middle-aged Scientific and Technological Innovation Leading Talents Program of the Corps/ ; 2020GXNSFAA297097//Natural Science Foundation of Guangxi Zhuang Autonomous Region/ ; },
mesh = {Animals ; *N-Acetylglucosaminyltransferases/genetics/metabolism ; Exostosin 1 ; Mice ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; Disease Models, Animal ; Phenotype ; Male ; Female ; *Exostoses, Multiple Hereditary/genetics/pathology ; Frameshift Mutation ; Exons ; Humans ; },
abstract = {Hereditary multiple exostoses (HME) is an autosomal dominant skeletal disease. Genetic linkage analyses have identified that mutations in the exostosin glycosyltransferase (EXT)1 and EXT2 genes are linked to HME pathogenesis, with EXT1 mutation being the most frequent. The aim of this study was to generate a mice model with Ext1 gene editing to simulate human EXT1 mutation and investigate the genetic pathogenicity of Ext1 through phenotypic analyses. We designed a pair of dual sgRNAs targeting exon 1 of the mice Ext1 gene for precise deletion of a 46 bp DNA fragment, resulting in frameshift mutation of the Ext1 gene. The designed dual sgRNAs and Cas9 proteins were injected into mice zygotes cytoplasm. A total of 14 mice were obtained via embryo transfer, among which two genotypic chimera mice had a deletion of the 46 bp DNA fragment in exon 1 of the Ext1 gene. By hybridization and breeding, we successfully generated heterozygous mice with edited Ext1 gene (Ext[+/-]). Off-target effect analysis did not reveal off-target mutations in Ext[+/-] mice caused by the two sgRNAs used. Compared to wild-type mice, Ext[+/-] mice exhibited lower body weights. X-ray imaging showed hyperplastic bone near caudal vertebrae only in male Ext[+/-] mice, with computed tomography values approximately at 200 HU for hyperplastic bone between ribs and spine regions. Furthermore, immunohistochemical analysis revealed fewer articular chondrocytes expressing EXT1 in edited mice compared to wild-type ones. Pathological section analysis demonstrated no structural or morphological abnormalities in heart, liver, lung, or kidney tissues from Ext[+/-] mice. In conclusion, we successfully generated an accurate DNA deletion model for studying Ext1 using dual sgRNAs/Cas9 systems. In conclusion, we successfully generated precise DNA deletions in the Ext1 mice model using the dual sgRNAs/Cas9 system. In conclusion, we observed significant phenotypic changes in Ext[+/-] mice, particularly bone hyperplasia in male individuals; however, no exostosis was detected in the gene-edited mice. The introduction of a frameshift mutation into the Ext1 gene through CRISPR/Cas9 technology resulted in novel phenotypic alterations, highlighting the genetic pathogenicity of Ext1. Therefore, our Ext[+/-] mice serve as a valuable model for further biomedical investigations related to the Ext1 gene.},
}
@article {pmid41148687,
year = {2025},
author = {Nass, NM and Zaher, KA},
title = {Beyond the Resistome: Molecular Insights, Emerging Therapies, and Environmental Drivers of Antibiotic Resistance.},
journal = {Antibiotics (Basel, Switzerland)},
volume = {14},
number = {10},
pages = {},
doi = {10.3390/antibiotics14100995},
pmid = {41148687},
issn = {2079-6382},
abstract = {Antibiotic resistance remains one of the most formidable challenges to modern medicine, threatening to outpace therapeutic innovation and undermine decades of clinical progress. While resistance was once viewed narrowly as a clinical phenomenon, it is now understood as the outcome of complex ecological and molecular interactions that span soil, water, agriculture, animals, and humans. Environmental reservoirs act as silent incubators of resistance genes, with horizontal gene transfer and stress-induced mutagenesis fueling their evolution and dissemination. At the molecular level, advances in genomics, structural biology, and systems microbiology have revealed intricate networks involving plasmid-mediated resistance, efflux pump regulation, integron dynamics, and CRISPR-Cas interactions, providing new insights into the adaptability of pathogens. Simultaneously, the environmental dimensions of resistance, from wastewater treatment plants and aquaculture to airborne dissemination, highlight the urgency of adopting a One Health framework. Yet, alongside this growing threat, novel therapeutic avenues are emerging. Innovative β-lactamase inhibitors, bacteriophage-based therapies, engineered lysins, antimicrobial peptides, and CRISPR-driven antimicrobials are redefining what constitutes an "antibiotic" in the twenty-first century. Furthermore, artificial intelligence and machine learning now accelerate drug discovery and resistance prediction, raising the possibility of precision-guided antimicrobial stewardship. This review synthesizes molecular insights, environmental drivers, and therapeutic innovations to present a comprehensive landscape of antibiotic resistance. By bridging ecological microbiology, molecular biology, and translational medicine, it outlines a roadmap for surveillance, prevention, and drug development while emphasizing the need for integrative policies to safeguard global health.},
}
@article {pmid41148514,
year = {2025},
author = {Tripathi, A and Vishwakarma, K and Tripathi, S and Jadaun, JS and Nayak, AK},
title = {Utilization of MADS-Box genes for agricultural advancement: current insights and future prospects.},
journal = {Molecular biology reports},
volume = {53},
number = {1},
pages = {20},
pmid = {41148514},
issn = {1573-4978},
mesh = {*MADS Domain Proteins/genetics/metabolism ; Gene Expression Regulation, Plant/genetics ; Plant Proteins/genetics/metabolism ; *Crops, Agricultural/genetics ; CRISPR-Cas Systems ; Oryza/genetics ; *Agriculture/methods ; Solanum lycopersicum/genetics ; Phylogeny ; Plants, Genetically Modified/genetics ; Gene Editing/methods ; Genes, Plant ; },
abstract = {MADS-box genes constitute a highly conserved family of transcription factors integral to the regulation of a diverse array of plant developmental processes, encompassing floral organ specification, fruit maturation, root architecture and adaptation to abiotic stresses. These transcription factors encode proteins containing the distinctive MADS (MCM1, AGAMOUS, DEFICIENS, and SRF) domain, which mediates DNA binding and orchestrates interaction with co-regulators, thereby enabling the precise transcriptional control of developmental gene networks. Functional characterization through transgenic approaches including overexpression, knockdown, and CRISPR/Cas9-based mutagenesis-has revealed the capacity of MADS-box gene manipulation to modulate key agronomic traits, such as yield potential, as well as resilience to salinity, drought, and temperature fluctuations. In rice, targeted editing of OsMADS18 using CRISPR/Cas9 generated a substantial quantitative variation in tiller and panicle number, demonstrating the direct contribution of MADS-box gene function to biomass and yield performance. Similarly, CRISPR/Cas9-mediated disruption of the RIPENING INHIBITOR (RIN) gene in tomato (Solanum lycopersicum) underscored its central role in regulating fruit ripening, linking MADS-box gene activity to postharvest quality and development. Phylogenomic studies reveal strong conservation of MADS-box gene lineages in monocot grasses, as evidenced by clustered short internal branches, whereas eudicots, particularly Solanaceae present well-differentiated subclades, reflecting lineage-specific diversification events. Notably, network analysis highlight the high connectivity and central regulatory position of many MADS-box proteins, underlining their roles as master integrators of developmental and environmental signalling involved in both floral and vegetative transitions. A mechanistic understanding of these regulatory circuits offers translational opportunities to engineer crops with improved performance and resilience, reinforcing the pivotal role of MADS-box genes in crop improvement.},
}
@article {pmid41148377,
year = {2025},
author = {Baars, J and Kurm, V and Scholten, B and Griekspoor, Y and Lavrijssen, B and Steens, JA and Smulders, MJM and van Peer, A},
title = {On site discrimination between two closely related commercial strains of oyster mushroom using a loop-mediated isothermal amplification (LAMP) test.},
journal = {Molecular biology reports},
volume = {53},
number = {1},
pages = {17},
pmid = {41148377},
issn = {1573-4978},
mesh = {*Nucleic Acid Amplification Techniques/methods ; *Pleurotus/genetics/classification ; DNA, Fungal/genetics ; Molecular Diagnostic Techniques ; },
abstract = {BACKGROUND: Protection of the intellectual property (IP) rights on new crop varieties is important as it allows the breeding company or entity that produced the variety to earn back (part of) the investment. Infringement on the IP rights of mushroom varieties is not uncommon. In order to combat infringement of the IP rights on two strains of Oyster mushroom (SPOPPO and ALLERPO) it is important to be able to readily recognize and discriminate the two strains in commercial practice. This article describes the development of tools for the on-site identification of two closely related sporeless strains of Oyster mushroom.<br><br>METHODS AND RESULTS: To develop a reliable method of discriminating between SPOPPO and ALLERPO, we used either the LAMP technique or a modification of that technique. It allows for fast (within 30&#xa0;min) identification of the commercially used strains SPOPPO and ALLERPO with high specificity. Fast on-site answers on strain identity can be important when experiencing unexpected strain behavior or when strains are of suspect origin. Both strains are discriminated from sporulating strains by a LAMP reaction on the intact version of the msh4 gene; sporeless strains contain a msh4 gene with a large insert that renders the associated protein inactive.<br><br>CONCLUSIONS: SPOPPO and ALLERPO are distinguished from each other and other commercially used Pleurotus ostreatus strains by LAMP reactions that target genomic regions with strain specific recombinations. To our knowledge, this is the first time LAMP reactions have been developed to discriminate between Pleurotus ostreatus strains.},
}
@article {pmid41145060,
year = {2025},
author = {Monteiro Belo Dos Santos, S and Van Tricht, C and Lammertyn, J and Spasic, D},
title = {Zoonotic disease detection at the point-of-care: the best of RPA and CRISPR-Cas.},
journal = {Biosensors &amp; bioelectronics},
volume = {293},
number = {},
pages = {118139},
doi = {10.1016/j.bios.2025.118139},
pmid = {41145060},
issn = {1873-4235},
abstract = {Biosensors are increasingly crucial in detecting biomarkers for emerging zoonotic diseases at the point-of-care (POC). This imminence was recently highlighted by the deficient response during the SARS-CoV-2 pandemic. While polymerase chain reaction (PCR) is the common nucleic acid (NA) testing method for zoonotic diseases in laboratory settings, it is impractical for the POC settings due to the equipment-related cost, lack of portability and user-friendliness. Recent advances in NA amplification introduced isothermal methods, such as recombinase polymerase amplification (RPA), which is known for its low temperature (37-42 °C), short incubation time (5-20 min) and suitability for integration in miniaturized, portable, low-cost, highly sensitive diagnostic platforms. However, RPA susceptibility to false positive results steered to its combination with CRISPR-Cas12/13, leading to the rise of SHERLOCK and DETECTR. This review first explores RPA-CRISPR-Cas bioassay development as either two- or one-step. This is followed by a discussion on the integration of canonical RPA, or its combination with CRISPR-Cas, into different diagnostic platforms towards NA amplification at the POC (e.g., mobile laboratories, centrifugal, or pump-free platforms). Finally, the advantages, limitations, and outlook for POC-based diagnostics of zoonotic diseases with RPA(-CRISPR-Cas) are discussed, highlighting the need for innovative technologies to address global health challenges. While promising, many of these approaches still require further research to achieve streamlined, single-step reactions and seamless integration into diagnostic platforms. Moreover, despite two decades of RPA(-CRISPR-Cas) development, technology readiness is limited, still missing validated platforms, integrated sample preparation, and AI-powered results analysis enabling real time epidemiological monitoring.},
}
@article {pmid41144208,
year = {2026},
author = {Shaw, S and Sateriale, A and Pawlowic, MC and Vinayak, S and Brooks, CF and Byerly, JH and Striepen, B},
title = {Genetic Manipulation of Cryptosporidium parvum.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2978},
number = {},
pages = {173-185},
pmid = {41144208},
issn = {1940-6029},
mesh = {*Cryptosporidium parvum/genetics ; Animals ; Mice ; CRISPR-Cas Systems ; *Cryptosporidiosis/parasitology ; Gene Editing/methods ; Transfection/methods ; Humans ; },
abstract = {Cryptosporidium parvum can be reliably genetically manipulated using CRISPR/Cas9 driven homologous repair coupled to in vivo propagation in immunodeficient mice. This chapter will guide through foundational procedures for excystation, transfection, infection, collection, and purification of transgenic Cryptosporidium parvum. The genetic tools for Cryptosporidium research were expanded significantly over the last 5 years. While we cannot cover all advances in detail, we will highlight novel selection markers, conditional mutagenesis strategies, and genetic crosses.},
}
@article {pmid41141466,
year = {2025},
author = {Mohiuddin, M},
title = {Monitoring and Assessment of Circulating Tumor DNA in Cancers Using Ultrarapid Sensitivity as an Innovative Practice.},
journal = {Health science reports},
volume = {8},
number = {10},
pages = {e71409},
pmid = {41141466},
issn = {2398-8835},
abstract = {BACKGROUND: Liquid biopsy with circulating tumor DNA (ctDNA) has rapidly emerged as a new paradigm for assessing tumor burden, genetic heterogeneity, and therapeutic response in a real-time, noninvasive manner. However, ctDNA is often visually low (sometimes < 0.1% of the total circulating cell-free DNA), creating a significant challenge for reliable detection (especially for early-stage disease and minimal residual disease).<br><br>DISCUSSION: New technologies for structural variant (SV)-based ctDNA assays, nanomaterial-based electrochemical sensors, magnetic nano-electrode platforms, and fragment-enriched library preparation have improved sensitivity to attomolar concentrations and less in some populations. In some cancers, ctDNA may provide early evidence of recurrence (i.e., >&#x2009;1 year) before being clinically evident using traditional metrics. These technologies allow for unprecedented opportunities and sensitivity for early detection, monitoring of treatment response, and early detection of molecular recurrence. Nevertheless, a barrier remains for widespread clinical application owing to pre-analytical technique variability, analytical platform variability, cost, and the necessity of large-scale, prospective trials.<br><br>CONCLUSION: This study will analyze new innovative technology-based ultrasensitive ctDNA assay detection and future research and clinical applications for breast, colorectal, lung, lymphoid, and gastroesophageal cancers, and studies assessing ctDNA for monitoring treatment. Prospects for ctDNA detection utilizing multiplexed CRISPR-Cas ctDNA assays, microfluidic point-of-care (POC) devices, and AI-based error suppression methods may be the next horizon for ctDNA liquid biopsy technology.},
}
@article {pmid41141388,
year = {2026},
author = {Wang, T and Yu, M and Liu, P and Song, Z and Li, C and Yang, J and Liu, N},
title = {In vivo gene therapy: A strategy for mutations, degenerations, and tumors.},
journal = {Genes &amp; diseases},
volume = {13},
number = {1},
pages = {101808},
pmid = {41141388},
issn = {2352-3042},
abstract = {Gene mutations, organ function degeneration, and carcinogenesis are the primary threats to human health. Gene therapy, which involves the addition, deletion, regulation, and editing of genes, as well as the development of genetic vaccines, can potentially cure genetic mutation disorders, degenerative diseases, and cancers. Ex vivo gene therapy has recently been used to treat monogenetic mutation diseases of the hematopoietic system and cancers. However, in vivo gene therapy remains inapplicable. The primary elements of in vivo gene therapy include deoxyribonucleic acid (DNA) nucleases (e.g., zinc finger nucleases, transcription activator-like effector nucleases), CRISPR-Cas system, base editors, prime editors, and delivery vectors (e.g., viral and non-viral vehicles). According to the development of DNA nucleases and delivery vectors, in vivo gene therapy can be made available for future clinical use. The current review summarizes the development of DNA nucleases and delivery vectors for in vivo gene therapy, emphasizing recent progress.},
}
@article {pmid41139369,
year = {2025},
author = {Wei, H and Li, D and Xie, K and Lou, S and Dong, G and Guo, F and Lian, G and Pan, X and Zeng, Z and Han, N and Gao, Z and Bian, H},
title = {Creation of new rice germplasm with cross-resistance to auxin herbicides picloram and dicamba by genome editing of OsAFB4.},
journal = {TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik},
volume = {138},
number = {11},
pages = {282},
pmid = {41139369},
issn = {1432-2242},
support = {LGN21C130006//Natural Science Foundation of Zhejiang Province/ ; 32201834//Innovative Research Group Project of the National Natural Science Foundation of China/ ; },
mesh = {*Oryza/genetics/drug effects/growth &amp; development ; *Herbicide Resistance/genetics ; *Gene Editing ; *Picloram/pharmacology ; *Herbicides/pharmacology ; *Dicamba/pharmacology ; Indoleacetic Acids ; *Plant Proteins/genetics/metabolism ; CRISPR-Cas Systems ; Mutation ; Plant Breeding ; Gene Expression Regulation, Plant ; Plants, Genetically Modified ; },
abstract = {Genome Editing of OsAFB4 in rice cultivar Jiahe212 confers cross-resistance to synthetic auxin herbicides picloram and dicamba without reducing grain yield, providing a novel germplasm for herbicide-resistant rice breeding. Synthetic auxin herbicides (SAHs) are widely employed in global agriculture owing to low toxicity and high efficiency in weed management. Mutations in OsAFB4, an AUXIN SIGNALING F-BOX gene, led to specific resistance to SAH picloram in rice. However, potential application of OsAFB4 modification for resistance to other SAHs and its effect on agronomic traits in rice remain uncharacterized. In this study, we employed CRISPR/Cas9-mediated genome editing to generate OsAFB4 mutations in the elite commercial rice cultivar Jiahe212 (JH212). Hydroponic-culture experiments showed that Osafb4 mutant lines exhibited significant resistance to both SAHs: picloram and dicamba. Promoter activity assays using a pOsAFB4:GUS reporter line revealed that SAH treatments induced significant upregulation of GUS expression specifically in shoot apices. Under picloram or dicamba treatment, expressions of OsIAA1, OsIAA3, OsIAA9 and OsIAA20 were significantly upregulated in wild-type plants, while remarkably suppressed in Osafb4 mutants, revealing a critical role for OsAFB4 in regulating auxin-responsive IAA genes during OsAFB4-mediated sensitivity to SAH. Selected for field trials, the Cas9-free Osafb4-13 mutant line exhibited robust resistance to both picloram and dicamba without reducing grain yield compared to untreated controls. This study provides a new cross-herbicide-resistant rice germplasm without reducing grain yield by targeted editing of OsAFB4.},
}
@article {pmid41104823,
year = {2025},
author = {Li, X and Ma, J and Luo, Y and Zhu, D and Wang, L and Su, S},
title = {Enhancing Performance in Electrochemical Early Diagnosis of African Swine Fever Based on CRISPR-Responsive DNA Nanoflowers.},
journal = {Analytical chemistry},
volume = {97},
number = {42},
pages = {23401-23408},
doi = {10.1021/acs.analchem.5c04188},
pmid = {41104823},
issn = {1520-6882},
mesh = {*African Swine Fever Virus/genetics/isolation &amp; purification ; *African Swine Fever/diagnosis ; Animals ; *Electrochemical Techniques/methods ; *Biosensing Techniques/methods ; *CRISPR-Cas Systems ; Swine ; *Nanostructures/chemistry ; *DNA, Viral/analysis/genetics ; Early Diagnosis ; Limit of Detection ; Nucleic Acid Amplification Techniques ; *DNA/chemistry ; },
abstract = {Accurate, ultrasensitive, and fast detection of the African swine fever virus (ASFV) can efficiently prevent its spread and reduce the losses. Herein, an electrochemical biosensor was designed for high-performance detection of ASFV DNA by coupling with CRISPR technology and signal amplification technology. Porous DNA nanoflowers (DNFs) were prepared by rolling circle amplification (RCA), which was preconjugated Cas12a-crRNA complex to improve detection sensitivity, shorten detection time, and simplify detection steps. In the presence of ASFV DNA, the trans-cleavage activity of Cas12a was activated, degrading DNFs into DNA fragments and causing a significant electrochemical signal change. Combined with the CRISPR-Cas12a system, the detection limit of the DNF-amplified biosensor (3.57 aM) is 3 orders of magnitude lower than that of the conventional RCA-amplified biosensor (2.90 fM). Moreover, the designed electrochemical biosensor showed excellent reproducibility, storage stability, and practical analysis ability, suggesting that it has a promising application in the early diagnosis of African swine fever (ASF).},
}
@article {pmid41084819,
year = {2025},
author = {Gao, Z and Yang, X and Ren, X and Ma, H and Wu, D and Du, Y and Fan, Q and Ma, Q and Wei, Q},
title = {Ultrasensitive Detection of m[6] A-Modified RNA Using CRISPR/Cas12a-Integrated Iontronic Biosensor with Hydrophobized Nanochannels: Toward Early Cancer Diagnosis by Machine Learning.},
journal = {Analytical chemistry},
volume = {97},
number = {42},
pages = {23609-23621},
doi = {10.1021/acs.analchem.5c05377},
pmid = {41084819},
issn = {1520-6882},
mesh = {*Biosensing Techniques/methods ; Humans ; *Machine Learning ; *Adenosine/analogs &amp; derivatives/analysis/chemistry ; *CRISPR-Cas Systems ; *Early Detection of Cancer/methods ; *RNA, Long Noncoding/genetics/analysis ; Hydrophobic and Hydrophilic Interactions ; *Neoplasms/diagnosis ; Limit of Detection ; Aluminum Oxide/chemistry ; },
abstract = {N[6] -methyladenosine (m[6] A), the most prevalent internal modification in eukaryotic RNAs, has emerged as a focal point of intensive research in recent years owing to its pivotal regulatory roles in carcinogenesis, progression, and metastasis. However, conventional methods for site-specific detection of m[6] A modifications are plagued by operational complexity, pose challenges for quantitative assessment of methylation levels, and exhibit elevated false-positive rates, severely limiting their utility in clinical and mechanistic studies. In this study, we engineered an ultrasensitive iontronic biosensor leveraging a hydrophobized anodic aluminum oxide (AAO) nanochannel platform, synergistically integrating the precise target recognition capability of the CRISPR/Cas12a system with the efficient signal amplification of the clamped hybridization chain reaction (CHCR). This integration enables ultrasensitive and specific detection of m[6] A-modified RNA with a low detection limit of 32 aM. Validation experiments targeting MALAT1 and HOTAIR lncRNAs demonstrated that the sensor achieves exceptional specificity in qualitative analysis of m[6] A modifications. Furthermore, combinatorial detection of these two lncRNAs enables robust discrimination between cancer patients and healthy individuals. Through in-depth mining of latent data patterns via machine learning, the random forest (RF) model yielded a cancer diagnostic accuracy of 96.7%. This study establishes a novel and potent paradigm for early cancer diagnosis, with far-reaching implications for epitranscriptomic research and clinical translation.},
}
@article {pmid41081763,
year = {2025},
author = {Wang, X and Feng, S and Chen, H and Zhou, B and Fan, T and Qin, Y and Zhao, L and Jiang, Y and Chen, Y},
title = {Development of an Aptamer/CRISPR-Cas12a-Based Dual-Modal Biosensor for Fusobacterium nucleatum Detection in Non-Invasive Colorectal Cancer Screening.},
journal = {Analytical chemistry},
volume = {97},
number = {42},
pages = {23360-23369},
doi = {10.1021/acs.analchem.5c04132},
pmid = {41081763},
issn = {1520-6882},
mesh = {*Fusobacterium nucleatum/isolation &amp; purification ; *Colorectal Neoplasms/diagnosis/microbiology ; *Biosensing Techniques/methods ; Humans ; *CRISPR-Cas Systems ; *Aptamers, Nucleotide/chemistry ; *Early Detection of Cancer/methods ; Feces/microbiology ; Limit of Detection ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {Colorectal cancer (CRC) is the third most common cancer and leading cause of cancer-related deaths worldwide. However, current CRC screening methods are complex, invasive, and tend to exhibit low sensitivity. Recent evidence has highlighted gut microbiota dysbiosis, especially elevated Fusobacterium nucleatum levels, as a promising biomarker for CRC. In this study, a sensitive and specific detection platform was developed for F. nucleatum by combining a highly specific aptamer with rolling circle amplification (RCA) and the CRISPR/Cas12a technology. The aptamer enables specific target recognition, while RCA amplifies the target signal, and the Cas12a-mediated cleavage of a fluorescence-quenching substrate generates a quantifiable fluorescence or grayscale signal. Using a microplate reader, this assay achieved a limit of detection (LOD) of 3.68 CFU/mL; furthermore, by incorporating smartphone-assisted ImageJ grayscale analysis, it elevated the LOD to 4.30 CFU/mL, thereby enabling a dual-mode output along with on-site applicability. Additionally, the strong correlation between the two signals allowed for mutual validation. Upon application to clinical fecal samples, the developed method sensitively distinguished CRC patients from healthy controls, and its results correlated with the quantitative polymerase chain reaction results. This triple-synergistic platform, integrating aptamer specificity, RCA amplification, and CRISPR/Cas12a sensitivity, enables the noninvasive, ultrasensitive detection of F. nucleatum, supporting early CRC screening, prognosis monitoring, and microbiome-targeted therapy. Moreover, this approach overcomes the challenges of detecting low-abundance bacteria in early stage CRC and advances the precision of microbiome-based diagnostics for CRC.},
}
@article {pmid40905611,
year = {2025},
author = {Hu, Y and Yan, H and Zhang, Y and Yu, Q and Xue, T and Zhang, X and Zeng, Q and Yang, H and Xia, X and Xu, Y and Deng, R and Li, J},
title = {In-Field Molecular Diagnostics of Plant Pathogens Using Bioluminescent CRISPR-Guided Caspase Assay.},
journal = {Angewandte Chemie (International ed. in English)},
volume = {64},
number = {44},
pages = {e202508870},
doi = {10.1002/anie.202508870},
pmid = {40905611},
issn = {1521-3773},
support = {2023YFB3208302//National Key Research and Development Program of China/ ; //New Cornerstone Investigator Program/ ; 2025NSFTD0001//Sichuan Science and Technology/ ; 22522408//National Natural Science Foundation of China/ ; 22074100//National Natural Science Foundation of China/ ; },
mesh = {*Plant Diseases/microbiology ; *Caspases/metabolism ; *Luminescent Measurements/methods ; CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {In-field molecular diagnostics of plant pathogens are critical for crop disease management and precision agriculture, but tools are still lacking. Herein, we present a bioluminescent molecular diagnostic assay capable of detecting viable pathogens directly in minimally processed plant samples, enabling rapid and precise in-field crop disease diagnosis. The assay, called bioluminescent craspase diagnostics (BioCrastics), leverages newly discovered RNA-activated protease of CRISPR (Craspase) with enzymatic luminescence to generate a cascaded amplification, thus bypasses nucleic acid purification and amplification while achieving sub-nanogram sensitivity for fungal pathogens. Using wheat stripe rust as a proof of concept, we demonstrate direct pathogen detection in crude leaf homogenates within 40 min, early identification of infections 6 days prior to symptom emergence. Notably, the assay, via targeting pathogenic RNAs, specifically quantifies viable fungi, overcoming false positives from dead pathogens-a limitation of PCR-based methods that impairs disease risk assessment. Featuring simplified sample processing, portable detection, and species-specific accuracy, BioCrastics establishes a field-deployable tool that bridges the gap between laboratory-level precision and on-farm diagnostic needs for crop disease management.},
}
@article {pmid41138779,
year = {2025},
author = {Afresham, S and Khan, MK and Mughal, MAS and Mehmood, MS and Ali, S and Bashir, M and Abbas, Z and Azeem, A and Ahmed, W and Imran, M and Abbas, RZ and Sindhu, ZU and Sajid, MS},
title = {Recent Advancements in the Diagnosis of Parasitic Diseases.},
journal = {Molecular and biochemical parasitology},
volume = {},
number = {},
pages = {111706},
doi = {10.1016/j.molbiopara.2025.111706},
pmid = {41138779},
issn = {1872-9428},
abstract = {Parasitic infections present a significant health risk to the public, affecting millions of people, particularly in underdeveloped and developing countries. In developing countries, these infections are also responsible for causing significant economic challenges due to elevated healthcare expenditure. Accurate diagnosis and effective treatment methods are essentially required to combat this global issue. For decades, traditional diagnostic methods such as microscopy, serological testing, histopathology, and culturing have been used for the diagnosis of these parasitic infections. While these methods can be effective and helpful in many ways, they often consume a lot of time, require an elevated level of expertise, and have limited applications particularly in endemic regions having issues like poor infrastructure and limited access to healthcare facilities. This review aims to highlight the urgent need for a revolution to replace these conventional techniques with more affordable, quick, and field-adjustable tools such as rapid diagnostic tests (RDTs) and molecular methods and provides a comprehensive picture of advanced diagnostic tools used in the identification of parasites. With the advancements in science and technology, molecular methods such as Polymerase chain reaction, Next generation sequencing, and isothermal loop-mediated amplification have remarkably enhanced the sensitivity and accuracy of parasite detection and identification. The range of these diagnostic methods has further extended by advanced serological methods, imaging techniques, and immunological methods. Moreover, the innovations in nanotechnology, CRISPR-Cas methods, and multi-omics techniques for identification of parasite DNA, antigens, metabolites, and host responses are invaluable for diagnostic accuracy, comprehensive understanding of parasite biology, and for the discovery of new therapeutic targets and diagnostic biomarkers. However, further research and developments are required for an effective and long-lasting impact of these advancements.},
}
@article {pmid41138228,
year = {2025},
author = {Hodge, CA and Donegan, NP and Armstrong, DA and Hayden, MS and Howell, AL},
title = {Enhanced cleavage of genomic CCR5 using CASX2[Max].},
journal = {RNA biology},
volume = {22},
number = {1},
pages = {1-18},
doi = {10.1080/15476286.2025.2577449},
pmid = {41138228},
issn = {1555-8584},
mesh = {Humans ; *CRISPR-Cas Systems ; *Receptors, CCR5/genetics/metabolism/chemistry ; *Gene Editing/methods ; RNA, Guide, CRISPR-Cas Systems/genetics ; HEK293 Cells ; },
abstract = {Development of novel CRISPR/Cas systems enhances opportunities for gene editing to treat infectious diseases, cancer, and genetic disorders. CasX2 (PlmCas12e) belongs to the class II CRISPR system derived from Planctomycetes, a non-pathogenic bacterium present in aquatic and terrestrial soils and offers several advantages as a potential therapeutic CRISPR system over Streptococcus pyogenes Cas9 (SpCas9) and Staphylococcus aureus Cas9 (SaCas9). These advantages include its smaller size, distinct protospacer adjacent motif (PAM) requirements, staggered cleavage cuts that promote homology-directed repair, and the absence of pre-existing immunity in humans. We compared the cleavage efficiency and double-stranded break repair characteristics between CasX2 and CasX2[Max], a recently generated CasX2 variant with three amino acid substitutions, for targeting CCR5, a gene that encodes the CCR5 receptor important for HIV-1 infection. Two single guide RNAs (sgRNAs) were designed that flank the 32 bases deleted in the natural CCR5 ∆32 mutation. Nanopore sequencing demonstrated that CasX2 using sgRNAs with spacers of 17 nucleotides (nt), 20 nt or 23 nt in length were ineffective at cleaving genomic CCR5. In contrast, CasX2[Max] using sgRNAs with 20 nt and 23 nt spacer lengths, enabled cleavage of genomic CCR5. Structural modelling indicated that two of the CasX2[Max] amino acid substitutions enhanced sgRNA-DNA duplex stability, while the third improved DNA strand alignment within the catalytic site. These structural changes likely underlie the increased activity of CasX2[Max] in cellular gene excision. In sum, CasX2[Max] consistently outperformed native CasX2 across all assays and represents a superior gene-editing platform for therapeutic applications.},
}
@article {pmid41137488,
year = {2025},
author = {Kang, YW and Park, HH},
title = {The anti-CRISPR protein AcrIE8.1 inhibits the type I-E CRISPR-Cas system by directly binding to the Cascade subunit Cas11.},
journal = {FEBS letters},
volume = {},
number = {},
pages = {},
doi = {10.1002/1873-3468.70201},
pmid = {41137488},
issn = {1873-3468},
support = {2024//Chung-Ang University/ ; RS-2025-02316334//National Research Foundation of Korea (NRF)/ ; },
abstract = {CRISPR-Cas systems provide adaptive immunity to bacteria by recognizing and destroying foreign genetic elements. The type I-E CRISPR-Cas system utilizes a multi-subunit Cascade complex to detect target DNA and recruit the Cas3 nuclease for degradation. To overcome this defense, bacteriophages have evolved anti-CRISPR (Acr) proteins that inhibit various steps of the CRISPR interference pathway. Here, we determined the crystal structure of AcrIE8.1, an uncharacterized Acr, revealing it binds to Cas11, a Cascade subunit, to disrupt function. AcrIE8.1 has a compact fold with a defined Cas11-binding interface, suggesting a unique inhibitory mechanism among AcrIE proteins. These findings highlight Cas11 as a critical target for Acr-mediated immune evasion. Impact statement Through a combination of structural and biochemical analyses, we demonstrate that AcrIE8.1 directly binds to the Cas11 subunit of the Cascade complex to inhibit the CRISPR-Cas system. This represents a novel inhibitory strategy not previously observed among AcrIE proteins.},
}
@article {pmid41137484,
year = {2025},
author = {Lee, SY and Park, HH},
title = {Investigating the molecular mechanisms underlying the anti-CRISPR function of AcrIIA13b protein.},
journal = {The FEBS journal},
volume = {},
number = {},
pages = {},
doi = {10.1111/febs.70304},
pmid = {41137484},
issn = {1742-4658},
support = {RS-2025-02316334//NRF-Korea/ ; },
abstract = {The CRISPR-Cas systems of adaptive immunity in bacteria and archaea provide resistance against phages and other mobile genetic elements. Counteractive anti-CRISPR (Acr) proteins in phages and archaeal viruses impede these CRISPR-Cas systems. Although CRISPR-Cas systems have revolutionized genome editing, potential off-target events remain a safety concern. Hence, a thorough comprehension of the structural and molecular basis of diverse Acrs is imperative to unravel the fundamental mechanisms governing CRISPR-Cas regulation. Here, we present the structure of AcrIIA13b from Staphylococcus haemolyticus and analyze its structural and functional features to reveal the molecular basis underlying the inhibition of Cas9 by AcrIIA13b. Our structural analysis shows that AcrIIA13b eliminates the cleavage activity of Staphylococcus aureus Cas9 (SauCas9) by blocking the PAM-binding region of Cas9 so that Cas9 cannot recognize the target DNA. In addition, we demonstrate that the 15 amino acid residues at the N terminus of AcrIIA13b, which were revealed to be important for its dimerization, are critical for its inhibitory activity against Cas9. Our findings shed light on the molecular basis of AcrIIA13b-mediated CRISPR-Cas inhibition and provide valuable insights into the arms race between bacteria and phages.},
}
@article {pmid41136894,
year = {2025},
author = {Liu, H and Zhu, G and Chen, L and Ye, H and Zhang, Y and Han, G},
title = {Machine learning prediction of bacterial optimal growth temperature from protein domain signatures reveals thermoadaptation mechanisms.},
journal = {BMC genomics},
volume = {26},
number = {1},
pages = {957},
pmid = {41136894},
issn = {1471-2164},
support = {32172769//National Natural Science Foundation of China/ ; },
abstract = {UNLABELLED: Cultivating the vast majority of uncultured microbes requires knowledge of their physiological preferences, particularly optimal growth temperature (OGT). We present a machine learning approach that utilizes protein domain frequencies from bacterial genomes to predict OGT across a wide continuous range (1–83 °C). Our Random Forest model, trained on a dataset of 1,498 genomes, achieved high predictive accuracy (R[2]=0.853 on test data, 82.4% of predictions within a ± 10 °C error margin), substantially advancing current capabilities and offering a practical tool to guide cultivation experiments. Analysis of the model identified key protein domain signatures associated with thermal adaptation. The enrichment of domains related to polyamine metabolism, the tRNA methyltransferase family, and CRISPR-Cas systems was positively correlated with higher OGTs, providing genomic evidence for their roles in thermotolerance. Conversely, domains involved in redox homeostasis, transport, and nucleic acid binding were more abundant at lower temperatures. These findings not only facilitate targeted cultivation efforts but also deepen our understanding of the molecular strategies bacteria employ to thrive across diverse thermal niches.<br><br>SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s12864-025-12162-z.},
}
@article {pmid40618724,
year = {2025},
author = {Botkin, JR and Curtin, SJ},
title = {CRISPR-Cas9 Mutagenesis and Gene Overexpression to Enhance Resistance to Ascochyta medicaginicola in Medicago truncatula.},
journal = {Molecular plant-microbe interactions : MPMI},
volume = {38},
number = {5},
pages = {751-761},
doi = {10.1094/MPMI-05-25-0053-R},
pmid = {40618724},
issn = {0894-0282},
mesh = {*Medicago truncatula/microbiology/genetics/immunology ; *CRISPR-Cas Systems/genetics ; *Plant Diseases/microbiology/immunology/genetics ; *Ascomycota/physiology ; *Disease Resistance/genetics ; Mutagenesis ; Gene Expression Regulation, Plant ; Plant Proteins/genetics/metabolism ; Plants, Genetically Modified ; },
abstract = {Alfalfa (Medicago sativa), the most widely cultivated forage legume globally, is vulnerable to Ascochyta medicaginicola, the fungus causing spring black stem and leaf spot (SBS) disease, which significantly reduces yield. SBS disease also affects Medicago truncatula, a diploid model legume with extensive genetic resources, including susceptible and resistant accessions. Using comparative genomics, four candidate genes for disease resistance were identified, MtTCAR1, MtPHO2A, MtCPR1-like, and MtPAM16. CRISPR/Cas9 mutagenesis was applied to generate independent mutant plants in the R108 accession, and disease resistance was evaluated by a detached leaf qPCR-based pathogen assay. MtCPR1-like mutant plants exhibited a 34% reduction in pathogen biomass, along with variable constitutive expression of pathogenesis-related genes. Additionally, a fifth candidate gene, MtKCS12, identified through transcriptomic analysis, was overexpressed in transformed plants, resulting in a 71.4 to 80.9% reduction in pathogen biomass compared with wild-type segregants. This study validates gene editing and transgenic approaches for improving SBS disease resistance in M. truncatula, and future research will focus on applying these strategies to enhance resistance in economically important alfalfa. [Formula: see text] Copyright © 2025 The Author(s). This is an open access article distributed under the CC BY 4.0 International license.},
}
@article {pmid41136797,
year = {2025},
author = {Basarali, MK and Daemi, A and Tahiraga, RG and Özbolat, G and Hooshiar, MH and Shirazi, MSR and Döğüş, Y},
title = {Artificial intelligence-driven epigenetic CRISPR therapeutics: a structured multi-domain meta-analysis of therapeutic efficacy, off-target prediction, and gRNA optimization.},
journal = {Functional &amp; integrative genomics},
volume = {25},
number = {1},
pages = {223},
pmid = {41136797},
issn = {1438-7948},
mesh = {*Artificial Intelligence ; Humans ; *Gene Editing/methods ; *CRISPR-Cas Systems ; *RNA, Guide, CRISPR-Cas Systems/genetics ; *Epigenesis, Genetic ; *Genetic Therapy/methods ; },
abstract = {CRISPR-based epigenetic editing enables reversible regulation of gene expression without permanent DNA modification. The integration of artificial intelligence (AI) enhances guide RNA (gRNA) design, off-target prediction, and delivery optimization. We conducted a systematic review and meta-analysis (2015-2025) in accordance with PRISMA 2020 guidelines to evaluate the impact of AI on the precision, safety, and therapeutic efficacy of epigenetic CRISPR tools. From 540 screened records, 58 studies met inclusion criteria, of which 41 provided extractable quantitative data for meta-analysis and 17 contributed to qualitative synthesis. Random-effects models, subgroup analyses, and bias assessments were applied. Pooled analyses demonstrated strong positive effects across three domains: therapeutic efficacy (SMD = 1.67), gRNA optimization (SMD = 1.44), and off-target prediction (AUC = 0.79). Publication bias was minimal, and subgroup analyses indicated the strongest impact in therapeutic applications. Deep learning models were consistently associated with higher effect sizes. Qualitative synthesis revealed trends in interpretable AI, omics integration, and delivery innovations, underscoring AI's role in safer and more precise CRISPR editing. Overall, AI significantly improves the precision and therapeutic performance of CRISPR-based epigenetic tools, with the strongest effects observed in therapeutic efficacy, supporting their potential for personalized gene editing.},
}
@article {pmid41136394,
year = {2025},
author = {Chow, JT and Desjardins, A and Lee, DKC and Grigore, IA and Lee, L and Fu, NJ and Chau, S and Lee, BY and Gabra, MM and Salmena, L},
title = {A microRNA CRISPR screen reveals microRNA-483-3p as an apoptotic regulator in prostate cancer cells.},
journal = {Cell death &amp; disease},
volume = {16},
number = {1},
pages = {752},
pmid = {41136394},
issn = {2041-4889},
mesh = {*MicroRNAs/genetics/metabolism ; Humans ; *Prostatic Neoplasms/genetics/pathology/metabolism ; Male ; *Apoptosis/genetics ; Cell Line, Tumor ; Gene Expression Regulation, Neoplastic ; *CRISPR-Cas Systems/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Cell Survival/genetics ; },
abstract = {The development of traditional protein-targeted cancer therapies is a slow and arduous process, often taking years or even decades. In contrast, RNA-based therapies targeting crucial microRNA (miRNA) offer a faster alternative due to the sequence-specific nature of miRNA inhibitor binding. This, combined with the capacity of individual miRNA to influence multiple cellular pathways, makes these small RNA attractive targets for cancer therapy. While miRNA are known to be dysregulated in prostate cancer (PCa), identifying their individual contributions to disease progression and the identification of therapeutically actionable miRNA targets in PCa has been challenging due to limited profiling and lack of screening tools. To address this need, we developed miRKOv2, a miRNA-only CRISPR knockout library enabling systematic, genome-wide loss-of-function screens to identify miRNA essential for PCa cell survival. Our screens uncovered 70 potential essential miRNA candidates, with miR-483 demonstrating the most significant impact on PCa cell viability. Functional characterization revealed that miR-483 disruption potentiated apoptosis in PCa cell lines. Mechanistically, we uncovered a novel regulatory axis wherein miR-483-3p directly modulates a BCLAF1/PUMA/BAK1 apoptotic signaling network, highlighting its critical role in maintaining PCa cell survival. Our findings provide novel insights into the complex regulatory role of miRNA in PCa progression and offer a potential therapeutic strategy for targeting miRNA-mediated pathways in metastatic disease.},
}
@article {pmid41135510,
year = {2025},
author = {Yang, Q and Sun, Y and Sun, L and Chi, T and Chen, Z},
title = {Cryo-EM structure of the RfxCas13d-crRNA-off-target-RNA complex.},
journal = {Structure (London, England : 1993)},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.str.2025.09.010},
pmid = {41135510},
issn = {1878-4186},
abstract = {The CRISPR-Cas system is crucial for the adaptive immune response of prokaryotes and has been widely applied for genetic engineering. Cas13d, a type VI-D CRISPR-Cas effector, functions as RNA-guided ribonuclease and has been engineered for programmable RNA editing, which is a commonly used, active, and well-characterized small type VI editor. Here, we determined cryoelectron microscopy (cryo-EM) structures of Ruminococcus flavefaciens Cas13d in a RfxCas13d-crRNA-off-target-RNA ternary complex and RfxCas13d-crRNA binary complex at 3.10 and 3.13 Å resolution. The ternary complex consists of RfxCas13d, crRNA, and a captured short off-target ssRNA at a complex state of binding proximal mismatched RNA. RfxCas13d undergoes conformational changes with or without the off-target RNA, but the catalytic sites remain unchanged. Mg[2+] aids in stabilizing the crRNA repeat region structure, which may be crucial for RNA binding. This discovery provides the foundation for developing RfxCas13d as a mature tool and offers a framework for advancing transcriptome engineering.},
}
@article {pmid41134772,
year = {2025},
author = {Xu, X and Zhu, L and Xu, X and Chen, J and Du, X and Zhu, L and Yu, S and Qian, L and Jiang, X and Zhou, L and Dong, Y and Wang, Y and Huang, Y and Wang, Y},
title = {TSSKL is essential for sperm mitochondrial morphogenesis and male fertility in moths.},
journal = {PLoS genetics},
volume = {21},
number = {10},
pages = {e1011914},
pmid = {41134772},
issn = {1553-7404},
mesh = {Animals ; Male ; *Spermatozoa/metabolism ; *Moths/genetics ; *Mitochondria/genetics/metabolism ; *Insect Proteins/genetics/metabolism ; Fertility/genetics ; Bombyx/genetics ; Female ; Infertility, Male/genetics ; Morphogenesis/genetics ; Testis/metabolism ; CRISPR-Cas Systems ; },
abstract = {Sperm deliver male genomic DNA to the ovum, playing a pivotal role in sexual reproduction across the animal kingdom. The molecular regulation of sperm morphogenesis has consequently become a focal point of genetic research, with dual implications for both reproductive medicine and sustainable agriculture. Here, we characterize the functional role of the testis-specific serine/threonine protein kinase-like (TSSKL) gene in the model lepidopteran insect Bombyx mori and the globally destructive crop pest Plutella xylostella. RNA-seq and qPCR analyses revealed TSSKL's testis-specific expression pattern. Using CRISPR/Cas9-mediated mutagenesis, we demonstrate that TSSKL knockout induces complete male sterility, while female fertility remains unaffected compared to wild-type. Fluorescence microscopy and ultrastructural analyses revealed that TSSKL deletion leads to severe morphological defects in both eupyrene and apyrene sperm, accompanied by impaired mitochondrial dynamics and aberrant autophagy. Comparative transcriptome and functional analyses linked these phenotypes to dysregulated energy metabolism pathways. Crucially, this sterility phenotype is conserved in P. xylostella, recapitulating the findings in B. mori. Our study demonstrates that TSSKL is crucial for male fertility, coordinating both structural and metabolic aspects of sperm development. Beyond advancing fundamental knowledge of insect reproductive biology, this work also identifies TSSKL as an ideal target for lepidopteran pest control through precision sterility induction.},
}
@article {pmid41133737,
year = {2025},
author = {Devine, R and Noble, K and Wilkinson, B and Hutchings, M},
title = {Microbe Profile: Streptomyces formicae KY5: an ANT-ibiotic factory.},
journal = {Microbiology (Reading, England)},
volume = {171},
number = {10},
pages = {},
pmid = {41133737},
issn = {1465-2080},
mesh = {*Streptomyces/genetics/metabolism/isolation &amp; purification/classification ; *Ants/microbiology ; Multigene Family ; Anti-Bacterial Agents/biosynthesis ; Animals ; Antifungal Agents/metabolism/pharmacology ; Genome, Bacterial ; Secondary Metabolism/genetics ; Biosynthetic Pathways/genetics ; CRISPR-Cas Systems ; Gene Editing ; },
abstract = {Streptomyces formicae KY5 was isolated from a Tetraponera penzigi plant-ant nest. It is primarily known for its production of the formicamycins, antibiotics with potent activity against Gram-positive pathogens including methicillin-resistant Staphylococcus aureus, and additionally produces an antifungal compound that inhibits multi-drug-resistant fungal pathogens including Lomentospora prolificans. S. formicae is genetically tractable using CRISPR-Cas9 gene editing, allowing for detailed analysis of the formicamycin biosynthetic gene cluster. AntiSMASH analysis predicts the genome to encode at least 45 secondary metabolite biosynthetic gene clusters, many of which appear to encode novel compounds. Current research efforts are focussing on characterising the regulation of secondary metabolism at a global level in order to switch on pathways that are not typically expressed under standard laboratory conditions with the aim of identifying novel antimicrobials.},
}
@article {pmid41132368,
year = {2025},
author = {Morais, C and Costa, SS and Hanke, D and Santos, A and Krüger-Haker, H and Pomba, C and Feßler, AT and Schwarz, S and Couto, I},
title = {Genomic analysis of the Staphylococcus pseudintermedius mobilome associated with antimicrobial resistance.},
journal = {Frontiers in microbiology},
volume = {16},
number = {},
pages = {1640322},
pmid = {41132368},
issn = {1664-302X},
abstract = {The increasing antimicrobial resistance (AMR) in Staphylococcus pseudintermedius causing skin and soft-tissue infections (SSTIs) in companion animals is a public health concern. The aim of this study was to verify if mobile genetic elements (MGEs), in particular plasmids, are related to the carriage of AMR genes among circulating and clinically relevant S. pseudintermedius. In total, 56 S. pseudintermedius, representing predominant and emerging clonal lineages associated with SSTIs in dogs and cats collected in Lisbon (Portugal), were subjected to plasmid DNA extraction and digestion with EcoRI and XbaI. Each unique restriction pattern was assigned to a plasmid profile. A subset of 17 strains was further selected for hybrid whole genome sequencing (WGS) on Oxford Nanopore MinION and Illumina MiSeq platforms. Thirty-one of the 56 S. pseudintermedius strains carried one or more plasmid(s), mostly of small or medium sizes, corresponding to eight plasmid profiles. Two of the identified plasmids carried AMR determinants; plasmid pSP-G3C4, isolated from ST71 strains, carried the tetracycline resistance gene tet(K) and plasmid pSP5912, isolated from a ST2061 strain, harbored the qacG biocide resistance gene. Other AMR determinants were detected as part of MGEs integrated into the bacterial chromosomal DNA, namely Tn552, Tn552-like, Tn553, Tn916, Tn5405-like, Tn5801, Tn5801-like GI6287 and pRE25-like elements. In addition, a new chromosomal cassette, carrying fusC, was identified in a ST1183 strain. The 12 methicillin-resistant S. pseudintermedius studied carried staphylococcal cassette chromosome mec (SCCmec) type III (n = 5), SCCmec type IVg (n = 3), SCCmec NA45 (n = 1), ΨSCCmec 57395 (n = 1), the recently described cassettes SCCmec 7017-61515 (n = 1), or SCCmec type V(T)SL/154 (n = 1). Most strains carried intact prophages without AMR determinants. Intact restriction-modification systems were detected in 12 out of the 17 strains and CRISPR/Cas in five strains, four of which were methicillin-susceptible. The results of this study suggest that the AMR content in S. pseudintermedius is mainly related to MGEs integrated into the chromosomal DNA rather than located on plasmids. These results provide important insights that may lead to a better understanding of multidrug resistance in S. pseudintermedius towards improved SSTIs treatment in companion animals.},
}
@article {pmid41129856,
year = {2025},
author = {Zhou, J and Ren, XM and Gao, J and Wu, RP and Chen, L and Li, Z},
title = {Amplification-free detection of mycoplasma pneumoniae via CRISPR-Cas12a and deep learning-optimized crRNAs on a lateral flow platform.},
journal = {Journal of pharmaceutical and biomedical analysis},
volume = {268},
number = {},
pages = {117196},
doi = {10.1016/j.jpba.2025.117196},
pmid = {41129856},
issn = {1873-264X},
abstract = {Accurate and rapid diagnosis of Mycoplasma pneumoniae infection is essential for reducing its significant health burden. An amplification-free CRISPR-Cas12a-mediated detection platform has been developed, incorporating a deep learning-optimized crRNA library (CCDLCL) targeting conserved regions of the MP P1 gene. The system enables visual readout via lateral flow strips, supporting its potential as a point-of-care testing (POCT) nucleic acid testing strategy. Through computational design and screening, 16 highly active crRNAs were identified from an initial set of over 50 candidates. Combinatorial use of these crRNAs demonstrated synergistic enhancement of fluorescence signal intensity and reaction kinetics. Compared to single-crRNA assays, the multiplexed crRNA library improved sensitivity by 16.8-fold, achieving a limit of detection (LOD) of 0.15 pM, and reduced time to signal saturation by 30 %. When deployed on lateral flow strips, the assay exhibited a tenfold increase in visual detection sensitivity, with a LOD of 100 pM. Clinical evaluations confirmed high specificity-showing no cross-reactivity with SARS-CoV-2, hepatitis B virus (HBV), or human genomic DNA-and over 95 % agreement with standard clinical results without target pre-amplification, delivering outcomes within 45 min. This study establishes a deep learning-facilitated crRNA design framework and a novel crRNA library-based detection system, offering a feasible approach for POCT nucleic acid testing in resource-limited settings and paving the way for streamlined clinical translation of CRISPR-Cas diagnostics.},
}
@article {pmid41125583,
year = {2025},
author = {Yang, J and Li, H and Li, M and Song, R and Shen, T and Wang, G and Xu, D and Hao, M and Jia, A and Rehman, SU and Hua, L and Liang, Y and Chi, C and Lan, C and Deng, XW and Dubcovsky, J and Song, B and Wang, X and Chen, S},
title = {Genome-assisted identification of wheat leaf rust resistance gene Lr.ace-4A/Lr30.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {9339},
pmid = {41125583},
issn = {2041-1723},
mesh = {*Triticum/genetics/microbiology/immunology ; *Plant Diseases/microbiology/genetics/immunology ; *Disease Resistance/genetics ; *Plant Proteins/genetics/metabolism ; Plants, Genetically Modified ; Plant Leaves/microbiology/genetics ; Basidiomycota ; Genome, Plant ; *Genes, Plant ; CRISPR-Cas Systems ; Puccinia ; },
abstract = {Leaf rust is a devastating disease of wheat. Growing rust-resistant wheat varieties is the best strategy to mitigate this threat. Here, we generate a 10.51-gigabase chromosome-scale assembly of the durum wheat landrace PI 192051. Using mutagenesis and transcriptome sequencing, we identify the leaf rust resistance gene Lr.ace-4A within a recombination-sparse region of PI 192051 and demonstrate that Lr.ace-4A is identical to the previously designated Lr30 gene in hexaploid wheat. Lr.ace-4A/Lr30 encodes a non-canonical coiled-coil nucleotide-binding leucine-rich repeat receptor, featuring tandem nucleotide-binding domains. This gene is both necessary and sufficient to confer resistance to leaf rust, as demonstrated by CRISPR/Cas9-induced mutations and transgenic complementation. Lr.ace-4A provides near-immunity resistance in durum wheat, though its effectiveness is diminished in hexaploid wheat. Two amino acid polymorphisms differentiate the resistant and susceptible Lr.ace-4A haplotypes, with transgenic plants carrying either susceptible variant showing susceptibility. The cloning of Lr.ace-4A will accelerate its deployment in wheat breeding programs.},
}
@article {pmid41125159,
year = {2025},
author = {Zhou, C and Zhu, S and Luo, C and Wang, W and Fan, H and Gao, Y and Xu, X and Wang, Q and You, Y and Xie, T},
title = {From IscB to Cas9: Engineering and advances in the next generation of miniature gene editing tools.},
journal = {Biotechnology advances},
volume = {},
number = {},
pages = {108743},
doi = {10.1016/j.biotechadv.2025.108743},
pmid = {41125159},
issn = {1873-1899},
abstract = {The CRISPR-Cas system, distinguished by its inherent modularity and broad programmability, has catalyzed a paradigm shift in genome engineering due to its unprecedented accuracy, specificity, and on-target efficiency, now serving as the cornerstone of modern genome manipulation. The efficient delivery of gene editing tools remains a major technical hurdle to clinical application, primarily due to the lack of compact editors. The recent identification of the transposon-associated nuclease IscB as an evolutionary ancestor of Cas9 has provided important insights into the molecular evolution of the CRISPR-Cas9 system. Notably, IscB is a highly compact nuclease, approximately one-third the size of Cas9, capable of precise nucleic acid cleavage in eukaryotic cells under the guidance of ωRNA. These features make it a promising candidate for the development of next-generation miniaturized genome editors. However, natural IscB exhibits limited editing performance in eukaryotic systems. This review first outlines the biochemical function of the transposon IscB and briefly traces the evolutionary origin of the Cas9 system. It then describes and compares the structural characteristics and cleavage mechanisms of OgeuIscB and Cas9. Subsequent sections summarize various engineering strategies for current IscB systems, including the development of base editors and recent advances in their application. Finally, the limitations of existing systems are discussed, and potential directions for future optimization are proposed, aiming to provide new insights and facilitate the advancement of IscB-based miniaturized editors.},
}
@article {pmid41124242,
year = {2025},
author = {Lu, X and Zhu, Y and Wei, C and Cheng, L and Goodier, KD and Kong, J and Gao, X and Yu, D and Liu, X and Long, Y and Lin, J and Ma, J and Su, Y and Mao, HQ},
title = {A multistep platform identifies spleen-tropic lipid nanoparticles for in vivo T cell-targeted delivery of gene-editing proteins.},
journal = {Science advances},
volume = {11},
number = {43},
pages = {eady5579},
pmid = {41124242},
issn = {2375-2548},
mesh = {Animals ; *Gene Editing/methods ; *Spleen/metabolism/cytology ; Mice ; *T-Lymphocytes/metabolism/immunology ; *Nanoparticles/chemistry ; *Lipids/chemistry ; *Gene Transfer Techniques ; Humans ; Receptors, CCR5/genetics ; CRISPR-Cas Systems ; Liposomes ; },
abstract = {Lipid nanoparticles (LNPs) are a promising nonviral delivery system for gene-editing proteins, but optimal formulations remain underexplored. Unlike messenger RNA-based approaches, ribonucleoprotein delivery enables immediate genome editing without relying on endogenous translation. However, intracellular delivery remains a major challenge due to protein size, charge variability, and susceptibility to denaturation and degradation. Here, we present a multistep screening platform to optimize LNP formulations for gene-editing protein delivery, focusing on in vivo T cell targeting. Through in vitro screening of a composition library, we identified top-performing candidates. In vivo screening in Ai9 mice revealed a spleen-tropic LNP formulation that preferentially targets T cells, enabling efficient gene editing in vivo. Using this LNP formulation, we achieved targeted knockout of CCR5 and PD-1 in splenic T cells, supporting potential applications in HIV resistance and cancer immunotherapy. Furthermore, a machine learning-guided mechanistic study revealed key design principles for LNP-based protein delivery, highlighting unexplored opportunities for in vivo genome-editing therapies.},
}
@article {pmid41123979,
year = {2025},
author = {Wang, S and Wei, Z and Feng, Y and Gan, Z and Yu, L and Cheng, H and Xiao, Y},
title = {Rationally Trapped Polycrystalline Perovskite in Lanthanide MOF Cages for Ammonia-Mediated Nucleic Acid Intelligent Visualization.},
journal = {Small (Weinheim an der Bergstrasse, Germany)},
volume = {},
number = {},
pages = {e07214},
doi = {10.1002/smll.202507214},
pmid = {41123979},
issn = {1613-6829},
support = {82273895//National Natural Science Foundation of China/ ; 82304440//National Natural Science Foundation of China/ ; 82073811//National Natural Science Foundation of China/ ; 2024M763727//China Postdoctoral Science Foundation/ ; },
abstract = {Perovskites nanoparticles (PNPs), promising materials in fluorescence biosensing, have has their practical applications stymied by poor stability in polar solvents. Integrating PNPs into metal-organic frameworks (MOFs) offers a solution by enhancing their compatibility with various environments. Lanthanide MOFs (Ln-MOFs) are particularly advantageous due to their customizable structure, enhanced stability, and intrinsic fluorescence. Herein, the mechanism by which hybrid materials achieve a balance between physical and fluorescence properties is elucidated. Through theoretical calculations, 2,2'-bipyridine-5,5'-dicarboxylic acid is selected as the ligand to sensitize Eu[3+] and facilitate Pb[2+] chemisorption. In addition, 4-bromobutyric acid is not only employed to create hierarchical Eu-MOFs and optimize the framework for in situ growth of polycrystalline perovskites but also utilized to generate zwitterionic ligands through an SN2 reaction with MOF cages restricted n-octylamine, ensuring the stable dispersion of hybrid materials in ethyl acetate (polar solvent). The prepared PNPs@Ln-MOF exhibits significantly enhanced fluorescence lifetime (50-fold) and stability in polar solvents. Besides, the highly sensitive fluorescence color shift of PNPs@Ln-MOF in response to ammonia offers a generalizable strategy for ammonia-mediated biosensing device. Supported by CRISPR/Cas technology, this device allows for precise on-site nucleic acid assay (LOD = 200 fM), pioneering advanced applications of perovskite-based hybrid materials in biosensing.},
}
@article {pmid41065718,
year = {2025},
author = {Zeng, B and Sheng, A and Zhang, X and Wang, X and Bao, Y and Huang, Y and Huang, Y and Shan, L and Xu, X and Qin, Y and Yang, Y and Deng, Y and Tian, L and Wang, J and Ma, L},
title = {CRISPR/Cas12a Integrated with a Microfluidic System Enhanced Analysis of Programmed Cell Death Ligand 1 Expression in Circulating Tumor Cells from Non-Small Cell Lung Cancer Patients.},
journal = {ACS sensors},
volume = {10},
number = {10},
pages = {7388-7402},
doi = {10.1021/acssensors.5c01152},
pmid = {41065718},
issn = {2379-3694},
mesh = {Humans ; *Carcinoma, Non-Small-Cell Lung/blood/pathology/metabolism ; *Neoplastic Cells, Circulating/metabolism/pathology ; *B7-H1 Antigen/genetics/metabolism/blood ; *Lung Neoplasms/blood/pathology/metabolism ; *CRISPR-Cas Systems ; *Lab-On-A-Chip Devices ; *Microfluidic Analytical Techniques/methods ; Cell Line, Tumor ; },
abstract = {The detection of programmed cell death ligand 1 (PD-L1) positive circulating tumor cells (CTCs) in peripheral blood has significant clinical value for predicting and evaluating the efficacy of immunotherapy in patients with non-small cell lung cancer (NSCLC). However, traditional methods remain limited by low sensitivity and the precise quantification remains a challenge. A dual-mode microfluidic analysis chip was constructed here that included clustered regularly interspaced short palindromic repeats/Cas12a quantification and immunofluorescence visualization. Quantification of the PD-L1 protein on the surface of CTCs (20 to 10[7] cell/mL) was achieved selectively and sensitively by amplifying the nucleic acid target to generate a strong fluorescent signal, even with very low levels of target cells. The system effectively detected PD-L1[+] CTCs expression in peripheral blood samples from patients with NSCLC and monitored the efficacy of PD-1/PD-L1 targeted immune checkpoint inhibitors in real time. It exhibited excellent performance for clinical applications in monitoring the prognosis in patients with NSCLC.},
}
@article {pmid41123685,
year = {2025},
author = {Yang, S and Liu, Y and Zhang, J and Xu, J and Li, T and Huang, H and Zhu, Z and Li, M and Wang, H and Yang, C},
title = {An integrated lab-in-a-tube platform for point-of-care detection of blaKPC in urinary tract infections.},
journal = {Mikrochimica acta},
volume = {192},
number = {11},
pages = {748},
pmid = {41123685},
issn = {1436-5073},
support = {82472374//National Natural Science Foundation of China/ ; YG2024LC02//Cross-Research Fund of Biomedical Engineering of Shanghai Jiaotong University/ ; 2024ZY008//The Clinical and Excellence Program at the Institute of Molecular Medicine, Shanghai Jiao Tong University School of Medicine/ ; },
mesh = {Humans ; *Urinary Tract Infections/microbiology/diagnosis/urine ; *Klebsiella pneumoniae/enzymology/genetics/drug effects/isolation &amp; purification ; *beta-Lactamases/genetics/urine ; *Bacterial Proteins/genetics/urine ; *Point-of-Care Systems ; Klebsiella Infections/urine/diagnosis/microbiology ; Nucleic Acid Amplification Techniques ; CRISPR-Cas Systems ; },
abstract = {Carbapenem-resistant Klebsiella pneumoniae (CRKP), predominantly mediated by the blaKPC carbapenemase gene, poses a critical therapeutic challenge for urinary tract infections (UTIs). To enable rapid identification at the point of need, we engineered TubeCARE (tube-integrated platform for carbapenem antimicrobial resistance evaluation), an integrated, disposable lab-in-a-tube system for direct blaKPC detection from urine samples. This self-contained system uniquely combines urine processing, nucleic acid extraction, recombinase polymerase amplification (RPA), and CRISPR/Cas12a reaction within a single sealed unit, offering dual-modality outputs: smartphone-based real-time fluorescence or naked-eye lateral flow strip interpretation. The full workflow delivers "sample-in, result-out" in 40 min, eliminating external multi-step nucleic acid extraction and reducing contamination risks. Both detection modes demonstrated laboratory-grade sensitivity (1 CFU/mL) with 100% specificity against non-target carbapenemase genes. Clinical validation using 24 urine samples (20 blaKPC-positive, 4 negative) showed 100% concordance with quantitative PCR. In a representative UTI case, TubeCARE enabled 40-min bedside blaKPC detection; early implementation would circumvent 72-h diagnostic delays, facilitating timely carbapenem avoidance and preventing bloodstream complications. Featuring integrated lab-in-a-tube operation, cost-effective manufacturing, user-friendly workflow, and laboratory-grade accuracy, TubeCARE provides actionable antimicrobial resistance surveillance in diverse clinical and resource-limited settings.},
}
@article {pmid40938646,
year = {2025},
author = {Lee, JH and Lee, E-S and Kyung, SM and Xiang, X-R and Park, H-E and Shin, M-K and Yoo, HS},
title = {Functional analysis of the intracellular survival of Mycobacterium avium subsp. paratuberculosis in THP-1 cells using CRISPR interference.},
journal = {Journal of bacteriology},
volume = {207},
number = {10},
pages = {e0024425},
doi = {10.1128/jb.00244-25},
pmid = {40938646},
issn = {1098-5530},
support = {//National Institute of Wildlife Disease Control and Prevention/ ; },
mesh = {Humans ; *Mycobacterium avium subsp. paratuberculosis/genetics/physiology/pathogenicity ; *Macrophages/microbiology ; THP-1 Cells ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Virulence/genetics ; Bacterial Proteins/genetics/metabolism ; Gene Expression Regulation, Bacterial ; *Microbial Viability ; CRISPR-Cas Systems ; Paratuberculosis/microbiology ; },
abstract = {UNLABELLED: Mycobacterium avium subsp. paratuberculosis (MAP) is a causative agent of Johne's disease in ruminants and a potential zoonotic agent linked with Crohn's disease in humans. Despite the possible risk to public health, few studies have focused on the virulence of MAP against human macrophages. Therefore, a functional analysis of mycobacterial genes associated with virulence, especially the intracellular survival of MAP, was performed after infection of MAP CRISPR interference (CRISPRi) mutants in the human THP-1 macrophages. MAP mutants were targeted to four genes (mdh, pknG, MAP1981c, and icl). The optimal concentration of anhydrotetracycline (ATc) was determined to be 5 µg/mL by measuring the survival of the cells and the downregulation of gene expression levels in the cells up to Day 3. The clump formation and intracellular survival of MAP were investigated using transmission electron microscopy and the colony-forming units, respectively. The clump formation of MAP mutants induced by CRISPRi was decreased in THP-1 macrophages at 24 and 72 h post-infection. The survival rates of the MAP mutants significantly decreased with increasing ATc concentration and time course of infection in MAP-mdhKD, MAP1981cKD, and MAP-iclKD. Conversely, the survival rate of THP-1 macrophages increased with increasing ATc concentration. Our results suggest that these genes might be closely related to MAP virulence along with intracellular survival in THP-1 macrophages. These data can provide novel insights into the utilization of CRISPRi in further research on MAP virulence by exploring intracellular survival using mycobacterial genes related to the virulence of MAP during host infection.<br><br>IMPORTANCE: Johne's disease, caused by Mycobacterium avium subsp. paratuberculosis (MAP) is a worldwide issue in the dairy industry and has a possible connection to Crohn's disease (CD) in humans. Despite its potential contribution to the etiology of CD, there have been few studies focusing on the virulence of MAP against human macrophages. In the current study, we investigated MAP virulence along with intracellular survival in human THP-1 macrophages using functional analysis of MAP CRISPR interference (CRISPRi) mutants at the knockdown of genes associated with mycobacterial virulence. The identified potential genes represent novel candidate classes that could be necessary for MAP virulence by exploring intracellular survival during host infection and could provide novel insights for future studies on the utilization of CRISPRi.},
}
@article {pmid40919935,
year = {2025},
author = {Alberts, ME and Kurtz, MP and Müh, U and Bernardi, JP and Bollinger, KW and Dobrila, HA and Duncan, L and Laster, HM and Orea, AJ and Pannullo, AG and Rivera-Rosado, JG and Torres, FV and Ellermeier, CD and Weiss, DS},
title = {Analysis of essential genes in Clostridioides difficile by CRISPRi and Tn-seq.},
journal = {Journal of bacteriology},
volume = {207},
number = {10},
pages = {e0022025},
doi = {10.1128/jb.00220-25},
pmid = {40919935},
issn = {1098-5530},
support = {DBI-1852070//National Science Foundation/ ; R01 AI155492/AI/NIAID NIH HHS/United States ; R21 AI159071/AI/NIAID NIH HHS/United States ; },
mesh = {*Clostridioides difficile/genetics/metabolism ; *Genes, Essential ; *DNA Transposable Elements ; Mutagenesis, Insertional ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Bacterial Proteins/genetics/metabolism ; CRISPR-Cas Systems ; },
abstract = {Essential genes are interesting in their own right and as potential antibiotic targets. To date, only one report has identified essential genes on a genome-wide scale in Clostridioides difficile, a problematic pathogen for which treatment options are limited. That foundational study used large-scale transposon mutagenesis to identify 404 protein-encoding genes as likely to be essential for vegetative growth of the epidemic strain R20291. Here, we revisit the essential genes of strain R20291 using a combination of CRISPR interference (CRISPRi) and transposon insertion site sequencing (Tn-seq). First, we targeted 181 of the 404 putatively essential genes with CRISPRi. We confirmed essentiality for >90% of the targeted genes and observed morphological defects for >80% of them. Second, we conducted a new Tn-seq analysis, which identified 346 genes as essential, of which 283 are in common with the previous report and might be considered a provisional essential gene set that minimizes false positives. We compare the list of essential genes to those of other bacteria, especially Bacillus subtilis, highlighting some noteworthy differences. Finally, we used fusions to red fluorescent protein (RFP) to identify 18 putative new cell division proteins, 3 of which are conserved in Bacillota but of largely unknown function. Collectively, our findings provide new tools and insights that advance our understanding of C. difficile.IMPORTANCEClostridioides difficile is an opportunistic pathogen for which better antibiotics are sorely needed. Most antibiotics target pathways that are essential for viability. Here, we use saturation transposon mutagenesis and gene silencing with CRISPR interference to identify and characterize genes required for growth on laboratory media. Comparison to the model organism Bacillus subtilis revealed many similarities and a few striking differences that warrant further study and may include opportunities for developing antibiotics that kill C. difficile without decimating the healthy microbiota needed to keep C. difficile in check.},
}
@article {pmid40897994,
year = {2025},
author = {Kumar, N},
title = {Genome Editing for Fertility: Unlocking the Promise of CRISPR/Cas9 in Addressing Male Infertility - A Narrative Review.},
journal = {Reproductive sciences (Thousand Oaks, Calif.)},
volume = {32},
number = {10},
pages = {3221-3239},
pmid = {40897994},
issn = {1933-7205},
mesh = {*Gene Editing/methods ; Humans ; Male ; *CRISPR-Cas Systems ; *Infertility, Male/genetics/therapy ; Animals ; *Fertility/genetics ; *Genetic Therapy/methods ; },
abstract = {Male infertility remains a significant global reproductive health challenge, frequently attributed to genetic mutations impairing spermatogenesis and sperm function. This narrative review aims to explore the genetic and molecular underpinnings of male infertility and evaluate the emerging role of Clustered Regularly Interspaced Short Palindromic Repeats-CRISPR-associated protein 9 (CRISPR/Cas9) genome editing as a diagnostic and therapeutic tool, while addressing its associated ethical, technical, and safety considerations. A Comprehensive literature search was conducted across PubMed, Scopus, Web of Science databases, covering studies published between September 1992 and April 2025. Keywords included "male infertility," "genetic causes of male infertility," "genome editing," "CRISPR/Cas9 and male infertility," "genome editing in male reproduction," "ethical concerns of CRISPR," and "future fertility treatments." Eligible studies focused on genetic correction strategies, spermatogonial stem cell applications, off-target effects, mosaicism, and ethical implications of gene editing. The review synthesizes current knowledge on genetic and epigenetic etiologies of male infertility. It discusses the therapeutic potential of CRISPR/Cas9 in correcting these defects and restoring fertility in preclinical models. Critical challenges, including off-target gene editing, germline mosaicism, long-term safety, and ethical debates surrounding human germline modification, were examined. The review also considers future advancements in genome editing and artificial sperm development. CRISPR/Cas9 represents a transformative platform in reproductive medicine with promising implications for treatment of genetically linked male infertility. However, its clinical translation demands rigorous validation, transparent ethical deliberation, and robust regulatory frameworks. Future innovations combining genome editing, regenerative biology, and precision diagnostics may revolutionize fertility care, but must proceed with caution to ensure safety, efficacy, and ethical integrity.},
}
@article {pmid40782836,
year = {2025},
author = {Agboola, OE and Agboola, SS and Odeghe, OB and Olaiya, OE and Ayinla, ZA and Akinsanya, PO and Ilesanmi, OS and Ibrahim, TK and Adegbuyi, TA and Kolawole, OA and Omotuyi, IO and Oyinloye, BE},
title = {Computational genome engineering through AI-CRISPR-precision medicine integration in modern therapeutics.},
journal = {Annales pharmaceutiques francaises},
volume = {83},
number = {6},
pages = {1073-1085},
doi = {10.1016/j.pharma.2025.08.001},
pmid = {40782836},
issn = {2772-803X},
mesh = {Humans ; *Precision Medicine/methods ; *Gene Editing/methods ; *Artificial Intelligence ; Drug Discovery ; *CRISPR-Cas Systems ; *Genetic Engineering/methods ; Machine Learning ; },
abstract = {The convergence of precision medicine strategies, CRISPR gene editing technologies, and artificial intelligence (AI) is causing a revolutionary change in the pharmaceutical industry in recent times. Latest trends and future directions of these integrated technologies in pharmaceutical science and molecular biology are presented in the present exhaustive review. With more than 250 gene-editing clinical trials being tracked internationally as of February 2025, the recent clinical successes point toward the therapeutic potency of CRISPR-based therapeutics. In parallel, AI-based drug discovery platforms are recording fantastic hit rates; compared to conventional industry benchmarks, AI-emerging drugs reflect 80-90% Phase I trial success rates. Therapeutic development paradigms are being transformed by the intersection of machine learning algorithms, multi-omics technologies, and precision medicine paradigms. The review provides insights into the revolutionary potential of these converging approaches in addressing unmet medical requirements and optimizing therapeutic benefits through syntheses of existing evidence from clinical trials, regulatory matters, and technological innovations.},
}
@article {pmid41123679,
year = {2025},
author = {Rocha, DC and Omoregbee, MO and Contiliani, DF and Mandlik, R and Li, G and Mascoveto, J and Coleman, G and Culver, JN and Leal, DR and de Souza, AA and Qi, Y},
title = {Transgene-free genome editing in citrus and poplar trees using positive and negative selection markers.},
journal = {Plant cell reports},
volume = {44},
number = {11},
pages = {244},
pmid = {41123679},
issn = {1432-203X},
support = {IOS-2132693//Division of Integrative Organismal Systems/ ; 2224203//Division of Integrative Organismal Systems/ ; IOS-2428015//Division of Integrative Organismal Systems/ ; 2021-67013-34554//National Institute of Food and Agriculture/ ; 2020-33522-32274//National Institute of Food and Agriculture/ ; 2024-33522-42755//National Institute of Food and Agriculture/ ; 2020-70029- 33161//National Institute of Food and Agriculture/ ; DE-SC0023011//Department of Energy/ ; 2023/09068-9//FAPESP/ ; },
mesh = {*Populus/genetics ; *Gene Editing/methods ; Plants, Genetically Modified/genetics ; *Citrus/genetics ; Transgenes/genetics ; CRISPR-Cas Systems/genetics ; Genome, Plant ; Genetic Markers ; Herbicide Resistance/genetics ; },
abstract = {Transgene-free genome editing of the gene of interest in citrus and poplar has been achieved by co-editing the ALS gene via transient transgene expression of an efficient cytosine base editor. CRISPR-Cas genome editing systems have been widely used in plants. However, such genome-edited plants are nearly always transgenic in the first generation when Agrobacterium-mediated transformation is used. Transgene-free genome-edited plants are valuable for genetic analysis and breeding as well as simplifying regulatory approval. It can be challenging to generate transgene-free genome-edited plants in vegetatively propagated or perennial plants. To advance transgene-free genome editing in citrus and poplar, we investigated a co-editing strategy using an efficient cytosine base editor (CBE) to edit the ALS gene to confer herbicide resistance combined with transient transgene expression and potential mobile RNA-based movement of CBE transcripts to neighboring, non-transgenic cells. An FCY-UPP based cytotoxin system was used to select non-transgenic plants that survive after culturing on 5-FC containing medium. While the editing efficiency is higher in poplar than in citrus, our results show that the CBE-based co-editing strategy works in both citrus and poplar, albeit with low efficiency for biallelic edits. Unexpectedly, the addition of the TLS mobile RNA sequence reduced genome editing efficiency in both transgenic and non-transgenic plants. Although a small fraction of escaping plants is detected in both positive and negative selection processes, our data demonstrate a promising approach for generating transgene-free base-edited plants.},
}
@article {pmid41123065,
year = {2025},
author = {Sohail, M and Ma, S and Mushtaq, B and Haider, MU and Li, B and Zhang, X and Huang, H},
title = {Fabricating Four-Element Doped Carbon Dots-Based Fluorescent Ratiometric Reporter Platform for CRISPR/Cas-Driven Precise Sensing of Nucleic Acids.},
journal = {Analytical chemistry},
volume = {},
number = {},
pages = {},
doi = {10.1021/acs.analchem.5c03228},
pmid = {41123065},
issn = {1520-6882},
abstract = {Conventional CRISPR/Cas sensing platforms exhibit poor efficiency concerning reporter-based demerits, including their interference-labile nature, photobleaching, low robustness due to a single output signal, and probe-concentration dependence. Herein, a carbon dots (CDs)-based dual-emissive fluorescent ratiometric CRISPR/Cas reporter platform was fabricated for biosensing and other analytical applications to bottleneck the demerits of conventional reporters, integrating the benefits of a ratiometric strategy and four-element doped carbon dots (4D CDs) as a transducer. Briefly, doping enhances the optical and physicochemical traits of CDs and minimizes the effect of the interfering species. A series of state-of-the-art N, P, S, and Cu codoped CDs (4D CDs) were synthesized using the hydrothermal approach and statistical tools, such as Box-Behnken design, analysis of variance, and others, enhancing photophysical traits, surface features, and sensitivity of CDs. The red-emissive CDs were prepared by using the same procedure but different precursors. The optimum 4D CDs (blue-emissive) and red-emissive CDs were used to unleash the principle of the fluorescent ratiometric CRISPR/Cas reporter system for diverse applications. Finally, the designed 4D CDs-based CRISPR/Cas biosensor was applied for nucleic acid monitoring, such as the COVID-19 nucleic acid. This project disclosed the controlled-doping principle to synthesize 4D CDs and unleashed the mechanism of ratiometric dual-emissive CRISPR/Cas-powered reporters for precise sensing applications. We anticipate the implementation of this technology in commercial analytical, biosensing, point-of-care, and other applications.},
}
@article {pmid41122064,
year = {2025},
author = {Balobaid, A and Waterworth, WM and Vila Nova, SF and Causier, B and Sharma, V and Park, MR and Pandey, MK and West, CE},
title = {Arabidopsis thaliana FANCONI ANAEMIA I (FANCI) has roles in the repair of interstrand crosslinks and CRISPR-Cas9 induced DNA double strand breaks.},
journal = {The Plant journal : for cell and molecular biology},
volume = {124},
number = {2},
pages = {e70533},
doi = {10.1111/tpj.70533},
pmid = {41122064},
issn = {1365-313X},
support = {//King Saud University/ ; BB/S002081/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; BB/Y010426/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; },
mesh = {*Arabidopsis/genetics/metabolism ; *Arabidopsis Proteins/metabolism/genetics ; *DNA Repair/genetics ; *DNA Breaks, Double-Stranded ; CRISPR-Cas Systems/genetics ; Mitomycin/pharmacology ; Cross-Linking Reagents ; Mutation ; },
abstract = {DNA repair is crucial for genome stability, in particular for plants which are exposed to high levels of damage arising from UV irradiation, soil pollutants and reactive oxygen species. Damage that affects both strands of the DNA duplex is harder to repair due to both the lack of a template strand and the potential for physical separation of fragmented chromosomes. As such, DNA double-strand breaks (DSBs) and interstrand DNA crosslinks (ICL) are particularly cytotoxic forms of damage. Here we report the functions of FANCONI ANAEMIA I (FANCI), an Arabidopsis thaliana homologue of the mammalian ICL repair protein. We show that in plant cells, as in mammals, FANCI forms a nuclear localised complex with FANCD2. Genetic analysis of plants lacking FANCI displays significant hypersensitivity to the DNA crosslinking reagent mitomycin C. Furthermore, mutation of FANCI in combination with mutations in a second ICL repair factor, METHYL METHANESULFONATE AND UV-SENSITIVE PROTEIN 81 (MUS81), results in increased levels of programmed cell death compared to the corresponding single mutants, revealing roles in maintaining plant genome stability. Sequence analysis of mutational repair of CRISPR-Cas9-induced DSBs revealed that FANCI promotes single nucleotide insertions and reduces longer deletions. This pattern of mutations may reflect roles for FA proteins in replication-coupled repair of a subset of DSBs. Taken together, this analysis finds evidence for multiple roles for FANCI in the maintenance of plant genome stability.},
}
@article {pmid41121375,
year = {2025},
author = {Amieva, R and Rico-San Román, L and Pastor-Fernández, I and Hemphill, A and Boubaker, G and Collantes-Fernández, E and Ortega-Mora, LM and Horcajo, P},
title = {Loss of NcBPK1 impairs bradyzoite differentiation and enhances virulence in Neospora caninum.},
journal = {Parasites &amp; vectors},
volume = {18},
number = {1},
pages = {422},
pmid = {41121375},
issn = {1756-3305},
support = {PRE2020-092101//Spanish Ministry of Science, Innovation and Universities/ ; },
mesh = {*Neospora/pathogenicity/genetics/growth &amp; development ; Animals ; Virulence ; Mice ; *Coccidiosis/parasitology/veterinary ; *Protozoan Proteins/genetics/metabolism ; Female ; Cattle ; Macrophages/parasitology ; Pregnancy ; Virulence Factors/genetics ; Cattle Diseases/parasitology ; CRISPR-Cas Systems ; Host-Parasite Interactions ; Disease Models, Animal ; Gene Deletion ; },
abstract = {BACKGROUND: Neospora caninum is an apicomplexan parasite responsible for bovine neosporosis, a disease that leads to substantial economic losses in cattle due to abortion and reduced productivity. The pathogenesis of N. caninum is shaped by complex host-parasite interactions, and virulence is known to vary between strains. BPK1 (Bradyzoite pseudokinase 1), a pseudokinase previously identified as a potential virulence factor in Toxoplasma gondii, has not yet been functionally characterized in N. caninum.<br><br>METHODS: To investigate the role of NcBPK1 in parasite virulence, a knockout strain (Nc&#x394;BPK1) was generated using CRISPR/Cas9 genome editing. The virulence of the mutant was evaluated in a pregnant mouse model by assessing neonatal survival and parasite burden in dam tissues. In vitro assays were conducted to examine parasite replication in bovine macrophages and to analyze the expression of stage-specific genes.<br><br>RESULTS: Deletion of NcBpk1 resulted in enhanced parasite virulence in vivo, as shown by a decrease in neonatal survival and higher parasite loads in maternal brain tissue. The Nc&#x394;BPK1 mutant also displayed enhanced replication in bovine macrophages and reduced expression of bradyzoite-specific genes, suggesting a defect in stage conversion.<br><br>CONCLUSIONS: These findings indicate that NcBPK1 is crucial for regulating the balance between acute replication and chronic persistence. Its absence promotes rapid tachyzoite proliferation and worsens disease outcomes. This study sheds light on the molecular mechanisms underlying N. caninum virulence. Further research is needed to elucidate the signaling pathways and protein interactions involving NcBPK1.},
}
@article {pmid41121349,
year = {2025},
author = {Safarzadeh Kozani, P and Safarzadeh Kozani, P},
title = {Preventing secondary primary malignancies (SPMs) in CAR-T cell therapy through site-specific transgene integration into genomic safe harbors (GSHs).},
journal = {Journal of translational medicine},
volume = {23},
number = {1},
pages = {1155},
pmid = {41121349},
issn = {1479-5876},
mesh = {Humans ; *Transgenes/genetics ; *Neoplasms, Second Primary/prevention &amp; control/genetics ; *Receptors, Chimeric Antigen/metabolism ; Animals ; *Immunotherapy, Adoptive/adverse effects ; Gene Editing ; *Genomics ; CRISPR-Cas Systems ; },
abstract = {Chimeric antigen receptor (CAR)-T cell therapy has revolutionized oncology by achieving durable remissions in refractory hematologic malignancies. However, emerging reports link this therapy to second primary malignancies, including CAR+ lymphomas and leukemias, driven by insertional mutagenesis from semi-random viral vector integration near oncogenes or tumor suppressor loci. These rare but serious complications underscore the dual challenge of eradicating primary tumors while mitigating delayed genotoxic risks. Conventional CAR-T cell manufacturing, reliant on gamma-retroviral or lentiviral vectors, introduces genomic instability through integration into fragile sites or transcriptionally active regions. CRISPR/Cas9-mediated genome editing further amplifies risks via off-target double-strand breaks and chromosomal rearrangements. This review evaluates genomic safe harbors (GSHs)-such as AAVS1, TRAC, CCR5, ROSA26 and CLYBL-as loci validated for stable, high-level CAR transgene expression without oncogenic disruption. GSHs meet stringent criteria: distal from cancer-related genes, resistant to epigenetic silencing, and transcriptionally permissive. Preclinical studies demonstrate that site-directed CAR integration into GSHs preserves antitumor efficacy while eliminating malignant transformation risks. Challenges persist in optimizing homology-directed repair efficiency, mitigating residual dsDNA toxicity, and standardizing regulatory frameworks for long-term genomic surveillance. Emerging technologies-base/prime editing, hybrid nucleases, and rigorous monitoring-promise enhanced precision and safety. By reconciling therapeutic innovation with genomic integrity, GSH-engineered CAR-T cells herald a paradigm shift toward precision immunotherapies, offering curative potential while preempting secondary oncogenesis. Collaborative efforts to refine manufacturing, harmonize global standards, and prioritize patient-specific risk stratification will be critical to advancing this transformative approach.},
}
@article {pmid41121307,
year = {2025},
author = {Li, C and Peng, W and Zhong, Z and Zhang, C and Wang, X and Qin, R and Lei, Q and Lv, J and Liu, F and Zhao, Y and Lv, Z and Li, C and Yang, S and Zhang, H and Tao, Z and Sun, C},
title = {CRISPR/Cas9 library screening reveals that STK19 has synergistic antitumor effects when combined with cisplatin on tongue squamous cell carcinoma.},
journal = {Journal of translational medicine},
volume = {23},
number = {1},
pages = {1142},
pmid = {41121307},
issn = {1479-5876},
support = {82360568, 81960543//Natural Science Foundation of China/ ; 82160452//Natural Science Foundation of China/ ; 202401AY070001-137//Applied Basic Research Foundation of Yunnan Province/ ; 202301AY070001-247//Yunnan Fundamental Research Projects/ ; 2023YJKTB01//Basic Research Program of the First People's Hospital of Qujing/ ; 202305AF150091//Yunnan Province Academician Expert Workstation/ ; },
mesh = {*Cisplatin/pharmacology/therapeutic use ; *Tongue Neoplasms/drug therapy/genetics/pathology ; Humans ; Cell Line, Tumor ; *CRISPR-Cas Systems/genetics ; Animals ; Drug Synergism ; *Carcinoma, Squamous Cell/drug therapy/genetics/pathology ; *Protein Serine-Threonine Kinases/metabolism/genetics ; Xenograft Model Antitumor Assays ; *Antineoplastic Agents/pharmacology/therapeutic use ; DNA Damage/drug effects ; Mice ; Mice, Nude ; Cell Proliferation/drug effects ; },
abstract = {BACKGROUND: Tongue squamous cell carcinoma (TSCC) is a common oral cancer that has a high propensity for recurrence and metastasis. Therefore, TSCC has a 50% 5-year survival rate. Platinum-based chemotherapy is an effective treatment for squamous cell carcinoma, however, chemotherapy resistance remains a major issue. Therefore, innovative and effective drug combinations are needed to improve TSCC patient prognosis.<br><br>METHODS: In this study, we conducted an in vitro CRISPR/Cas9 library screen using two TSCC cell lines (Tscca and Cal27) to identify specific genes that, when inhibited, synergize with cisplatin to effectively suppress tumor growth.<br><br>RESULTS: We identified STK19 as a potential drug target. Inhibition of STK19 enhances the response of TSCC to cisplatin. Through genetic and pharmacological methods, it has been demonstrated that reducing STK19 activity enhances cisplatin-induced DNA damage. The mechanism involves the depletion of MGMT with STK19 inhibition, leading to conditional lethality and synergistic reduction of tumors in vivo when combined with cisplatin. Overall, in this study, unbiased genetic testing was used to successfully identify synthetic lethal drug combinations for TSCC.<br><br>CONCLUSION: STK19 was identified as a promising target that could enhance the killing effects of cisplatin on tongue squamous carcinoma cells, offering a novel therapeutic option for individuals who are insensitive to conventional treatment methods.},
}
@article {pmid41121296,
year = {2025},
author = {Shi, X and Lu, S and Tang, Q and Zhao, X},
title = {Targeted modification of cis-elements in the CUL3 gene to restore exon 9 inclusion for treating Gordon syndrome.},
journal = {Human genomics},
volume = {19},
number = {1},
pages = {119},
pmid = {41121296},
issn = {1479-7364},
support = {ZR2022MH300//the Shandong Province Natural Science Foundation/ ; 82271540//the National Natural Science Foundation of China/ ; QDFY+X2024131//the Natural Science Foundation of the Affiliated Hospital of Qingdao University/ ; },
mesh = {*Cullin Proteins/genetics ; Humans ; *Exons/genetics ; RNA Splice Sites/genetics ; Introns/genetics ; RNA Splicing/genetics ; CRISPR-Cas Systems/genetics ; HEK293 Cells ; },
abstract = {BACKGROUND: The weak splice acceptor site (AS) of exon 9 underlies almost all pathogenic variants of Cullin3 (CUL3) causing exon 9 skipping in Gordon syndrome, emphasizing the need for splicing-targeted therapeutic strategies. This study explored universal therapeutic targets to modulate AS and investigated their potential and mechanisms for restoring normal splicing.<br><br>RESULTS: Through bioinformatic prediction, minigene assays, EMSA, CRISPR/Cas9-mediated construction of mutant cell lines and RIP, three rescue sites in the polypyrimidine (Py) tract of intron 8 were identified, including A(-9)T, A(-10)T and AA(-9, -10)TT, with AA(-9, -10)TT most effectively promoting exon inclusion by extending the Py-tract to increase U2AF2 binding. Additionally, previous candidate target A18G was confirmed to rescue exon 9 skipping by weakening hnRNP A1 splicing inhibition in endogenous cell models.<br><br>CONCLUSIONS: Our findings highlight the therapeutic potential of AA(-9, -10)TT and A18G in CUL3-related Gordon syndrome, suggesting the targeted modification of cis-elements could be an ideal and universal strategy to develop treatments for splicing-related diseases.},
}
@article {pmid41120304,
year = {2025},
author = {Wang, Z and Liu, F and Chen, N and Wu, J and Li, X and Fang, M and Yan, M and Zhang, J and Deng, B and Wang, L and Wang, X and Liu, M and Zeng, D and Zou, Z and Wang, B and Songyang, Z and He, B and Liu, Q},
title = {Chromatin looping-based CRISPR screen identifies TLK2 as chromatin loop formation regulator in cancer stemness plasticity.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {9288},
pmid = {41120304},
issn = {2041-1723},
support = {82321003//National Natural Science Foundation of China (National Science Foundation of China)/ ; 82341020//National Natural Science Foundation of China (National Science Foundation of China)/ ; 82473123//National Natural Science Foundation of China (National Science Foundation of China)/ ; 82173367//National Natural Science Foundation of China (National Science Foundation of China)/ ; 82403653//National Natural Science Foundation of China (National Science Foundation of China)/ ; 82302929//National Natural Science Foundation of China (National Science Foundation of China)/ ; 82303009//National Natural Science Foundation of China (National Science Foundation of China)/ ; 2022A1515010915//Natural Science Foundation of Guangdong Province (Guangdong Natural Science Foundation)/ ; },
mesh = {Humans ; *Chromatin/metabolism/genetics ; Animals ; *Neoplastic Stem Cells/metabolism/pathology ; Female ; Mice ; Kruppel-Like Factor 4 ; *Breast Neoplasms/genetics/pathology/metabolism ; Cell Line, Tumor ; *Protein Serine-Threonine Kinases/metabolism/genetics ; CCCTC-Binding Factor/metabolism/genetics ; Cell Plasticity/genetics ; Kruppel-Like Transcription Factors/genetics/metabolism ; CRISPR-Cas Systems ; Gene Expression Regulation, Neoplastic ; Cell Cycle Proteins/metabolism ; Chromosomal Proteins, Non-Histone/metabolism ; Lung Neoplasms/secondary/genetics ; },
abstract = {Targeting cancer cell plasticity through chromatin organization is an emerging research area, yet the molecular mechanisms that govern chromatin loop formation remain unclear. Here, we develop a CRISPR screen based on our engineered live-cell CTCF-cohesin contact reporters to identify regulators of chromatin loops. Our findings reveal that tousled-like kinase 2 (TLK2) functions as a key regulator of chromatin loop formation during the cancer stemness transition. Mechanistically, TLK2 phosphorylates DYNLL1, enhancing its interaction with CTCF to promote CTCF-cohesin hub formation at the KLF4 locus. Suppressing TLK2 impairs cancer stemness plasticity, sensitizes cancer cells to cytotoxic stress in vitro, and reduces lung metastases and enhances immunotherapy response in breast cancer mouse models. Clinically, elevated TLK2 expression correlates with poor prognosis in breast cancer patients. Collectively, these findings identify TLK2 as a potential therapeutic target for mitigating cancer stemness plasticity, highlighting chromatin loop-targeting therapy as a promising strategy to eradicate cancer stem cells.},
}
@article {pmid41118579,
year = {2025},
author = {Hanai, Y and Hilario, PLL and Shiraishi, Y and Yoshida, N and Murakami, S and Shimizu, Y and Kano, N and Kojima, M and Murai, K and Kawai, T and Okamura, K},
title = {The knock-in atlas: a web resource for targeted protein trap by CRISPR/Cas9 in human and mouse cell lines.},
journal = {Nucleic acids research},
volume = {53},
number = {19},
pages = {},
doi = {10.1093/nar/gkaf1050},
pmid = {41118579},
issn = {1362-4962},
support = {//NAIST for the Creation of Innovation in Science and Technology/ ; 24KJ1692//JSPS Research Fellowship/ ; 25KJ1828//NAIST Granite Program/ ; 17K20145//JSPS Found for the Promotion of Joint International Research/ ; //Academic Assistant Grant by Office for Gender Equality at NAIST/ ; //Takeda Science Foundation/ ; 20H03468//KAKENHI/ ; 23K14546//JSPS/ ; },
mesh = {Humans ; Animals ; *CRISPR-Cas Systems/genetics ; Mice ; *Gene Knock-In Techniques/methods ; HEK293 Cells ; RNA, Guide, CRISPR-Cas Systems/genetics ; Internet ; Cell Line ; RNA-Binding Proteins/genetics ; HeLa Cells ; *Databases, Genetic ; },
abstract = {Various cell engineering techniques have been developed by leveraging the CRISPR-Cas9 technology, but large-scale resources for targeted gene knock-in are still limited. Here we introduce the Knock-in Atlas, a web resource for gene tagging by fluorescent proteins by inserting artificial exons in target gene introns. To produce knock-in cells efficiently and reproducibly, we carefully chose and catalogued guide RNAs (gRNAs) for targeting genes in the human and mouse genomes by taking the gRNA efficacy scores and protein structures around the insertion sites into account. As of August 2025, we have characterized knock-in cell lines for 350 proteins, with a focus on RNA binding proteins, by flow cytometry and confocal microscopy. The transfection and flow cytometry protocols were optimized for several cell lines including HEK293T, eHAP1, HeLa, THP-1, Neuro2a, mouse embryonic fibroblast (MEF) and mouse embryonic stem cell (mESC). A website has been launched to organize the results of initial characterization including flow cytometry data after transfection, confocal microscopy, and western blot results for the genes for which knock-in HEK293T cell lines were already made. The site also provides a database to organize the information of pre-designed gRNAs for the human and mouse genomes. <https://rnabio.naist.jp/atlas/>.},
}
@article {pmid41118576,
year = {2025},
author = {Shu, WJ and Ma, Z and Jia, L and Guo, B and Tian, X and He, C and Wang, F},
title = {MiR-ON-CRISPR: a microRNA-activated CRISPR-dCas9 system for precise gene therapy in living cells and mouse models of sepsis.},
journal = {Nucleic acids research},
volume = {53},
number = {19},
pages = {},
doi = {10.1093/nar/gkaf1037},
pmid = {41118576},
issn = {1362-4962},
support = {32271512//National Natural Science Foundation of China/ ; 82572281//National Natural Science Foundation of China/ ; 32201199//National Natural Science Foundation of China/ ; 2023-JC-ZD-43//Natural Science Basic Research Program of Shaanxi/ ; 2025JC-YBQN-1129//Natural Science Basic Research Program of Shaanxi/ ; 2022JC-56//Natural Science Basic Research Program of Shaanxi/ ; 2024SF-YBXM-138//Key Research and Development Program of Shaanxi/ ; 0959202513035//Young Talent Fund of Xi'an Association for Science and Technology/ ; 24YXYJ0031//Xi'an Science and Technology Research Program/ ; },
mesh = {Animals ; *MicroRNAs/genetics/metabolism ; *Sepsis/therapy/genetics/pathology ; *CRISPR-Cas Systems ; Mice ; Disease Models, Animal ; *Genetic Therapy/methods ; Humans ; Gene Editing/methods ; Endoplasmic Reticulum Stress/genetics ; Mice, Inbred C57BL ; RNA, Guide, CRISPR-Cas Systems/genetics ; NF-E2-Related Factor 2/genetics/metabolism ; },
abstract = {The CRISPR-dCas9 technology is a powerful tool for manipulating the expression of target genes in a variety of biomedical applications. Nevertheless, it is imperative that the activity of the CRISPR-dCas9 system be tightly controlled to improve its safety and applicability. In this study, we successfully designed a microRNA-activated CRISPR-dCas9 system, termed miR-ON-CRISPR, in which the core components (dCas9 and sgRNA) are both regulated by endogenous miRNA. Our findings demonstrated that the miR-ON-CRISPR system can regulate firefly luciferase reporter gene expression to faithfully visualize miRNA activity and image the differentiation status of neural cells. Moreover, the miR-ON-CRISPR was designed as an AND/OR gate system, thereby enabling the simultaneous detection of two distinct miRNAs. Furthermore, the system was adapted to achieve cell type-specific killing by activating the exogenous DTA genes or endogenous BAX genes. Finally, in mouse models of sepsis, the miR-ON-CRISPR system was shown to alleviate the sepsis-induced liver injury as well as the associated oxidative stress damage and endoplasmic reticulum stress via activating the nuclear erythroid 2-related factor 2 gene. In conclusion, this proof-of-concept study demonstrates the feasibility of the miR-ON-CRISPR system for cell type-specific control of CRISPR-dCas9 activity and its therapeutic applications in the treatment of genetic diseases.},
}
@article {pmid41118570,
year = {2025},
author = {Braithwaite, J and Cannon, C and Chalmers, R and Edwards, H},
title = {Single-colony resolution of CRISPR-Cas adaptation in E. coli reveals altered spacer-source bias during solid-phase growth.},
journal = {Nucleic acids research},
volume = {53},
number = {19},
pages = {},
doi = {10.1093/nar/gkaf1044},
pmid = {41118570},
issn = {1362-4962},
support = {RPG-2020-079//Leverhulme Trust/ ; //University of Nottingham/ ; },
mesh = {*Escherichia coli/genetics/growth &amp; development ; *CRISPR-Cas Systems ; Plasmids/genetics ; Escherichia coli Proteins/genetics/metabolism ; CRISPR-Associated Proteins/genetics/metabolism ; Lac Operon ; Adaptation, Physiological/genetics ; *DNA, Intergenic/genetics ; },
abstract = {CRISPR-Cas systems provide adaptive immunity by integrating short DNA fragments from mobile genetic elements into host arrays. While the core biochemical mechanism of adaptation is well defined, its modulation by physiological contexts is less well understood. Here, we present a visual papillation assay that enables single-colony detection of CRISPR-Cas adaptation in Escherichia coli. Spacer acquisition restores the reading frame of a disrupted lacZ gene, forming blue papillae on lactose X-gal plates. The assay is semi-quantitative, highly sensitive, capable of detecting single events among 109 cells, and responds predictably to Cas1-Cas2 expression levels. Spacer mapping revealed a major shift in source bias: in liquid culture, 64% of spacers were plasmid-derived, but on solid medium this dropped to ∼9%. Adjusting inducer concentration to match liquid conditions did not restore plasmid bias, indicating a physiological basis linked to colony growth. Accounting for the molar excess of chromosomal DNA, the 9% plasmid share reflects near-neutral DNA source sampling rather than plasmid overrepresentation. These findings suggest that the spatial and metabolic structure of colonies strongly shapes the adaptation landscape. The assay provides a scalable platform for dissecting condition-specific features of CRISPR-Cas adaptation, including spacer origin, sequence features, and growth context.},
}
@article {pmid41118394,
year = {2025},
author = {Gouin, Y and Wilcockson, A and Chan, AM and Suttle, CA and Zhong, KX},
title = {The genome of Gallaecimonas pentaromativorans strain 10A, isolated from a Pacific oyster, sheds light on an environmentally widespread genus with remarkable metabolic potential.},
journal = {PloS one},
volume = {20},
number = {10},
pages = {e0334406},
pmid = {41118394},
issn = {1932-6203},
mesh = {Animals ; *Genome, Bacterial ; Phylogeny ; *Ostreidae/microbiology ; },
abstract = {Bacteria in the genus Gallaecimonas are known for their ability to breakdown complex hydrocarbons, making them of particular ecological and biotechnological significance. However, few species have been isolated to date, and their ecological distribution has yet to be examined. Here, we report a novel strain of G. pentaromativorans, designated as strain 10A, which was isolated from a Pacific oyster (Magallana gigas, a.k.a. Crassostrea gigas) collected from a farm experiencing a mass mortality event in British Columbia (BC), Canada. Gallaecimonas pentaromativorans strain 10A is a rod-shaped, motile bacterium and has a circular genome of 4,322,156 bp encoding 3,928 protein-coding sequences (CDS). Phylogenetic analysis showed that strain 10A is closely related to members of G. pentaromativorans. Like other Gallaecimonas members, strain 10A is predicted to harbor specific pathways involved in degrading xenobiotic compounds including polycyclic aromatic hydrocarbons (PAHs), producing biosurfactants, and assimilating nitrate and sulfate; however, it is uniquely equipped with an additional 166 genes belonging to 147 protein families, including a putative higB-higA that likely contributes to enhanced stress response. Strain 10A also possesses Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) and CRISPR-associated (Cas) system (CRISPR-Cas), prevalent in Gallaecimonas (detected in three out of four species), implying a potential defense mechanism against exogenous mobile genetic elements such as plasmids and viruses. We also mined publicly available databases to establish the widespread distribution of bacteria in the genus Gallaecimonas in seawater, sediments, and freshwater across latitude, suggesting its versatility and importance to environmental processes. Ultimately, this study demonstrates that the genome of G. pentaromativorans strain 10A, isolated from a Pacific oyster, may encode a suite of putative functions, including xenobiotic breakdown, biosurfactant production, and CRISPR-Cas defense. This plasticity and breadth in metabolic function help to explain the cosmopolitan distribution of members of this genus.},
}
@article {pmid41068553,
year = {2025},
author = {Wang, YM and Xu, T and Duan, JQ and Zhao, L and You, D and Lai, SY and Qing, Y and Ge, LP and Liu, ZH and Sun, J and Zeng, X and Xu, ZW and Zhu, L},
title = {An Integrated One-Tube RPA-CRISPR/Cas13d Assay Coupled with Lateral Flow for Rapid PRRSV-1 Detection.},
journal = {Journal of agricultural and food chemistry},
volume = {73},
number = {42},
pages = {27080-27088},
doi = {10.1021/acs.jafc.5c07919},
pmid = {41068553},
issn = {1520-5118},
mesh = {*CRISPR-Cas Systems ; *Nucleic Acid Amplification Techniques/methods/instrumentation ; Animals ; *Porcine respiratory and reproductive syndrome virus/genetics/isolation &amp; purification ; Swine ; *Porcine Reproductive and Respiratory Syndrome/virology/diagnosis ; RNA, Viral/genetics ; },
abstract = {Rapid and scalable diagnostic technologies are essential for controlling infectious diseases. We present STEP (Single-Tube Extraction-free Platform for CRISPR/Cas13d detection), a streamlined, equipment-minimal CRISPR-based platform enabling rapid, sensitive, and accurate viral RNA detection. STEP integrates isothermal RPA amplification with CRISPR-based nucleic acid cleavage, providing multimodal readouts including lateral flow strips, in-tube fluorescence, and fluorescence quantification. Lab-free extraction reagents and lyophilized formulations enhance user friendliness of STEP and stability for point-of-care testing (POCT), reduce cost, and eliminate cold-chain requirements. Optimization of baseline time and the ability to operate at both ambient and body temperatures minimize temporal and equipment constraints. Clinical evaluation showed 100% sensitivity and specificity versus RT-qPCR, delivering a sample-to-answer workflow within 35 min. STEP provides a robust platform for decentralized infectious disease diagnostics and rapid public health response, combining speed, user friendliness, and minimal instrumentation requirements.},
}
@article {pmid41015034,
year = {2025},
author = {Fontana, L and Martinucci, P and Amistadi, S and Felix, T and Mombled, M and Tachtsidi, A and Corre, G and Chalumeau, A and Hardouin, G and Martin, J and Romano, O and Amendola, M and Antoniou, P and Miccio, A},
title = {Multiplex base editing of BCL11A regulatory elements to treat sickle cell disease.},
journal = {Cell reports. Medicine},
volume = {6},
number = {10},
pages = {102376},
doi = {10.1016/j.xcrm.2025.102376},
pmid = {41015034},
issn = {2666-3791},
mesh = {*Anemia, Sickle Cell/genetics/therapy ; *Gene Editing/methods ; Humans ; Fetal Hemoglobin/metabolism/genetics ; Animals ; *Repressor Proteins/genetics ; CRISPR-Cas Systems/genetics ; Mice ; Hematopoietic Stem Cells/metabolism ; *Regulatory Sequences, Nucleic Acid/genetics ; DNA Breaks, Double-Stranded ; *Carrier Proteins/genetics/metabolism ; },
abstract = {Sickle cell disease (SCD) is a genetic anemia caused by the production of an abnormal adult hemoglobin. Elevated levels of fetal hemoglobin (HbF) in adulthood reduce disease severity. A promising therapy involves the treatment of hematopoietic stem/progenitor cells (HSPCs) with CRISPR-Cas9 to downregulate the HbF repressor BCL11A via generation of double-strand breaks (DSBs) in the +58-kb enhancer. To improve safety and HbF induction, we use base editors to target both the +58-kb and +55-kb enhancers without generating DSBs. We dissect key DNA motifs recognized by transcriptional activators and identify critical nucleotides. Multiplex base editing efficiently disrupts these sites, reactivating HbF to levels exceeding those achieved with CRISPR-Cas9-induced editing, while minimizing DSBs and genomic rearrangements. Base editing is effective in long-term repopulating HSPCs and results in robust HbF reactivation in vivo. These findings demonstrate that multiplex base editing of BCL11A enhancers is a safe, efficient, and durable strategy to treat SCD.},
}
@article {pmid40993398,
year = {2025},
author = {Datlinger, P and Pankevich, EV and Arnold, CD and Pranckevicius, N and Lin, J and Romanovskaia, D and Schaefer, M and Piras, F and Orts, AC and Nemc, A and Biesaga, PN and Chan, M and Neuwirth, T and Artemov, AV and Li, W and Ladstätter, S and Krausgruber, T and Bock, C},
title = {Systematic discovery of CRISPR-boosted CAR T cell immunotherapies.},
journal = {Nature},
volume = {646},
number = {8086},
pages = {963-972},
pmid = {40993398},
issn = {1476-4687},
mesh = {Humans ; Animals ; Mice ; *Receptors, Chimeric Antigen/immunology/genetics/metabolism ; *T-Lymphocytes/immunology/metabolism/cytology ; *CRISPR-Cas Systems/genetics ; *Immunotherapy, Adoptive/methods ; Female ; Gene Editing ; Gene Knockout Techniques ; rho GTP-Binding Proteins/genetics/deficiency ; Xenograft Model Antitumor Assays ; Male ; Leukemia/therapy/immunology/genetics ; *Immunotherapy/methods ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; },
abstract = {Chimeric antigen receptor (CAR) T cell therapy has shown remarkable success in treating blood cancers, but CAR T cell dysfunction remains a common cause of treatment failure[1]. Here we present CELLFIE, a CRISPR screening platform for enhancing CAR T cells across multiple clinical objectives. We performed genome-wide screens in human primary CAR T cells, with readouts capturing key aspects of T cell biology, including proliferation, target cell recognition, activation, apoptosis and fratricide, and exhaustion. Screening hits were prioritized using a new in vivo CROP-seq[2] method in a xenograft model of human leukaemia, establishing several gene knockouts that boost CAR T cell efficacy. Most notably, we discovered that RHOG knockout is a potent and unexpected CAR T cell enhancer, both individually and together with FAS knockout, which was validated across multiple in vivo models, CAR designs and sample donors, and in patient-derived cells. Demonstrating the versatility of the CELLFIE platform, we also conducted combinatorial CRISPR screens to identify synergistic gene pairs and saturation base-editing screens to characterize RHOG variants. In summary, we discovered, validated and biologically characterized CRISPR-boosted CAR T cells that outperform standard CAR T cells in widely used benchmarks, establishing a foundational resource for optimizing cell-based immunotherapies.},
}
@article {pmid40993381,
year = {2025},
author = {Knudsen, NH and Escobar, G and Korell, F and Kienka, T and Nobrega, C and Anderson, S and Cheng, AY and Zschummel, M and Armstrong, A and Bouffard, A and Kann, MC and Goncalves, S and Pope, HW and Pezeshki, M and Rojas, A and Suermondt, JSMT and Phillips, M and Berger, TR and Park, S and Salas-Benito, D and Darnell, EP and Birocchi, F and Leick, MB and Larson, RC and Doench, JG and Sen, D and Yates, KB and Manguso, RT and Maus, MV},
title = {In vivo CRISPR screens identify modifiers of CAR T cell function in myeloma.},
journal = {Nature},
volume = {646},
number = {8086},
pages = {953-962},
pmid = {40993381},
issn = {1476-4687},
mesh = {*Multiple Myeloma/therapy/immunology/genetics/pathology ; Animals ; Humans ; Mice ; *Receptors, Chimeric Antigen/immunology/metabolism/genetics ; *T-Lymphocytes/immunology/cytology/metabolism ; *CRISPR-Cas Systems/genetics ; Female ; *Immunotherapy, Adoptive ; Male ; Cell Proliferation ; Cell Line, Tumor ; B-Cell Maturation Antigen/immunology ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; },
abstract = {Chimeric antigen receptor (CAR) T cells are highly effective in haematological malignancies[1]. However, progressive loss of CAR T cells contributes to relapse in many patients[2-4]. Here we performed in vivo loss-of-function CRISPR screens in CAR T cells targeting B cell maturation antigen to investigate genes that influence CAR T cell persistence and function in a human multiple myeloma model. We tracked the expansion and persistence of CRISPR library-edited T cells in vitro and at early and late time points in vivo to track the performance of gene-modified CAR T cells from manufacturing to survival in tumours. The screens revealed context-specific regulators of CAR T cell expansion and persistence. Ablation of RASA2 and SOCS1 enhanced T cell expansion in vitro, whereas loss of PTPN2, ZC3H12A and RC3H1 conferred early growth advantages to CAR T cells in vivo. Notably, we identified cyclin-dependent kinase inhibitor 1B (encoded by CDKN1B), a cell cycle regulator, as the most important factor limiting CAR T cell fitness at late time points in vivo. CDKN1B ablation increased CAR T cell proliferation and effector function, significantly enhancing tumour clearance and overall survival. Our findings reveal differing effects of gene perturbation on CAR T cells over time and in different environments, highlight CDKN1B as a promising target to generate highly effective CAR T cells for multiple myeloma and underscore the potential of in vivo screening for identifying genes to enhance CAR T cell efficacy.},
}
@article {pmid40993380,
year = {2025},
author = {Mittler, E and Tse, AL and Tran, PT and Florez, C and Janer, J and Varnaite, R and Kasikci, E and Mv, VK and Loomis, M and Christ, W and Cazares, E and Bakken, RR and Martin, CK and Zeng, X and Raymond, JL and Shahsavani, M and Khanal, S and Wilkinson, ER and Oktavia, RM and Slough, MM and Haslwanter, D and Han, J and Berrigan, J and Rosendal, E and Kielian, M and Manicassamy, B and Överby, AK and Falk, A and Barba-Spaeth, G and Rey, FA and Klingström, J and Gavathiotis, E and Herbert, AS and Chandran, K and Gredmark-Russ, S},
title = {LRP8 is a receptor for tick-borne encephalitis virus.},
journal = {Nature},
volume = {646},
number = {8086},
pages = {945-952},
pmid = {40993380},
issn = {1476-4687},
mesh = {Animals ; Mice ; Humans ; *Encephalitis Viruses, Tick-Borne/physiology/metabolism/pathogenicity ; Reelin Protein ; *LDL-Receptor Related Proteins/metabolism/genetics ; *Encephalitis, Tick-Borne/virology/prevention &amp; control/metabolism ; *Receptors, Virus/metabolism/genetics ; Female ; Cell Line ; Virus Internalization ; Viral Envelope Proteins/metabolism ; Male ; Neurons/virology/metabolism ; Brain/metabolism/virology ; Virus Attachment ; HEK293 Cells ; CRISPR-Cas Systems/genetics ; },
abstract = {Tick-borne encephalitis virus (TBEV) causes tick-borne encephalitis (TBE), a severe and sometimes life-threatening disease characterized by viral invasion of the central nervous system with symptoms of neuroinflammation[1,2]. As with other orthoflaviviruses-enveloped, arthropod-borne RNA viruses-host factors required for TBEV entry remain poorly defined. Here we used a genome-scale CRISPR-Cas9-based screen to identify LRP8, an apolipoprotein E and reelin receptor with high expression in the brain, as a TBEV receptor. LRP8 downregulation reduced TBEV infection in human cells, and its overexpression enhanced infection. LRP8 bound directly to the TBEV E glycoprotein and mediated viral attachment and internalization into cells. An LRP8-based soluble decoy blocked infection of human cell lines and neuronal cells and protected mice from lethal TBEV challenge. LRP8's role as a TBEV receptor has implications for TBEV neuropathogenesis and the development of antiviral countermeasures.},
}
@article {pmid40963013,
year = {2025},
author = {Tamura, S and Nelson, AD and Spratt, PWE and Hamada, EC and Zhou, X and Kyoung, H and Li, Z and Arnould, C and Barskyi, V and Krupkin, B and Young, K and Zhao, J and Holden, SS and Sahagun, A and Keeshen, CM and Lu, C and Ben-Shalom, R and Taloma, SE and Schamiloglu, S and Li, YC and Min, L and Jenkins, PM and Pan, JQ and Paz, JT and Sanders, SJ and Matharu, N and Ahituv, N and Bender, KJ},
title = {CRISPR activation for SCN2A-related neurodevelopmental disorders.},
journal = {Nature},
volume = {646},
number = {8086},
pages = {983-991},
pmid = {40963013},
issn = {1476-4687},
mesh = {Animals ; Mice ; *NAV1.2 Voltage-Gated Sodium Channel/genetics/deficiency ; Humans ; *Neurodevelopmental Disorders/genetics/therapy/physiopathology ; Haploinsufficiency/genetics ; Dependovirus/genetics ; Male ; Female ; *CRISPR-Cas Systems/genetics ; Seizures/genetics/therapy/chemically induced ; Pyramidal Cells/metabolism/pathology ; Gene Knock-In Techniques ; Phenotype ; Synapses/metabolism ; Disease Models, Animal ; Proof of Concept Study ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; *Genetic Therapy/methods ; },
abstract = {Most neurodevelopmental disorders with single gene diagnoses act via haploinsufficiency, in which only one of the two gene copies is functional[1]. SCN2A haploinsufficiency is one of the most frequent causes of neurodevelopmental disorder, often presenting with autism spectrum disorder, intellectual disability and, in a subset of children, refractory epilepsy[2]. Here, using SCN2A haploinsufficiency as a proof-of-concept, we show that upregulation of the existing functional gene copy through CRISPR activation (CRISPRa) can rescue neurological-associated phenotypes in Scn2a haploinsufficient mice. We first show that restoring Scn2a expression in adolescent heterozygous Scn2a conditional knock-in mice rescues electrophysiological deficits associated with Scn2a haploinsufficiency (Scn2a[+/-]). Next, using an adeno-associated virus CRISPRa-based treatment in adolescent mice, we show that we can correct intrinsic and synaptic deficits in neocortical pyramidal cells, a major cell type that contributes to neurodevelopmental disorders and seizure aetiology in SCN2A haploinsufficiency. Furthermore, we find that systemic delivery of CRISPRa protects Scn2a[+/-] mice against chemoconvulsant-induced seizures. Finally, we also show that adeno-associated virus CRISPRa treatment rescues excitability in SCN2A haploinsufficient human stem-cell-derived neurons. Our results showcase the potential of this therapeutic approach to rescue SCN2A haploinsufficiency and demonstrates that rescue even at adolescent stages can ameliorate neurodevelopmental phenotypes.},
}
@article {pmid40919940,
year = {2025},
author = {Le, Y and Liu, X and Zhou, S and Wu, P and Zhang, M and Sun, J and Ni, J and Wang, H},
title = {A thermostable Cas9-based genome editing system for thermophilic acetogenic bacterium Thermoanaerobacter kivui.},
journal = {Applied and environmental microbiology},
volume = {91},
number = {10},
pages = {e0117025},
doi = {10.1128/aem.01170-25},
pmid = {40919940},
issn = {1098-5336},
support = {32470130//National Natural Science Foundation of China/ ; BK20231326//Natural Science Foundation of Jiangsu Province/ ; 2020YFA0906800//National Key Research and Development Program of China/ ; M2022-10//State Key Laboratory of Microbial Technology Open Projects Fund/ ; },
mesh = {*Thermoanaerobacter/genetics/metabolism ; *Gene Editing/methods ; *CRISPR-Cas Systems ; Ethanol/metabolism ; Alcohol Dehydrogenase/genetics/metabolism ; Metabolic Engineering ; Genome, Bacterial ; *CRISPR-Associated Protein 9/genetics/metabolism ; Bacterial Proteins/genetics/metabolism ; Hot Temperature ; },
abstract = {Thermoanaerobacter kivui is a thermophilic acetogenic bacterium capable of thriving at elevated temperatures up to 66°C. It metabolizes carbohydrates such as glucose, mannose, and fructose and can also grow lithotrophically utilizing hydrogen (H2) and carbon dioxide (CO2) or carbon monoxide (CO), with acetate serving as its main product. A simple and efficient genome editing system for T. kivui would not only facilitate the understanding of the physiological function of enzymes involved in energy and carbon metabolism but also enable metabolic engineering. To address this issue, we developed a thermostable Cas9-based genome editing system for targeted gene knockout and gene integration into the T. kivui genome. Gene knockout assays were conducted on the adh gene, responsible for encoding alcohol dehydrogenase, and the ldh gene, encoding lactate dehydrogenase. Furthermore, the adhE gene from Thermoanaerobacter ethanolicus, which encodes a bifunctional aldehyde/alcohol dehydrogenase enzyme, was successfully integrated into the T. kivui genome. As a result, the engineered strain was able to produce ethanol. Following a liquid culturing period with kanamycin sulfate for about 72 hours, the efficiency of gene editing was enhanced, resulting in a ratio of mutants out of all colonies obtained of 90%. The results confirm the validity and efficiency of the thermostable Cas9-based genome editing system in T. kivui for gene editing.IMPORTANCEThermophilic acetogenic microorganisms represent an emerging metabolic engineering platform for the production of various biochemicals from hydrogen and carbon dioxide, or synthesis gas, under conditions of high-temperature fermentation. Gas fermentation has gained significant research interest due to its excellent thermodynamics, economic feasibility, and multisubstrate utilization. However, a major obstacle to the use of thermophilic acetogenic microorganisms as metabolic engineering platforms is the scarcity of genetic tools. This study demonstrates a proof of concept for a thermostable Cas9-based genome editing of the thermophilic acetogenic bacterium T. kivui. The system is an important expansion to the genetic toolbox of T. kivui, enabling a better understanding of key enzyme functions and the construction of cell factories for the biotechnological conversion of carbon dioxide and organic substrates into value-added products.},
}
@article {pmid40891847,
year = {2025},
author = {Li, L and Luo, K and Zhang, S and Wang, X and Wang, S and Liu, X and Zang, S and Liu, Y and Zhou, C and Luo, C},
title = {A three-plasmid-containing CRISPR-Cas9 platform to engineer Bacillus velezensis 916 as an efficient biocontrol agent.},
journal = {Applied and environmental microbiology},
volume = {91},
number = {10},
pages = {e0138925},
doi = {10.1128/aem.01389-25},
pmid = {40891847},
issn = {1098-5336},
mesh = {*Bacillus/genetics ; *CRISPR-Cas Systems ; *Gene Editing/methods ; *Plasmids/genetics ; *Biological Control Agents ; Plant Diseases/prevention &amp; control/microbiology ; Lipopeptides/genetics ; Bacterial Proteins/genetics ; Peptide Synthases/genetics ; Oryza/microbiology ; },
abstract = {Bacillus velezensis (Bv) is a widely used biocontrol agent against plant diseases, mainly because its genome contains numerous non-ribosomal peptide synthetases (NRPS) gene clusters for the synthesis of various cyclic lipopeptides (CLPs). The domesticated strain Bv916, capable of co-producing four CLPs, has been successfully applied for green control of rice sheath blight and angular leaf spot. To enhance Bv916's biological control efficacy while maintaining environmental safety, it is essential to establish a food-grade gene editing platform in Bv916. Here, a three-plasmid CRISPR-Cas9 platform for Bv916 was constructed using the thermosensitive origin pET194ts, constitutive P43 promoters for Cas9, the specific promoter Psrf for single guide RNAs (sgRNAs), and three resistance gene expression cassettes. By replacing the native promoters of ComX and RecA in Bv916 with the strong promoters P43 and PrepU, respectively, this platform achieved a single-gene editing efficiency of 96%, while the simultaneous dual-gene editing efficiency reached 61%, with each round completed within five business days. Furthermore, this gene editing platform is used to replace promoters of four NRPS gene clusters (loc, srf, bl, and fen) in Bv916 with strong constitutive promoters (PB, PA, P43, and PrepU), generating the derivative BvLSBF. Compared to Bv916, BvLSBF showed 6.8-fold, 5.9-fold, 10.9-fold, and 6.2-fold increases in locillomycin, surfactin, bacillomycin L, and fengycin, respectively. Its antagonistic activity against plant pathogens was also significantly enhanced. This system enables further development of Bv916 as a cell factory and integration of multiple biocontrol factors, offering significant potential for sustainable agriculture.IMPORTANCEIn this study, a food-grade three-plasmid CRISPR-Cas9 platform for Bv916 was established by incorporating the optimized BvCas9 under the constitutive promoter P43, single guide RNAs (sgRNAs), and homologous recombination fragments into three thermosensitive shuttle vectors. This gene editing system was used to achieve gene insertion, deletion, and replacement in Bv916, particularly by editing four non-ribosomal peptide synthetase (NRPS) gene clusters. This resulted in increased production of four cyclic lipopeptides and significantly enhanced antibacterial and antifungal activity.},
}
@article {pmid41118255,
year = {2025},
author = {Ong, CJN and Elesho, OE and Bramwell, BB and Cabuhat, KSP and Bacalzo, GD and Nuevo, JJM and Fortaleza, JAG},
title = {Staphylococcus aureus: Antimicrobial resistance, quorum sensing, and antibiofilm approaches.},
journal = {European journal of microbiology &amp; immunology},
volume = {},
number = {},
pages = {},
doi = {10.1556/1886.2025.00050},
pmid = {41118255},
issn = {2062-509X},
abstract = {Staphylococcus aureus is a clinically important bacterial pathogen causing infections from superficial skin lesions to life-threatening systemic diseases. The emergence of methicillin-resistant S. aureus (MRSA) has compounded the global health burden, particularly in low- and middle-income countries, as its quorum-sensing (QS) mediated mechanisms contribute to its persistence, resistance, and evasion from host immune responses and antimicrobial treatments. Thus, these features compromise the effectiveness of conventional antibiotics, urging the need for alternative therapeutic approaches. To resolve these issues, several non-antibiotic antibiofilm approaches have been developed. Bacteriophages and phage-derived enzymes show promising specificity in lysing bacterial cells and disrupting biofilms. Antimicrobial peptides (AMPs), with their broad-spectrum activity, destabilize bacterial membranes and modulate immune responses. Monoclonal antibodies can neutralize toxins or inhibit adhesion molecules within biofilms. Phytochemicals have demonstrated activity against QS pathways and efflux pumps. Metal ion chelators like deferiprone interfere with iron acquisition, which is essential for biofilm stability. Nanoparticles (NPs), ranging from metallic and polymeric to lipid-based and cyclodextrin-based systems, enhance drug delivery and biofilm penetration. CRISPR-Cas systems provide precise genome editing to target resistance genes and virulence factors. Rhamnolipids disrupt biofilm matrix integrity, while enzymes such as dispersin B degrade extracellular polymeric substances. Photodynamic and laser therapies offer localized disruption of biofilm structures through oxidative stress. Collectively, this review offers a transformative complementary approach to traditional antibiotics, enhancing treatment efficacy while potentially reducing the emergence of resistance. Continued research on delivery systems, safety profiles, and synergistic combinations will be pivotal for their clinical translation against S. aureus infections.},
}
@article {pmid41117937,
year = {2025},
author = {Menge, S and Segura, I and Hartmann, M and Decker, L and Kiran, S and Danzer, KM and Iben, S and Harbauer, AB and Oeckl, P and Freischmidt, A},
title = {Comparing loss of individual fragile X proteins suggests strong links to cellular senescence and aging.},
journal = {Cellular and molecular life sciences : CMLS},
volume = {82},
number = {1},
pages = {358},
pmid = {41117937},
issn = {1420-9071},
mesh = {Humans ; *Cellular Senescence/genetics ; *Fragile X Mental Retardation Protein/genetics/metabolism ; *Aging/genetics/metabolism ; *RNA-Binding Proteins/metabolism/genetics ; CRISPR-Cas Systems ; Autophagy ; Cell Line, Tumor ; Proteomics/methods ; Mitochondria/metabolism ; Fragile X Syndrome/metabolism/genetics/pathology ; Neurodegenerative Diseases/metabolism/genetics/pathology ; },
abstract = {Members of the fragile X protein (FXP) family (FMR1, FXR1 and FXR2) are differentially expressed in most types of cancer and major neurodegenerative diseases. While increased expression of FXR1 in cancer has been linked to senescence evasion and consequently tumor initiation and progression, decreased expression of FXPs in neurodegeneration may contribute to pathogenic protein aggregation and death of vulnerable neurons. However, due the causal role in fragile x syndrome, most data are available about loss of FMR1 in neurons while functions of FXR1 and especially FXR2 remain largely unexplored. To address this knowledge gap, and to directly compare functions of the FXPs, we used proteomics of CRISPR/Cas9 edited HAP1 cells carrying knockouts of the individual FXPs for identification of cellular mechanisms associated with these proteins. Further exploration of proteomic findings suggests roles of the FXPs in ribosome biogenesis, autophagy and mitochondrial health linked to organismal aging, and cellular senescence. Validation of FXP induced defects relevant for neurodegenerative diseases in neuroblastoma cell line SH-SY5Y upon FXP knockdown revealed high cell type specificity of individual FXP functions. Overall, we provide a comprehensive overview and comparison of cellular mechanisms related to the individual FXPs, as well as starting points for further studying this protein family in respective cell types of FXP associated diseases, and in aging in general.},
}
@article {pmid41117602,
year = {2025},
author = {Keshry, SS and Nayak, U and Mamidi, P and Mohanty, S and Ghorai, U and Swain, RK and Chattopadhyay, S},
title = {CRISPR-Induced Mutations of mk2b and mk3 Host Proteins Enhance Chikungunya Virus Susceptibility and Modulate Host Immune Responses in Zebrafish.},
journal = {FASEB journal : official publication of the Federation of American Societies for Experimental Biology},
volume = {39},
number = {20},
pages = {e71112},
doi = {10.1096/fj.202501236RR},
pmid = {41117602},
issn = {1530-6860},
support = {113-2334-6704/2K23/1//CSIR | Human Resource Development Centre, Council of Scientific And Industrial Research (HRDC)/ ; IF180156//Department of Science and Technology, Ministry of Science and Technology, India (DST)/ ; BT/PR42322/TRM/120/525/2021//Department of Biotechnology, Ministry of Science and Technology, India (DBT)/ ; },
mesh = {Animals ; *Zebrafish/genetics/virology/immunology ; *Chikungunya virus/immunology ; *Chikungunya Fever/immunology/genetics/virology ; *Protein Serine-Threonine Kinases/genetics/metabolism ; *Zebrafish Proteins/genetics/metabolism ; *Intracellular Signaling Peptides and Proteins/genetics/metabolism ; CRISPR-Cas Systems ; *Mutation ; Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {Host factors are essential at every stage of the viral life cycle and therefore represent attractive and potentially effective targets for the development of antiviral therapeutics. This study highlights the crucial roles of host factors, specifically mitogen-activated protein kinase 2 (mk2) and mitogen-activated protein kinase 3 (mk3), both of which are stress-stimulated serine/threonine kinases. The roles of mk2 and mk3 were investigated by generating single (mk2b-/- and mk3-/-) and double knockouts (mk2b-/-mk3-/-) in a zebrafish model using the CRISPR-Cas9 technique, followed by chikungunya virus (CHIKV) infection. All knockout lines exhibited significantly higher CHIKV titers and severe phenotypes compared to the WT control, with mk3[-]/[-] showing the greatest susceptibility. After CHIKV infection, expression levels of TNF-α changed across all knockout models. Notably, mk2b[-]/[-] and mk2b[-]/[-]mk3[-]/[-] double knockout larvae exhibited reduced TNF-α expression, suggesting that higher levels of TNF-α may be associated with viral clearance via the p38-MK2-TNF-α signaling axis. In contrast, mk3-/- zebrafish exhibited increased vulnerability to CHIKV through alternative, yet unidentified, pathways. Furthermore, an increase in viral titer corresponded with an enhanced host immune response, as indicated by significantly higher expression levels of ifnɸ1 and rsad2 in all knockout groups. In conclusion, this study confirms that the mk2b and mk3 host proteins are essential in controlling CHIKV infection at the organism level. These findings might have implications towards designing strategies for future antiviral therapeutics. Furthermore, the knockout model of mk2b and mk3 in zebrafish could serve as a valuable tool for studying their roles in other viral infections.},
}
@article {pmid41116722,
year = {2025},
author = {Lin, Y and Jiang, D and Dong, X and Li, Y and Wu, X and Li, R and Li, F and Sun, D and Yu, Y},
title = {A label-free orthogonal dual-channel CRISPR-Cas platform for simultaneous detection of Mycobacterium tuberculosis and respiratory syncytial virus.},
journal = {Chemical communications (Cambridge, England)},
volume = {},
number = {},
pages = {},
doi = {10.1039/d5cc05269f},
pmid = {41116722},
issn = {1364-548X},
abstract = {A label-free, orthogonal dual-channel CRISPR-Cas platform is developed for the simultaneous detection of diverse pathogens. Cas12a is reported by a G4-PPIX complex, and Cas13 by a split Broccoli-DFHBI aptamer, which ensure exclusive channel specificity and eliminate optical cross-talk. Clinical tests on 50 samples show 100% concordance with PCR.},
}
@article {pmid41115973,
year = {2025},
author = {Mladenov, E and Kallies, M and Stuschke, M and Gkika, E and Iliakis, G},
title = {CRISPR/Cas9 generated DSB clusters mimic complex lesions induced by high-LET radiation and shift repair from c-NHEJ to mutagenic repair pathways.},
journal = {Scientific reports},
volume = {15},
number = {1},
pages = {36480},
pmid = {41115973},
issn = {2045-2322},
mesh = {*CRISPR-Cas Systems ; Humans ; *DNA Breaks, Double-Stranded/radiation effects ; *DNA End-Joining Repair/radiation effects/genetics ; *Linear Energy Transfer ; Hypoxanthine Phosphoribosyltransferase/genetics ; Animals ; *DNA Repair ; Cell Line ; Poly (ADP-Ribose) Polymerase-1/genetics/metabolism ; Mutagenesis ; },
abstract = {DNA double-strand break (DSB) clusters are a hallmark of high-linear energy transfer (high-LET) radiation and are associated with pronounced biological effects, including reduced cell survival and elevated genomic instability. Our previous work in Chinese hamster cells, engineered with variably designed clusters of I-SceI recognition sites, integrated at multiple genomic locations, revealed that DSB clusters suppress classical non-homologous end-joining (c-NHEJ) and induce chromosomal translocations that ultimately increase cell lethality. Here, we extend this line of investigation to human cell lines and generate DSB clusters using alternative approaches that do not require prior genetic manipulation of the test cell lines. We employ CRISPR/Cas9-technology to generate DSB clusters of specific design at a selected genomic locus and examine their consequences on locus integrity. We target Exon 3 of the human HPRT (hHPRT) gene and introduce single DSBs or DSB clusters of varying numbers and inter-DSB distances. Alterations at the locus reflecting hHPRT gene inactivation, are quantified as mutations causing resistance to 6-thioguanine (6TG). Our results show that DSB clusters are markedly more potent inducers of mutations than single DSBs and that DSBs spaced within ~ 600 base pairs synergize in mutation induction. Mechanistic analyses using small-molecule inhibitors and engineered gene knockout cell lines reveal that the increased mutagenicity of clustered DSBs is primarily mediated by DNA end resection and PARP1-dependent alternative end-joining (alt-EJ) pathways. These findings reinforce the biological relevance of DSB clusters as a severe form of complex DNA damage and provide mechanistic insights into high-LET radiation-induced increased cell killing and genomic instability.},
}
@article {pmid41114873,
year = {2025},
author = {Su, CW and Tsai, LC and Hsu, YC and Lu, YS and Lee, JC and Linacre, A and Hsieh, HM},
title = {Saliva identification by RT-LAMP integrated with CRISPR-Cas and LFA.},
journal = {Forensic science, medicine, and pathology},
volume = {},
number = {},
pages = {},
pmid = {41114873},
issn = {1556-2891},
support = {MOST 110-2320-B-015-001 and MOST 111-2320-B-015-001-MY2//National Science and Technology Council/ ; },
abstract = {Saliva is a frequently encountered body fluid at crime scenes, however currently there are no definite means to rapidly identify a body fluid as being saliva. In this study, a novel detection method for saliva using a modified Loop-mediated Isothermal Amplification (LAMP) integrated with CRISPR-Cas (Clustered Regularly Interspaced Short Palindromic Repeat-CRISPR associated protein) and LFA (Lateral Flow Assay) was developed to detect the expression of a saliva-specific gene: follicular dendric cell secreted protein (FDCSP). To determine the specificity of the assay, RNA from saliva plus other commonly encountered body fluids was tested (peripheral blood, semen, vaginal fluid, and menstrual blood): positive results were only observed from RNA extracted from known saliva samples and RNA from all the other body fluids exhibited a negative result. To assess the reproducibility, triplicates were used from one saliva sample, and the assay was performed on three different days: positive results were observed from all triplicates. The limit of detection was 2[-6] (0.3906 ng RNA) or 2[-7] (0.1953 ng RNA). This preliminary study for the identification of saliva requires no complex equipment and is easy to perform, offering an alternative means for body fluid identification.},
}
@article {pmid41113410,
year = {2025},
author = {Feng, L and Huang, Y and Zhao, R and Zhang, K and Yang, W},
title = {[Effect of Different Caenorhabditis elegans U6 Promoters on the Efficiency of CRISPR/Cas9-Mediated Gene Editing].},
journal = {Sichuan da xue xue bao. Yi xue ban = Journal of Sichuan University. Medical science edition},
volume = {56},
number = {4},
pages = {1038-1044},
pmid = {41113410},
issn = {1672-173X},
mesh = {Animals ; *Caenorhabditis elegans/genetics ; *Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; *Promoter Regions, Genetic/genetics ; *RNA, Small Nuclear/genetics ; Plasmids/genetics ; Caenorhabditis elegans Proteins/genetics ; },
abstract = {OBJECTIVE: To investigate the effects of Caenorhabditis elegans (C. elegans) endogenous U6 promoters on dpy-10 gene editing efficiency.<br><br>METHODS: We screened endogenous U6 small nuclear RNA (snRNA) genes of C. elegans from the WormBase database and constructed 14 editing plasmids targeting dpy-10 by replacing the U6 r07e5.16 promoter in the pSX524 plasmid (Peft-3::cas9::tbb-2 terminator::U6 r07e5.16::dpy-10 sgRNA) through molecular cloning. Gene editing was performed in wild-type C. elegans using a standardized microinjection protocol. Gene editing efficiency and the high-efficiency gene editing index were quantified based on the screening of dpy-10 mutant phenotypes in the F1 progeny.<br><br>RESULTS: A total of 15 U6 snRNA genes (r07e5.16, f35c11.9, t20d3.13, k09b11.15, k09b11.16, w05b2.8, c28a5.7, f54c8.9, k09b11.11, k09b11.12, k09b11.14, t20d3.12, f54c8.8, f54c8.10, and k09b11.13) were identified from the WormBase database. Based on the editing efficiency and high-efficiency gene editing index, the activity of these promoters was evaluated, and 4 U6 promoters (w05b2.8, c28a5.7, f54c8.9, and k09b11.11) were found to have significantly enhanced gene editing success rates, outperforming other promoters, including U6 r07e5.16 and U6 k09b11.12 , which are commonly used in the C. elegans research community. Notably, the gRNA[F+E] scaffold did not show superior editing efficiency over the gRNA scaffold when paired with the optimal U6w05b2.8 promoter.<br><br>CONCLUSION: In this study, U6 promoters that significantly improve gene editing efficiency in C. elegans are identified and the critical role of promoter optimization in CRISPR-Cas9 systems is highlighted. These findings provide a valuable foundation for improving genome editing strategies and offer new ideas for optimizing the CRISPR technology applied in nematode research.},
}
@article {pmid41112309,
year = {2025},
author = {Cruz-Cárdenas, JA and López-Arredondo, A and Cázares-Preciado, JA and Rodríguez-Gonzalez, M and Palomares, LA and Brunck, MEG},
title = {Development of CRISPR/Cas9-mediated CD16b[-/-] and CD32a[-/-] promyelocytic cell lines to study FcγR signaling in human neutrophils.},
journal = {Frontiers in immunology},
volume = {16},
number = {},
pages = {1633609},
pmid = {41112309},
issn = {1664-3224},
mesh = {Humans ; *Receptors, IgG/genetics/metabolism/immunology ; *Neutrophils/immunology/metabolism ; *CRISPR-Cas Systems ; *Signal Transduction/immunology ; HL-60 Cells ; Phagocytosis ; GPI-Linked Proteins/genetics ; Reactive Oxygen Species/metabolism ; Cytokines/metabolism ; },
abstract = {INTRODUCTION: Neutrophils use Fc gamma receptors (FcγRs) to recognize IgG-opsonized pathogens, triggering antimicrobial functions including phagocytosis, ROS production, and cytokine release. CD16b, the most abundant FcγR on neutrophils, plays a key role in initiating these responses, while CD32a is another abundant FcγR on neutrophils that contributes to modulating immune functions. CD16b lacks an intracellular domain and its signaling mechanisms remain unclear. The prevalence of the CD16b-deficient phenotype on donor neutrophils is estimated at <1% of the global population, which complicates its study. To address this, we employed CRISPR/Cas9 to generate HL-60-derived neutrophil-like cells deficient for CD16b or CD32a, that facilitate investigation of their respective roles in neutrophil biology.<br><br>METHODS: We disrupted the FCGR3B or FCGR2A genes using CRISPR/Cas9 in the HL-60 cell line and differentiated clones into neutrophil-like cells using 1.3% DMSO. Functional assays were performed, including phagocytosis, ROS production, SYK phosphorylation, and cytokine responses.<br><br>RESULTS AND DISCUSSION: Both CD16b[-/-] and CD32a[-/-] HL-60-derived clones maintained neutrophilic differentiation and phagocytic capacity but displayed impaired Fc&#x3b3;R-mediated ROS production and SYK phosphorylation, with more pronounced defects in CD16b[-/-] cells. Cytokine production was altered in both lines, with CD16b[-/-] cells producing less IL-6 and IL-1&#x3b2;, and CD32a[-/-] cells producing less TNF-&#x3b1; and IL-10. This model provides new insights into the distinct roles of CD16b and CD32a in neutrophil activation and immune responses.},
}
@article {pmid41110011,
year = {2025},
author = {De La Mora, CL and Havey, MJ and Krysan, PJ},
title = {Efficient production of gene-edited onion (Allium cepa) plants using biolistic delivery of cas9 RNPs and transient expression constructs.},
journal = {Plant cell reports},
volume = {44},
number = {11},
pages = {243},
pmid = {41110011},
issn = {1432-203X},
support = {1844304//Division of Integrative Organismal Systems/ ; },
mesh = {*Onions/genetics ; *Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; Plants, Genetically Modified/genetics ; *Biolistics/methods ; *CRISPR-Associated Protein 9/metabolism/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; *Ribonucleoproteins/genetics/metabolism ; Plant Proteins/genetics/metabolism ; },
abstract = {Delivery of Cas9/sgRNA RNPs and DNA vectors designed to transiently express morphogenic regulatory genes or an antibiotic resistance gene enabled efficient recovery of gene edited onion lines. We developed a protocol for efficiently producing gene edited onion plants that does not depend on stable transformation. The process makes use of transient gene expression to enrich for gene editing among plants regenerated from immature embryos bombarded with ribonucleoprotein (RNP) complexes composed of CRISPR-associated protein 9 (Cas9) and single guide RNAs (sgRNAs). We used the Allium cepa Downy Mildew Resistant 6 (AcDMR6) gene as the target gene for our studies and produced a total of 47 onion plants with edited AcDMR6 alleles, including 13 homozygous plants, 12 biallelic plants, 7 heterozygous plants, and 15 chimeric plants. The most effective strategy for producing gene edited onion plants involved co-delivering plasmids encoding a hygromycin-resistance protein and plant developmental regulator genes along with the Cas9/sgRNA RNPs followed by transient Hygromycin selection for 48 h. Using this approach, up to 12% of the regenerated onion plants carried edited alleles of AcDMR6. By comparison, no editing was observed among the 146 plants regenerated from explants transfected with the Cas9/sgRNA RNPs alone. The strategy we describe here for using transient gene expression to enrich for gene editing in onion could potentially be extended to other crop species as well.},
}
@article {pmid41071600,
year = {2025},
author = {Zhang, W and Feng, ZY and Feng, Z and Lian, R and Liu, Z and Zhang, J},
title = {A CRISPR-Customizable Copper-Coordinated DNA Nanoplatform Potentiates Cuproptosis Through Circadian and Metabolic Pathway Manipulation.},
journal = {ACS nano},
volume = {19},
number = {41},
pages = {36701-36717},
doi = {10.1021/acsnano.5c12641},
pmid = {41071600},
issn = {1936-086X},
mesh = {*Copper/chemistry/pharmacology ; Humans ; Animals ; *DNA/chemistry/pharmacology ; *Circadian Rhythm/drug effects ; Mice ; Metabolic Networks and Pathways/drug effects ; Apoptosis/drug effects ; *Antineoplastic Agents/pharmacology/chemistry ; Cell Line, Tumor ; *Clustered Regularly Interspaced Short Palindromic Repeats ; ARNTL Transcription Factors/genetics/metabolism ; CRISPR-Cas Systems ; },
abstract = {Despite the promise of cuproptosis in antitumor therapy, developing strategies to enhance its therapeutic efficacy within the tumor microenvironment remains a challenge. Inspired by the chronotherapy that manipulate circadian rhythms to enhance drug effectiveness, herein we report for a CRISPR-customized copper-DNA nanoplatform (Cu-RNP) that synergistically induces multimodal cell death, including potentiated cuproptosis, by manipulating circadian and metabolic pathways. Cu-RNP integrates coordination-driven self-assembly of Cu[2+]-DNA nanospheres with Cas13d/crRNA ribonucleoproteins targeting BMAL1. Upon cellular internalization, the acidic and reducing endo/lysosomal environment triggers Cu-RNP disassembly, releasing RNP to silence BMAL1 and disrupt circadian oscillations, leading to WEE1 downregulation and p21 upregulation, thereby inducing apoptosis. Simultaneously, liberated Cu[2+] generates cytotoxic hydroxyl radicals for chemodynamic therapy (CDT) and concurrently depletes GSH, promoting mitochondrial copper overload for cuproptosis. Importantly, we demonstrate that silencing BMAL1 disrupts circadian rhythms, inhibits glycolysis, enhances mitochondrial respiration, and redirects metabolic flux to the TCA cycle, thereby amplifying the cell's vulnerability to copper-induced cuproptosis. In vitro and in vivo results demonstrate that Cu-RNP sensitizes cancer cells to cuproptosis and elicit strong antitumor response through the synergistic combination of cuproptosis, CDT, apoptosis, and circadian-metabolic modulation. This study demonstrates a mechanistic link between BMAL1-regulated circadian rhythms and cuproptosis sensitivity, suggesting a potential treatment strategy for multimodal, cuproptosis-potentiating cancer therapies.},
}
@article {pmid41024711,
year = {2025},
author = {Sanchez, A and Zhou, C and Tulaiha, R and Ramirez, F and Wang, L and Zhang, X},
title = {CRISPR Screen Identifies BAP1 as a Deubiquitinase Regulating SPIN4 Stability.},
journal = {Biochemistry},
volume = {64},
number = {20},
pages = {4318-4326},
pmid = {41024711},
issn = {1520-4995},
support = {R35 GM146979/GM/NIGMS NIH HHS/United States ; R35 GM154945/GM/NIGMS NIH HHS/United States ; T32 GM149439/GM/NIGMS NIH HHS/United States ; },
mesh = {*Ubiquitin Thiolesterase/metabolism/genetics ; Humans ; *Tumor Suppressor Proteins/metabolism/genetics ; *CRISPR-Cas Systems ; *Cell Cycle Proteins/metabolism/genetics ; Ubiquitination ; HEK293 Cells ; Protein Stability ; Ubiquitin-Protein Ligases/metabolism ; Proteolysis ; Microtubule-Associated Proteins ; Phosphoproteins ; },
abstract = {Protein homeostasis is tightly controlled by the coordinated actions of E3 ubiquitin ligases and deubiquitinases (DUBs). We previously identified Spindlin-4 (SPIN4), a histone H3K4me3 reader, as a degradation substrate of DCAF16. In this study, we confirmed this degradation pathway using an E3 ligase-focused CRISPR-Cas9 knockout screen. Furthermore, through a DUB-focused CRISPR-Cas9 knockout screen and biochemical analyses, we demonstrated that the deubiquitinase BAP1 interacts with and stabilizes SPIN4 via its deubiquitination activity. Inhibition or loss of BAP1 reduces SPIN4 levels, highlighting its critical role in maintaining SPIN4 homeostasis. Proteomics and interactome analyses further support this regulatory axis. These findings reveal a dynamic balance controlling SPIN4 stability, with potential implications for epigenetic regulation and disease processes.},
}
@article {pmid41109516,
year = {2025},
author = {Pandya, K and Kumar, D},
title = {CRISPR/Cas Genome Editing for Neurodegenerative Diseases: Mechanisms, Therapeutic Advances, and Clinical Prospects.},
journal = {Ageing research reviews},
volume = {},
number = {},
pages = {102922},
doi = {10.1016/j.arr.2025.102922},
pmid = {41109516},
issn = {1872-9649},
abstract = {Neurodegenerative diseases such as Alzheimer's disease (AD), Parkinson's disease (PD), and Huntington's disease (HD) are major public health challenges. Current treatments are only symptomatic and do not address the underlying pathogenic genetic mechanisms. The development of the CRISPR/Cas genome editing tool has increased possibilities for targeted repair of pathological mutations. CRISPR/Cas9, Cas12, and Cas13 systems enable targeted editing and transcriptome modulation in various preclinical models. CRISPR/Cas9 disruption of mutant APP and Tau genes in AD models has restored normal genetic function. This technique reduces toxic protein aggregation. Results in neurodegenerative phenotype in these models are ameliorated. Correction of CAG nucleotide repeats in HD, and reduction of alpha-synuclein expression in PD. Advancements in engineered Cas variants with enhanced specificity, such as SpCas9-HF1 and prime editors, with innovative delivery strategies including adeno-associated virus (AAVs) and nanoparticle-based systems, have improved genome editing. However, challenges remain, including off-target effects, mosaicism, and delivery across the BBB, and long-term safety. Ethical consideration focuses on somatic versus germline editing, equitable access, and regulatory oversight. While somatic editing shows acceptance in treating neurological disorders. Germline interventions face strict regulations due to potential multigeneration impacts. Collectively, these technologies are the vanguard of precision molecular medicine, advancing from symptom management towards potentially curative gene therapies for neurological disorders.},
}
@article {pmid41109509,
year = {2025},
author = {Gujjar, RS and Sherin, S and Batra, S and Mahto, A and Goswami, SK and Kumar, R and Tiwari, RK and Singh, A and Kumari, M},
title = {Emerging trends in the development of efficient CAS nucleases for meticulous gene editing in plants.},
journal = {Plant science : an international journal of experimental plant biology},
volume = {},
number = {},
pages = {112820},
doi = {10.1016/j.plantsci.2025.112820},
pmid = {41109509},
issn = {1873-2259},
abstract = {CRISPR-Cas (clustered regularly interspaced short palindromic repeats/CRISPR-associated nuclease) is a formidable and precise gene-editing system, invented as a natural immune system of bacterial defense. The system relies on a guide RNA/CrRNA (Crispr RNA) sequence, specifically designed to bind to the target nucleic acid, enabling the Cas nuclease to act like a molecular scissor and cleave the target sequence. Recent research has substantially advanced the development, optimization, and application of Cas nucleases, particularly Cas9, Cas12, and their engineered orthologs to enhance the fidelity, flexibility, and proficiency of specific edits at the target site. This review focuses on the latest advancements in the research on Cas nucleases such as Cas9, Cas12a, Cas12b, CasΦ, Cas13, and Cas14, along with their mode of function and the exclusive features of each nuclease. Further, we have elaborated on the emergence of redesigned Cas9 orthologs, such as dCas9 (deactivated Cas9) and nCas9 (Cas9 nickase). The modified Cas9 orthologs have empowered the CRISPR-based editing with sophisticated techniques such as base editing and prime editing, which are capable of providing preferred edits with more precision and specificity. These advanced genome editing approaches have been favorably employed to improve the desirable agronomic traits such as stress tolerance, herbicide resistance, and yield stability in a wide range of crops, including rice, maize, wheat, tomato, and cotton. KEY MESSAGE: Our manuscript offers the recent advancement in the research on Cas nucleases, and their modified orthologs to perform CRISPR-based gene edits with enhanced more precision and specificity.},
}
@article {pmid41108631,
year = {2025},
author = {Harzli, I and Mostafa, K and Mutlu, VA and Shamsi, IH and Kavas, M},
title = {Exploring Novel Microbial Approaches for Enhancing Crop Resilience to Abiotic Stress: Mechanisms and Applications.},
journal = {Physiologia plantarum},
volume = {177},
number = {5},
pages = {e70582},
doi = {10.1111/ppl.70582},
pmid = {41108631},
issn = {1399-3054},
support = {121O463//T&#xfc;bitak/ ; },
mesh = {*Crops, Agricultural/microbiology/physiology ; *Stress, Physiological ; Mycorrhizae/physiology ; Gene Editing ; Plant Breeding ; },
abstract = {The current state of agriculture heavily relies on chemical fertilizers and pesticides, which can negatively impact plant nutritional quality, plant health, and productivity. Additionally, abiotic stresses pose significant challenges to global agricultural productivity, threatening food security and crop sustainability. Therefore, developing and implementing sustainable alternatives to chemical fertilizers and pesticides is crucial to enhance agricultural productivity and resilience. Recent research highlights the potential of microorganisms, such as plant growth-promoting rhizobacteria (PGPR), mycorrhizal fungi, and endophytes, as sustainable solutions to improve plant resilience under abiotic stress conditions. However, challenges including scalability, ecological impacts, and the need for standardized application methods persist. This review explores novel microbial approaches to improving crop resilience against abiotic stress, focusing on how microorganisms interact with plants to mitigate stress impacts. Key mechanisms include the production of stress-alleviating compounds, enhanced nutrient uptake, and modulation of plant stress response pathways. We also examine advanced strategies in plant breeding, emphasizing CRISPR/Cas-mediated genome editing technologies as powerful tools for elucidating plant-microbe interactions. A thorough understanding of these interactions is essential for effectively applying genome editing to enhance the functional capacities of plants or associated microbes, ultimately improving key agronomic traits. This review provides a comprehensive overview of these innovative microbial approaches and their practical applications in sustainable agriculture, offering insights into future research directions, such as developing novel microbial strains and optimizing field applications.},
}
@article {pmid41108328,
year = {2025},
author = {Liu, C and Yao, D and Jiang, Y and Huo, C and Zhang, J and Yi, S and Yang, W and Han, Y and Liu, Y and Wang, X},
title = {Electroporation-based CRISPR/Cas9 Gene Editing in Haliotis Discus Hannai.},
journal = {Marine biotechnology (New York, N.Y.)},
volume = {27},
number = {6},
pages = {148},
pmid = {41108328},
issn = {1436-2236},
support = {ZR2023QC107//Natural Science Foundation of Shandong Province/ ; ZR2022QC233//Natural Science Foundation of Shandong Province/ ; 2022LZGC015//Key Research and Development Program of Shandong Province/ ; SDAIT-14-01//Modern Agricultural Industry Technology System of Shandong Province/ ; tstp20240518//Special Funds for the Taishan Scholar Project of Shandong Province/ ; 202203//Special Supporting Funds for Leading Talents above Provincial Level in Yantai City/ ; 2023HD//Aquaculture Seed Industry Promotion Project of Yantai City/ ; },
mesh = {Animals ; *CRISPR-Cas Systems ; *Gene Editing/methods ; *Electroporation/methods ; *Myostatin/genetics ; *Gastropoda/genetics/embryology ; Aquaculture ; RNA, Guide, CRISPR-Cas Systems/genetics ; },
abstract = {Abalone, a marine mollusk with significant economic and ecological value, plays a crucial role in sustainable aquaculture. The development and application of CRISPR-Cas9 gene-editing technology have opened up a new path for improving breeding efficiency. CRISPR/Cas9-mediated gene editing has been achieved in abalones via microinjection. In this study, a gene encoding myostatin MSTN in H. discus hannai; was selected as target for conducting the CRISPR-Cas9 gene editing experiment in combination with an electroporation delivery system. Our results showed that all three sgRNAs effectively targeted and cleaved the target segment, with sgRNA1 and sgRNA2 exhibiting high in vitro activity. After electroporation, the effects of transfection on embryonic development of fertilized eggs were observed and statistically analyzed. 12.7 ± 5.4% of the fertilized eggs were damaged and deformed after electroporation. Twenty-four hours after electroporation, surviving larvae were collected for DNA extraction and sequencing. Two potential mutations within the target region of MSTN were identified by sequencing. These results provide a reference for the improvement and development of CRISPR-mediated gene editing methods in marine mollusks such as abalones.},
}
@article {pmid41107544,
year = {2025},
author = {Whyms, C and Zhao, Y and Addo-Yobo, D and He, H and Whittington, AC and Trasanidou, D and Salazar, CRP and Staals, RHJ and Li, H},
title = {The twist-and-squeeze activation of CARF-fused adenosine deaminase by cyclic oligoadenylates.},
journal = {The EMBO journal},
volume = {},
number = {},
pages = {},
pmid = {41107544},
issn = {1460-2075},
support = {R35 GM152081/GM/NIGMS NIH HHS/United States ; },
abstract = {The recently identified CARF (CRISPR-associated Rossman-fold) family of proteins play a critical role in prokaryotic defense, mediating cOA (cyclic oligoadenylate)-stimulated ancillary immune responses in the type III CRISPR-Cas systems. Whereas most previously characterized CARF proteins contain nucleic acids or protein degradation effectors, a subset of the family, including the CARF-fused adenosine deaminase (ADA) (Cad1), has recently been shown to convert ATP to ITP. The enzymatic mechanism and the activation process of Cad1, however, remain incompletely understood. Here we present biochemical and structural analyses of a ring nuclease Cad1, revealing its substrate binding specificity and a sequential activation process by cOAs. Despite an overall structural similarity to canonical ADA enzymes, the ADA domain of Cad1 possesses unique structural features that confer a specificity for ATP. Supported by mutational analysis, our structural work demonstrates an allosteric link between the cOA-binding CARF and the ADA domain through a protein network within the hexameric enzyme assembly. Binding of a cA4 molecule to paired CARF domains induces a twisting of the linked ADA domains around one another, which remodels their active sites and alters interactions with neighboring ADA domains, thereby driving a sequential conformational activation mechanism.},
}
@article {pmid41005541,
year = {2025},
author = {Kristof, A and Karunakaran, K and Ferry, Y and Mizote, P and Allen, C and Briggs, S and Blazeck, J},
title = {A next-generation platform for highly optimized CRISPR-mediated transcriptional repression.},
journal = {Journal of biotechnology},
volume = {408},
number = {},
pages = {192-200},
doi = {10.1016/j.jbiotec.2025.09.012},
pmid = {41005541},
issn = {1873-4863},
mesh = {*CRISPR-Cas Systems/genetics ; Humans ; HEK293 Cells ; Methyl-CpG-Binding Protein 2/genetics/metabolism ; RNA, Guide, CRISPR-Cas Systems/genetics ; Gene Knockdown Techniques ; Protein Engineering/methods ; Clustered Regularly Interspaced Short Palindromic Repeats ; *Repressor Proteins/genetics/metabolism ; Nuclear Localization Signals/genetics ; },
abstract = {CRISPR interference (CRISPRi), the fusion of transcriptional repressor domains to nuclease-inactive Cas9, is a powerful genetic tool enabling site-specific suppression of gene expression. However, its performance remains inconsistent across cell lines, gene targets, or single guide RNAs (sgRNAs) employed. This study described the development process of a novel, highly optimized CRISPRi repressor for mammalian gene regulation through a multi-pronged protein engineering approach: (1) truncating established domains, (2) characterizing candidate domains, (3) creating combinatorial domain fusion libraries, and (4) optimizing NLS configuration. First, by evaluating several truncations of MeCP2, a well-established repressor, we see that the ultra-compact NCoR/SMRT interaction domain (NID) significantly enhances CRISPRi gene knockdown performance, exceeding levels observed with canonical MeCP2 subdomains by an average of ∼40 %. Incorporating this optimized MeCP2 NID truncation with a diverse panel of authenticated repressor domains, we next assemble and screen combinatorial multi-domain libraries, discovering four new repressor fusions. Upon follow-up nuclear localization signal (NLS) configuration analysis, we see that affixing one carboxy-terminal NLS enhances gene knockdown efficiency of the repressors by an average of ∼50 %. Through rigorous validation of NLS-tagged repressor fusions across several cell lines, multiple sgRNA targets, and genome-wide dropout screens, we establish that our strongest system, dCas9-ZIM3-NID-MXD1-NLS, achieves superior gene silencing capabilities over alternative CRISPRi platforms. In addition to developing dCas9-ZIM3-NID-MXD1-NLS, a uniquely potent transcriptional repressor, we envision that the multi-domain engineering approach utilized in this study will be valuable framework enabling future strides in CRISPR platform development.},
}
@article {pmid40962145,
year = {2025},
author = {Gao, Y and Zhou, Y and Wang, L and Zhang, N and Qin, W and Meng, W and Zhou, C},
title = {Synergistic strategy for high-yield 2,3-butanediol and acetoin production in Bacillus licheniformis MW03 based on metabolic engineering.},
journal = {Journal of biotechnology},
volume = {408},
number = {},
pages = {232-243},
doi = {10.1016/j.jbiotec.2025.09.006},
pmid = {40962145},
issn = {1873-4863},
mesh = {*Acetoin/metabolism/analysis ; *Butylene Glycols/metabolism ; *Bacillus licheniformis/genetics/metabolism ; *Metabolic Engineering/methods ; Bacterial Proteins/genetics/metabolism ; Fermentation ; Alcohol Oxidoreductases/genetics/metabolism ; CRISPR-Cas Systems ; Gene Knockout Techniques ; Gene Editing ; },
abstract = {Bacillus licheniformis is an efficient platform for 2,3-butanediol (2,3-BD) and acetoin production due to its rapid glucose utilization rate and adaptability to industrial fermentation conditions. Here, we isolated the B. licheniformis strain MW03 with high yield of acetoin and 2,3-BD, which carried genetic mutations in acoR and budC, respectively encoding an acetoin dehydrogenase regulator and meso-2,3-BD dehydrogenase. To further confirm the physiological effects on acetoin and 2,3 BD biosynthesis, gene editing was performed using the CRISPR-Cas9 system, followed by phenotypic screening and genotype validation. The knockout of acoR and budC increased the acetoin maximum titer by 21.2 % and 49.2 %, respectively. Moreover, the optical purity of D-(-)-2,3-BD reached 92.7 % following the knockout of budC. Heterologous expression of acoR from B. licheniformis 2709 in both the wild type and acoR knockout mutant strongly inhibited acetoin accumulation compared to native acoR, which emphasized the regulatory role of AcoR in acetoin accumulation. Conversely, complementation of budC restored the synthesis of meso-2,3-BD synthesis, emphasizing its importance in this process. Overexpression of alsD in the acoR mutant increased the 2,3-BD titer by 61.9 % to 121.97 g/L, while the productivity reached 2.03 g/L·h. Finally, co-expression of bdhA and gldA increased 2,3-BD production by 25.6 %. This study elucidated the dual regulatory roles of acoR and budC in acetoin and 2,3-BD metabolism, establishing a "knockout-overexpression" synergic strategy, which offers theoretical support and practical guidance for further strain optimization.},
}
@article {pmid41106392,
year = {2025},
author = {Yang, M and Liu, S and Chen, G and Liu, X and Sun, D and Zhang, J and Wang, Y and Chen, S and Tian, R and Hu, Z},
title = {Structural and functional bases of F. rodentium Cas9 provide insights into CRISPR-Cas protein engineering.},
journal = {Cell genomics},
volume = {},
number = {},
pages = {101039},
doi = {10.1016/j.xgen.2025.101039},
pmid = {41106392},
issn = {2666-979X},
abstract = {The Faecalibaculum rodentium (Fr) CRISPR-Cas9 system exhibits enhanced gene-editing precision and efficiency compared to SpCas9, with distinctive advantages in targeting the TATA box in eukaryotic promoters. However, the underlying molecular mechanisms remained unexplored. Here, we present cryo-electron microscopy structures of the FrCas9-single guide RNA (sgRNA)-DNA complex in both the R-loop expansion and pre-catalytic states, shedding light on its specialized recognition of the 5'-NRTA-3' protospacer adjacent motif (PAM) and the unusual overwinding of the sgRNA-DNA heteroduplex. Our investigations into the structure and extensive mutational analyses reveal that the phosphate lock loop plays a pivotal role in finely adjusting FrCas9's off-target sensitivity and catalytic efficiency. Remarkably, targeted residue substitutions in the phosphate lock loop and the PAM-distal region were found to synergistically enhance both the editing precision and efficiency of FrCas9. These findings advance our understanding of Cas9's accuracy and potency mechanisms while providing a molecular foundation for the rational design and development of next-generation CRISPR technologies.},
}
@article {pmid41106357,
year = {2025},
author = {Gehrke, F and Puchta, H},
title = {CRISPR meets AI-based robotics: Advancing sustainable agriculture.},
journal = {Cell},
volume = {188},
number = {21},
pages = {5785-5787},
doi = {10.1016/j.cell.2025.09.011},
pmid = {41106357},
issn = {1097-4172},
mesh = {*Artificial Intelligence ; *Robotics/methods ; Gene Editing/methods ; *Crops, Agricultural/genetics ; *Agriculture/methods ; *CRISPR-Cas Systems ; },
abstract = {In this issue of Cell, Xu and colleagues develop an approach integrating genome editing, artificial intelligence, and robotics to enhance crop improvement. By reconfiguring reproductive traits for automated pollination in crops such as tomatoes and soybeans, their approach accelerates hybrid seed production and yields crops with better stress tolerance, flavor, and resilience, supporting sustainable agriculture and crop diversity.},
}
@article {pmid41104521,
year = {2025},
author = {Braunreiter, K and Kempton, A and Mejia-Guerra, MK and Murray, A and Baine, S and Adegboye, K and Haile, A and Kumar Ahuja, SJ and Fedoce, A and Liu, C and Burch, P and Kabadi, AM},
title = {Characterization of a humanized mouse model of Duchenne muscular dystrophy to support the development of genetic medicines.},
journal = {Disease models &amp; mechanisms},
volume = {18},
number = {10},
pages = {},
doi = {10.1242/dmm.052182},
pmid = {41104521},
issn = {1754-8411},
support = {//Sarepta Therapeutics/ ; },
mesh = {Animals ; *Muscular Dystrophy, Duchenne/therapy/genetics/physiopathology/pathology/blood ; Disease Models, Animal ; Humans ; Dystrophin/metabolism/genetics ; Muscle, Skeletal/pathology/physiopathology ; Mice, Inbred mdx ; *Genetic Therapy ; Mice ; Male ; Diaphragm/pathology ; Biomarkers/blood/metabolism ; Gene Editing ; Muscle Fibers, Skeletal/pathology/metabolism ; CRISPR-Cas Systems/genetics ; Myocardium/pathology/metabolism ; Fibrosis ; },
abstract = {Duchenne muscular dystrophy (DMD) is a rare, progressive neuromuscular disease resulting from DMD variants, leading to loss of functional dystrophin. To evaluate human-targeted genetic medicines for functional dystrophin restoration, humanized genetic models containing the full human locus are required. This study characterized the hDMDΔ52/mdx mouse model previously reported by Pickar-Oliver and colleagues. Genomic characterization confirmed complete DMD duplication with identical exon 52 deletion junctions on both copies. Histological analysis showed increased diaphragm fibrosis and skeletal muscle central nuclei in hDMDΔ52/mdx mice versus hDMD/mdx controls. hDMDΔ52/mdx mice demonstrated reduced tibialis anterior specific force, decreased skeletal muscle fiber diameter, decreased resistance to eccentric contraction-induced damage and cardiac defects. Multiple serum biomarkers of disease were identified. Using a CRISPR/Cas9 gene-editing strategy to restore human functional dystrophin protein expression, detectable dystrophin expression in the heart and skeletal muscle and increased resistance to injury in the tibialis anterior muscle were observed. In summary, hDMDΔ52/mdx mice display multiple physiological and functional deficits associated with DMD pathology, which can be restored by human-targeted therapy, confirming the suitability of this model for developing human-targeted genetic medicines.},
}
@article {pmid41104129,
year = {2025},
author = {Pan, L and Wei, L and Luo, S and Ren, B and Li, M and Liang, L and Li, X and Wei, G},
title = {Klebsiella pneumoniae detection by a light-controlled one-pot RPA-CRISPR/Cas12a method.},
journal = {Frontiers in cellular and infection microbiology},
volume = {15},
number = {},
pages = {1669860},
pmid = {41104129},
issn = {2235-2988},
mesh = {*Klebsiella pneumoniae/isolation &amp; purification/genetics ; *CRISPR-Cas Systems ; Humans ; *Klebsiella Infections/diagnosis/microbiology ; Sensitivity and Specificity ; *Nucleic Acid Amplification Techniques/methods ; Bacterial Proteins/genetics ; Ultraviolet Rays ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {BACKGROUND: Klebsiella pneumoniae (KP) is a significant pathogenic bacterium responsible for severe infections in hospitals. However, existing traditional detection techniques, such as culture and PCR, are relatively inefficient. Therefore, this study aims to establish a rapid and convenient method for detecting KP.<br><br>METHODS: This study developed a single-tube detection method combining recombinant polymerase amplification (RPA) and light-controlled CRISPR/Cas12a. RPA primers were designed and screened for the rcsA gene of KP to effectively amplify the target. A light-controlled CRISPR/Cas12a system was created using crRNA modified with a photocleavable group (NPOM). The two systems were integrated into a single tube. Following RPA amplification, UV light-controlled release of crRNA inhibition activates CRISPR-mediated target recognition and Cas12a trans-cleavage, detecting fluorescent signals (FD) in conjunction with UV analysis.<br><br>RESULTS: The light-controlled RPA-CRISPR/Cas12a detection platform developed in this study uses a 15 &#x3bc;L reaction system. By optimizing key parameters such as RPA amplification time (20&#xa0;min), primer concentration (400 nM), UV light activation time (30 s), and crRNA/Cas12a concentration (300 nM), the platform achieves optimal detection efficiency. The platform has a fluorescence detection limit of 4.072&#xd7;10[2] copies/reaction and can specifically identify KP in seven common clinical strains. Clinical sample validation demonstrated that the method yields results fully consistent with PCR detection (30/30 agreement rate of 100%), showcasing excellent detection performance and clinical application potential.<br><br>CONCLUSION: We have successfully developed a light-controlled RPA-CRISPR/Cas12a detection system capable of rapidly and highly sensitively detecting KP. This system demonstrates significant advantages in terms of detection speed (completed in as little as 50 minutes), sensitivity (as low as 4.072&#xd7;10[2] copies/reaction), and ease of use, providing an efficient and reliable solution for clinical pathogen detection.},
}
@article {pmid41039096,
year = {2025},
author = {Johnston, M and Dissanayake-Perera, S and Collins, JJ and Stevens, MM and Dincer, C},
title = {Convergence of nanotechnology and CRISPR-based diagnostics.},
journal = {Nature nanotechnology},
volume = {20},
number = {10},
pages = {1365-1373},
pmid = {41039096},
issn = {1748-3395},
support = {13GW0493//Bundesministerium f&#xfc;r Bildung und Forschung (Federal Ministry of Education and Research)/ ; CiET2021\94//Royal Academy of Engineering/ ; },
mesh = {*Nanotechnology/methods ; Humans ; *CRISPR-Cas Systems/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Gene Editing ; },
abstract = {In addition to its broad application in genome engineering and therapeutics, clustered regularly interspaced short palindromic repeats (CRISPR) technology provides field-deployable methods for the highly sensitive and selective detection of nucleic acids. From a diagnostic perspective, CRISPR-based assays hold clear clinical potential for identifying a range of both infectious and non-communicable diseases. In this Perspective we evaluate recent nanotechnologies and nanomaterials that have been engineered to interface with CRISPR systems on a nanoscale level to realize the full potential of this versatile diagnostic tool. We assess biomolecules such as enzymes and oligonucleotides, some of the more commonly used synthetic nanoparticles and detection platforms that integrate nanotechnologies in new and innovative ways. We discuss current trends and look ahead to future challenges and opportunities, including non-nucleic acid target detection, pre-amplification-free detection of nucleic acids, the development of wearable devices and integration with artificial intelligence workflows.},
}
@article {pmid40975295,
year = {2026},
author = {Zheng, X and Xu, H and Huang, Y and Liu, X and Zhu, S and Liu, H and Gao, S},
title = {Development of an RT-RAA-CRISPR-Cas12a assay for rapid, sensitive and visual detection of Tilapia Lake Virus (TiLV).},
journal = {Journal of virological methods},
volume = {339},
number = {},
pages = {115266},
doi = {10.1016/j.jviromet.2025.115266},
pmid = {40975295},
issn = {1879-0984},
mesh = {Animals ; *Fish Diseases/virology/diagnosis ; *CRISPR-Cas Systems ; Sensitivity and Specificity ; *Tilapia/virology ; *Nucleic Acid Amplification Techniques/methods ; CRISPR-Associated Proteins/genetics ; Aquaculture ; RNA, Viral/genetics ; DNA, Single-Stranded/genetics ; Bacterial Proteins ; Endodeoxyribonucleases ; },
abstract = {In this study，we developed a new, highly efficient, and sequence-specific method for detecting Tilapia Lake Virus (TiLV) based on the clustered regularly interspaced short palindromic repeats (CRISPR) - CRISPR-associated protein 12a (Cas12a) system. TiLV is a highly contagious virus that has caused significant damage to the global aquaculture industry. Specific primers, CRISPR RNA (crRNA), and single-stranded DNA (ssDNA) reporters were designed to detect TiLV genome segment 3, with the ssDNA reporters modified at the 5' and 3' ends with fluorophore and quencher groups, respectively. The assay showed no cross-reactivity with other bacterial and viral pathogens in fish. The detection limit was 9.10 copies per reaction for recombinant plasmid standards and 91.82 fg/μL for TiLV RNA, demonstrating high sensitivity. The reverse transcription recombinase aided amplification (RT-RAA) coupled CRISPR/Cas12a method showed 100 % concordance with the standard fluorescence method, indicating its accuracy and suitability for clinical testing. This study innovatively combined the RT-RAA technique with the CRISPR/Cas12a reaction system, offering a new diagnostic method for TiLV that is fast, portable, highly specific, and sensitive. This enables on-site rapid screening for TiLV, ensuring aquaculture safety and the secure circulation of aquatic animal products.},
}
@article {pmid40935117,
year = {2026},
author = {Terada, T and Fujii, S and Yamanishi, N and Kajihara, R and Watanabe, T and Ezaki, R and Horiuchi, H and Matsuzaki, M},
title = {Potential of recombinant avian adeno-associated virus as a viral vector for CRISPR/Cas9 delivery to avian cells.},
journal = {Journal of virological methods},
volume = {339},
number = {},
pages = {115263},
doi = {10.1016/j.jviromet.2025.115263},
pmid = {40935117},
issn = {1879-0984},
mesh = {Animals ; *CRISPR-Cas Systems ; *Genetic Vectors/genetics ; Chickens/genetics ; *Gene Editing/methods ; *Dependovirus/genetics ; Cell Line ; Green Fluorescent Proteins/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; Fibroblasts/virology ; Staphylococcus aureus/genetics/enzymology ; },
abstract = {While genome editing has been established in chickens, where cultured primordial germ cell (PGC) systems are available, the implementation of genome editing remains a major challenge in many other birds due to the lack of robust PGC culture methods. Therefore, the development of reliable and efficient tools can significantly accelerate precision genome modification in avian species. Here, we evaluated the applicability of recombinant avian adeno-associated virus (rA3V) as a delivery vector for a CRISPR/Cas9 construct in avian cells using Staphylococcus aureus-derived Cas9 (SaCas9) and single-guide RNA (sgRNA). Infection with rA3V particles carrying an EGFP expression cassette (rA3V-EGFP) successfully induced EGFP expression in chicken fibroblasts (DF-1) cells, with approximately 80 % EGFP-positive cells at the maximum multiplicity of infection (MOI = 10,000). In plasmid-based transfection experiments, sgRNAs targeting the chicken tyrosinase locus and SaCas9 exhibited DNA cleavage activity in DF-1 cells. Furthermore, infection with rA3V particles encoding these CRISPR components successfully introduced indel mutations into the tyrosinase gene in DF-1 cells, with a calculated indel frequency of approximately 5.4 % at MOI = 40,000 without drug selection. Although EGFP expression was observed in quail fibrosarcoma cells, the percentage of EGFP-positive cells was much lower than that in DF-1 cells. In addition, in vivo infection with rA3V-EGFP of the chicken blastoderm failed to induce EGFP expression in germline cells, even at the highest applicable viral dose. In summary, rA3V can be used as a genome-editing vector in birds, although further investigation of its infectivity and tropism is necessary to expand its applicability to diverse avian species.},
}
@article {pmid40915071,
year = {2026},
author = {Li, Z and Jiao, Y and Tang, J and Dong, X and Thomas, R and Xie, B and Li, Y and Jacques, P},
title = {The pH-responsive regulator PlPacC and GATA transcription factor PlAreB are involved in the regulation of the biosynthesis of the antifungal lipopeptaibols leucinostatins in Purpureocillium lilacinum.},
journal = {Microbiological research},
volume = {302},
number = {},
pages = {128324},
doi = {10.1016/j.micres.2025.128324},
pmid = {40915071},
issn = {1618-0623},
mesh = {Hydrogen-Ion Concentration ; *Transcription Factors/genetics/metabolism ; *Antifungal Agents/metabolism ; *Fungal Proteins/genetics/metabolism ; *Hypocreales/metabolism/genetics/growth &amp; development ; Gene Expression Regulation, Fungal ; Nitrogen/metabolism ; Culture Media/chemistry ; CRISPR-Cas Systems ; },
abstract = {The biocontrol fungus Purpureocillium lilacinum PLBJ-1 produces leucinostatins, a class of non-ribosomal peptides (NRPs) with broad-spectrum antimicrobial activities. However, the molecular mechanisms underlying the optimization of culture conditions for leucinostatin production remain unexplored. Previous research showed that PLBJ-1 synthesizes leucinostatins more effectively in hand-made Potato Dextrose Broth (PDB-M) than in commercially available PDB (PDB-C). Elementary analysis of these two media indicated that the difference in leucinostatin yield was correlated with variations in pH dynamics and nitrogen content. Subsequent experiments under different initial pH and nitrogen levels confirmed that an alkaline environment and reduced nitrogen availability could enhance leucinostatin production. To investigate the regulators involved, CRISPR-Cas9-mediated gene disruptions were performed on the pH-responsive transcription factor PlPacC and the nitrogen regulator PlAreB. The disruption of either PlPacC or PlAreB resulted in a marked reduction in biomass and sporulation in P. lilacinum PLBJ-1. Specifically, PlPacC disruption impaired environmental pH regulation and significantly decreased leucinostatin production. In contrast, PlAreB disruption led to an increased leucinostatin yield. Overall, these findings demonstrate that environmental pH and nitrogen availability are the critical factors governing leucinostatin biosynthesis, acting through two key transcriptional regulators, PlPacC and PlAreB. This study lays a molecular foundation for future large-scale optimization of leucinostatin fermentation.},
}
@article {pmid40789509,
year = {2025},
author = {Tsai, HY and Chen, MH and Yun, J and Lai, LA and Valentine, JF and Bronner, MP and Brentnall, TA and Pan, S and Chen, R},
title = {Restricting metabolic plasticity enhances stress adaptation through the modulation of PDH and HIF1A in TRAP1-depleted colon cancer.},
journal = {Cancer letters},
volume = {632},
number = {},
pages = {217977},
doi = {10.1016/j.canlet.2025.217977},
pmid = {40789509},
issn = {1872-7980},
support = {P30 DK056338/DK/NIDDK NIH HHS/United States ; R01 CA211892/CA/NCI NIH HHS/United States ; R01 CA276173/CA/NCI NIH HHS/United States ; },
mesh = {*Colonic Neoplasms/metabolism/genetics/pathology ; Humans ; *Hypoxia-Inducible Factor 1, alpha Subunit/metabolism/genetics ; Glycolysis ; Glucose Transporter Type 1/genetics/metabolism ; Pyruvate Dehydrogenase Acetyl-Transferring Kinase ; Cell Line, Tumor ; Oxidative Phosphorylation/drug effects ; HCT116 Cells ; Reactive Oxygen Species/metabolism ; Gene Expression Regulation, Neoplastic ; Monocarboxylic Acid Transporters/genetics/metabolism ; Adaptation, Physiological ; CRISPR-Cas Systems ; HSP90 Heat-Shock Proteins ; Symporters ; },
abstract = {Metabolic plasticity allows cancer cells to survive under adverse conditions. To investigate the role of mitochondrial chaperone tumor necrosis factor receptor-associated protein 1 (TRAP1) in this process, we used CRISPR/Cas9 mediated genetic deletion to knock out (KO) TRAP1 in colon cancer cells. Depletion of TRAP1 triggered a series of events: induced metabolic reprogramming, increased glycolytic flux, downregulation of mitochondrial complex I, and elevated ROS generation. TRAP1-deficient cells showed tolerance to Oxidative Phosphorylation (OXPHOS) inhibitors and exhibited a higher extracellular acidification rate (ECAR). Additionally, TRAP1 depletion activated hypoxia response elements (HREs) and upregulated HIF1A target genes such as GLUT1 and MCT1. Furthermore, pyruvate dehydrogenase kinases 1 (PDK1) was upregulated in KO cells, leading to the inactivation of the tricarboxylic acid (TCA) cycle enzyme, pyruvate dehydrogenase (PDH). This metabolic shift towards glycolytic metabolism resulted in increased glycolytic metabolism, elevated lactic acid production, and higher glucose consumption, making TRAP1-depleted cancer cells more dependent on this altered metabolism for survival. Treatment with DCA, a PDK inhibitor, restored PDH activity, exacerbated oxidative stress, and increased cell death in KO cells. Our study here sheds light on how TRAP1 depletion affects metabolic plasticity, driving colon cancer cells to adapt to metabolic and oxidative stress. These findings highlight TRAP1 as a promising therapeutic target for manipulating metabolic plasticity and overcoming drug resistance in cancer therapy.},
}
@article {pmid41103153,
year = {2025},
author = {Chen, L and Yun, M and Chen, B and Xie, S and Liu, W and Wang, M and Yan, J and Cai, J and Yang, S and Peng, Q and Xie, D and Lin, Y and Jiang, B},
title = {Loss of CsCLV2 function causes dwarfism and determinates growth in cucumber.},
journal = {The Plant journal : for cell and molecular biology},
volume = {124},
number = {1},
pages = {e70525},
doi = {10.1111/tpj.70525},
pmid = {41103153},
issn = {1365-313X},
support = {32202477//National Natural Science Foundation of China/ ; },
mesh = {*Cucumis sativus/genetics/growth &amp; development/metabolism ; *Plant Proteins/genetics/metabolism/physiology ; *Dwarfism/genetics ; Phenotype ; Meristem/growth &amp; development/genetics ; Gene Expression Regulation, Plant ; CRISPR-Cas Systems ; },
abstract = {Cucumber (Cucumis sativus L.) is a globally important vegetable crop. Ideal plant architecture optimizes spatial utilization, enhances economic coefficient, and facilitates mechanized cultivation. In this study, we identified a dwarf mutant, csdw3, exhibiting reduced plant height, shortened internodes, and fewer internodes. Genetic analysis showed that this dwarf phenotype is controlled by a single recessive gene. Fine-mapping localized the causal locus to an 80 kb region on chromosome 1, where we discovered a 102 bp deletion in CsCLV2, a gene encoding a leucine-rich repeat receptor-like protein homologous to Arabidopsis CLAVATA2. CRISPR-Cas9-generated loss-of-function mutants recapitulated the dwarf phenotype, confirming the role of CsCLV2 in plant height regulation. Histological examination revealed that CsCLV2 disruption causes premature termination of shoot apical meristem (SAM) development, reducing both internode number and length. Protein interaction assays further demonstrated that CsCLV2 associates with receptor-like kinase CsCIK1 (CLAVATA3 INSENSITIVE RECEPTOR KINASES 1), indicating their cooperative function in the CLV-WUS signaling pathway to maintain meristem activity. Our findings uncover a regulator of plant height in cucumber and provide valuable genetic resources for breeding ideotypes optimized for yield and cultivation efficiency.},
}
@article {pmid41102733,
year = {2025},
author = {Marchenko, N and Nesbitt, NM and Alexandrova, E and Reisz, JA and D'Alessandro, A and Suh, J and Uryasev, S and Pennacchia, L and Bahou, WF},
title = {Biliverdin reductase B as a new target in breast cancer.},
journal = {Breast cancer research : BCR},
volume = {27},
number = {1},
pages = {179},
pmid = {41102733},
issn = {1465-542X},
support = {NCI CA284999/CA/NCI NIH HHS/United States ; P30CA046934/CA/NCI NIH HHS/United States ; },
mesh = {Humans ; *Breast Neoplasms/genetics/pathology/metabolism/drug therapy ; Female ; Animals ; Cell Line, Tumor ; Mice ; *Oxidoreductases Acting on CH-CH Group Donors/genetics/metabolism/antagonists &amp; inhibitors ; Cell Proliferation ; Oxidation-Reduction ; Xenograft Model Antitumor Assays ; CRISPR-Cas Systems ; },
abstract = {BACKGROUND: Enhanced metabolic and mitochondrial activity inherent in actively proliferating cancer cells is associated with intracellular redox imbalance that impacts cellular viability. To restore redox homeostasis cancer cells evolve to activate redox protective mechanisms. This differential activation of redox defense pathways compared to normal cells provides a therapeutic window for novel targeted therapies in cancer. Although heme metabolism emerges as a crucial regulator of redox homeostasis and iron metabolism in cancer cells with frequent alteration in breast cancer, it remains largely unexplored, and no targeted translational approaches have been developed. Heme-regulated redox homeostasis is coordinately maintained through biosynthetic and degradation pathways. As a byproduct of TCA cycle, cytotoxic heme is initially derivatized by heme oxygenases and progressively metabolized to the potent antioxidant bilirubin by two non-redundant biliverdin reductases, BLVRA and BLVRB. BLVRB overexpression has been observed in breast cancers, although its function in breast cancer pathogenesis remains unknown.<br><br>METHODS: CRISPR/Cas9 deletion of BLVRB in multiple breast cancer cell lines demonstrated its profound effect on intracellular redox state and cell proliferation in vitro and in xenograft models. Integrated proteomic, metabolomic, and lipidomic studies identified and validated BLVRB-mediated adaptive metabolic responses required for breast cancer cell cytoprotection.<br><br>RESULTS: We have established BLVRB as a requisite component of the pro-survival redox defense mechanism in breast cancer cells. Targeted deletion of BLVRB induces reductive stress, leading to alterations in endoplasmic reticulum proteostasis and lipid composition. These defects impact plasma membrane functionality and endosomal recycling of multiple oncogenic receptors, such as HER2 and transferrin receptors.<br><br>CONCLUSIONS: These data collectively identify BLVRB as a novel metabolic target in breast cancer, distinct from other redox-regulating pathways. This study, along with our recent progress in developing novel specific BLVRB inhibitors, offers a unique translational opportunity for targeted therapies in personalized breast cancer medicine.},
}
@article {pmid41102523,
year = {2025},
author = {Savage, N and Danis, E and Chokshi, CR and Custers, S and Shaikh, MV and Miletic, P and Venugopal, C and Brown, KR and Vibhakar, R and Moffat, J and Singh, SK},
title = {CRISPR screen reveals SOX2 as a critical regulator of CD133 and cellular stress response in glioblastoma.},
journal = {Scientific reports},
volume = {15},
number = {1},
pages = {36228},
pmid = {41102523},
issn = {2045-2322},
support = {1065//Terry Fox Research Institute Program Project Grant/ ; 1065//Terry Fox Research Institute Program Project Grant/ ; 1065//Terry Fox Research Institute Program Project Grant/ ; 1065//Terry Fox Research Institute Program Project Grant/ ; 1065//Terry Fox Research Institute Program Project Grant/ ; 1065//Terry Fox Research Institute Program Project Grant/ ; 1065//Terry Fox Research Institute Program Project Grant/ ; 1065//Terry Fox Research Institute Program Project Grant/ ; 1065//Terry Fox Research Institute Program Project Grant/ ; },
mesh = {*AC133 Antigen/metabolism/genetics ; *SOXB1 Transcription Factors/genetics/metabolism ; *Glioblastoma/genetics/metabolism/pathology ; Humans ; *CRISPR-Cas Systems ; Neoplastic Stem Cells/metabolism/pathology ; Cell Line, Tumor ; *Brain Neoplasms/genetics/metabolism/pathology ; Gene Expression Regulation, Neoplastic ; *Stress, Physiological ; Animals ; Mice ; },
abstract = {Glioblastoma (GBM) remains a formidable challenge in clinical settings due to limited treatments available. The surface protein CD133 marks glioblastoma stem cells (GSCs), cells capable of overcoming therapeutic pressures and correlate with more aggressiveness tumor phenotypes. In this study, we employed a CRISPR-Cas9 functional screen to deconvolute CD133 dynamics in tumors. This led us to establish that SOX2 is a key player in controlling the PROM1 gene, which in turn influences how cells react to stress factors, including those induced by chemoradiation treatment. The discoveries in this study shed light on the complex web of mechanisms that control the survival and resistance of GSCs, offering promising new avenues for targeting and potentially overcoming therapy resistance.},
}
@article {pmid41101942,
year = {2025},
author = {Lim, SL and Chin, CH and Chiou, YJ and Hsu, MT and Chiang, PW and Chen, HJ and Tu, YC and Tzou, WS and Tang, SL},
title = {Unveiling Unusual Ecofunctional Traits of Endozoicomonas Species Through Comprehensive Comparative Genomics.},
journal = {Environmental microbiology},
volume = {27},
number = {10},
pages = {e70191},
doi = {10.1111/1462-2920.70191},
pmid = {41101942},
issn = {1462-2920},
support = {AS-IA-109-L05//Academia Sinica/ ; },
mesh = {*Genome, Bacterial ; Genomics ; Phylogeny ; Prophages/genetics ; Bacterial Proteins/genetics ; *Gammaproteobacteria/genetics ; Quorum Sensing ; },
abstract = {Endozoicomonas is an omnipresent marine bacterial genus, associated with various marine organisms, that contributes to host health, nutrient cycling and disease resistance. Nonetheless, its genomic features remain poorly characterised due to a paucity of high-quality genomes. In this study, we sequenced 5 novel Endozoicomonas strains and re-sequenced 1 known strain to improve genomic resolution. By integrating these 6 high-quality genomes with 31 qualified published genomes, our pan-genomic analysis revealed variation in genetic traits among clades. Notably, Endozoicomonas lacks quorum-sensing capabilities, suggesting resistance to quorum quenching mechanisms. It also lacks the capacity to synthesise and transport vitamin B12, indicating that it does not supply this nutrient to holobionts. Remarkably, Endozoicomonas genomes encode 92 identified giant proteins (15-65 kbp). These proteins cluster into three major groups associated with antimicrobial peptide synthesis, exotoxin production and cell adhesion. Additionally, we found that Endozoicomonas has acquired prophages from diverse sources via infection or other types of gene transfer. Notably, CRISPR-Cas sequences suggest evolutionary trajectories independent of both prophage acquisition and phylogenetic lineage, implying potential geographic influences or environmental pressures. This study provides new insights into the genomic diversity of Endozoicomonas and its genetic adaptations to diverse hosts.},
}
@article {pmid41100915,
year = {2025},
author = {Kim, I and Suh, JY},
title = {Old and new tactics of CRISPR-centric competition between bacteria and bacteriophages.},
journal = {Current opinion in structural biology},
volume = {95},
number = {},
pages = {103168},
doi = {10.1016/j.sbi.2025.103168},
pmid = {41100915},
issn = {1879-033X},
abstract = {The CRISPR-Cas system provides adaptive immunity for prokaryotes against mobile genetic elements (MGEs) such as bacteriophages and plasmids. As a countermeasure, MGEs have evolved various anti-CRISPR (Acr) mechanisms that neutralize the CRISPR-mediated immunity. Canonical Acr proteins block target binding of Cas proteins in a stoichiometric or enzymatic manner. New findings reveal that Acr also disintegrates functional Cas complexes, induces promiscuous target binding, and mimics Cas proteins and crRNA with defective mutations. Here, we summarize a broad repertoire of structural and functional mechanisms underlying CRISPR-centric competition, highlighting recent discoveries of molecular machinery that modulates CRISPR function.},
}
@article {pmid41100703,
year = {2025},
author = {Liao, X and Li, Y and Wu, Y and Wen, L and Jing, M and Chen, B and Li, X and Shang, X},
title = {TEMC-Cas: Accurate Cas Protein Classification via Combined Contrastive Learning and Protein Language Models.},
journal = {ACS synthetic biology},
volume = {},
number = {},
pages = {},
doi = {10.1021/acssynbio.5c00631},
pmid = {41100703},
issn = {2161-5063},
abstract = {The accurate classification of Cas proteins is crucial for understanding CRISPR-Cas systems and developing genome-editing tools. Here, we present TEMC-Cas, a deep learning framework for accurate classification of Cas proteins that combines a finely tuned ESM protein language model with contrastive learning. Unlike traditional methods that rely on sequence similarity (e.g., BLAST, HMMs) or structural prediction, TEMC-Cas leverages evolutionary-scale modeling to capture distant homology while employing contrastive learning to distinguish closely related subtypes. The framework incorporates LoRA for efficient parameter adaptation and addresses class imbalance through weighted loss functions. TEMC-Cas achieves superior performance in classifying the Cas1-Cas13 families and 17 Cas12 subtypes, demonstrating particular strength in identifying remote homology. This approach provides a robust tool for the discovery of the CRISPR system and expands the toolbox for genome engineering applications. TEMC-Cas is now freely accessible at https://github.com/Xingyu-Liao/TEMC-Cas.},
}
@article {pmid41099996,
year = {2025},
author = {Shafi, Z and Shahid, M and Ilyas, T and Singh, K},
title = {Next-generation perspectives on microbially synthesized siderophores: molecular engineering, multi-omics insights, and applications for smart climate-resilient crops.},
journal = {World journal of microbiology &amp; biotechnology},
volume = {41},
number = {10},
pages = {393},
pmid = {41099996},
issn = {1573-0972},
mesh = {*Siderophores/biosynthesis/genetics/metabolism ; *Crops, Agricultural/growth &amp; development/microbiology ; Iron/metabolism ; *Bacteria/metabolism/genetics ; Metabolomics ; Gene Editing ; Genomics ; Proteomics ; CRISPR-Cas Systems ; Multiomics ; },
abstract = {Siderophores, low-molecular-weight iron-chelating compounds synthesized by microbes, play a crucial role in iron (Fe) acquisition under Fe-limited conditions. In recent years, their significance in sustainable agriculture has gained increasing attention due to their multifaceted roles in plant growth promotion, stress alleviation, and disease suppression. This review presents next-generation insights into the biosynthesis, regulation, and applications of microbial siderophores, with a focus on advanced molecular and omics-based approaches. Innovations in synthetic biology and CRISPR/Cas-mediated genome editing have enabled precise manipulation of siderophore biosynthetic gene clusters, enhancing their production and functionality. Multi-omics platforms-genomics, transcriptomics, proteomics, and metabolomics-have revealed complex regulatory networks, unveiling cryptic pathways and inter-microbial variability in siderophore synthesis. Furthermore, the use of siderophore-producing plant growth-promoting rhizobacteria (PGPR) has shown promise in improving nutrient uptake, inducing systemic resistance, and mitigating abiotic stresses in crops. The integration of nano-formulations and encapsulation technologies has enhanced the stability and field efficacy of siderophore-based bioinoculants. This review also explores emerging strategies for developing microbial consortia and smart delivery systems to meet the challenges of climate-resilient agriculture. By bridging molecular insights with field-level applications, this article underscores the potential of siderophores as eco-friendly tools for next-generation sustainable farming practices.},
}
@article {pmid41097718,
year = {2025},
author = {Lye, SH and Polycarp, N and Durojaye, TJ and Tollefsbol, TO},
title = {Functional Heterogeneity and Context-Dependent Roles of LncRNAs in Breast Cancer.},
journal = {Cancers},
volume = {17},
number = {19},
pages = {},
pmid = {41097718},
issn = {2072-6694},
support = {R01CA178441/CA/NCI NIH HHS/United States ; },
abstract = {As with other non-coding RNAs (ncRNAs), the aberrant expression of long non-coding RNAs (lncRNAs) can be associated with different forms of cancers, including breast cancer (BC). Various lncRNAs may either promote or suppress cell proliferation, metastasis, and other related cancer signaling pathways by interacting with other cellular machinery, thus affecting the expression of BC-related genes. However, lncRNAs are characterized by features that are unlike protein-coding genes, which pose unique challenges when it comes to their study and utility. They are highly diverse and may display contradictory functions depending on factors like the BC subtype, isoform diversity, epigenetic regulation, subcellular localization, interactions with various molecular partners, and the tumor microenvironment (TME), which contributes to the intratumoral heterogeneity and phenotypic plasticity. While lncRNAs have potential clinical utility, their functional heterogeneity coupled with a current paucity of knowledge of their functions present challenges for clinical translation. Strategies to address this heterogeneity include improving classification systems, employing CRISPR/Cas tools for functional studies, utilizing single-cell and spatial sequencing technologies, and prioritizing robust targets for therapeutic development. A comprehensive understanding of the lncRNA functional heterogeneity and context-dependent behavior is crucial for advancing BC research and precision medicine. This review discusses the sources of lncRNA heterogeneity, their implications in BC biology, and approaches to resolve knowledge gaps in order to harness lncRNAs for clinical applications.},
}
@article {pmid41027589,
year = {2025},
author = {Jiang, W and Georgiadis, I and Fumagalli, T and Wang, S and Vasileiou, C and Dahlin, J and Borodina, I},
title = {In Vivo DNA Assembly in Yarrowia lipolytica.},
journal = {ACS synthetic biology},
volume = {14},
number = {10},
pages = {4116-4121},
doi = {10.1021/acssynbio.5c00296},
pmid = {41027589},
issn = {2161-5063},
mesh = {*Yarrowia/genetics/metabolism ; CRISPR-Cas Systems/genetics ; Rad52 DNA Repair and Recombination Protein/genetics/metabolism ; Saccharomyces cerevisiae/genetics ; Homologous Recombination/genetics ; *DNA/genetics/metabolism ; Gene Editing/methods ; Green Fluorescent Proteins/genetics/metabolism ; },
abstract = {The oleaginous yeast Yarrowia lipolytica is an important platform organism for biotechnology applications. In this study, we established an in vivo DNA assembly system leveraging CRISPR-Cas9 for efficient genomic integration of multiple DNA fragments into the genome of Y. lipolytica. Using the green fluorescent protein mNeonGreen as a model, we demonstrated 53% correct assembly of three DNA fragments with homology arms as short as 50 bp. The system was further validated by constructing 2-3 step biosynthetic pathways for pigments betaxanthin and betanin. To improve the homologous recombination efficiency of Y. lipolytica, we expressed S. cerevisiae RAD52 (ScRAD52) or a Cas9-hBrex27 fusion protein. While ScRAD52 expression impaired growth, the cas9-hBrex27 fusion enhanced integration efficiency, particularly for multifragment pathway assemblies. The in vivo assembly method simplifies pathway construction and gene overexpression in Y. lipolytica.},
}
@article {pmid41021780,
year = {2025},
author = {Golla, SA and Abo-Hashesh, M and Gupta, D and Liu, Y and Mahadevan, R},
title = {Model-Based Optimization of a qCRISPRi Circuit for Dynamic Control of Metabolic Pathways.},
journal = {ACS synthetic biology},
volume = {14},
number = {10},
pages = {3890-3898},
doi = {10.1021/acssynbio.5c00095},
pmid = {41021780},
issn = {2161-5063},
mesh = {Escherichia coli/genetics/metabolism ; *Metabolic Engineering/methods ; *Metabolic Networks and Pathways/genetics ; Quorum Sensing/genetics ; Repressor Proteins/genetics/metabolism ; Trans-Activators/genetics/metabolism ; CRISPR-Cas Systems/genetics ; Promoter Regions, Genetic/genetics ; Gene Expression Regulation, Bacterial ; },
abstract = {Metabolic engineering enables sustainable chemical production but often imposes metabolic burdens that reduce cellular viability and productivity. Dynamic control strategies, such as quorum sensing (QS)-based circuits, can mitigate these effects by autonomously regulating gene expression in response to cell density. In this study, we investigated a QS-regulated CRISPR interference (qCRISPRi) circuit for the dynamic control of metabolic pathways, focusing on the role of leaky expression and regulator stringency. Using a combination of mathematical modeling and experiments, we evaluated how promoter leakiness and LuxR stringency influence key switching characteristics including maximum gene expression, switching density, fold repression, and transition time. Our results show that high leaky expression of dCas9 reduces switching density and represses GFP prematurely, whereas a high-stringency LuxR variant enhances switching precision by reducing leakiness and enabling sharper transitions. These model predictions were validated experimentally in E. coli, confirming that LuxR stringency improves dynamic circuit performance. Together, this work provides a quantitative framework for optimizing QS-based regulatory systems and offers generalizable design insights for implementing dynamic control in metabolic engineering.},
}
@article {pmid40992138,
year = {2026},
author = {Xiao, G and Shi, H and Lin, Q and Li, S and He, J and Zhang, G},
title = {A rapid CRISPR-Cas12a/T7EI integrated workflow for high-throughput screening of homozygous mutant cell lines.},
journal = {Journal of pharmaceutical and biomedical analysis},
volume = {267},
number = {},
pages = {117152},
doi = {10.1016/j.jpba.2025.117152},
pmid = {40992138},
issn = {1873-264X},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *High-Throughput Screening Assays/methods ; Gene Editing/methods ; *Mutation/genetics ; Homozygote ; Workflow ; THP-1 Cells ; Cell Line ; },
abstract = {Efficient screening for homozygous mutant cell lines, particularly those resulting from low-efficiency CRISPR-Cas9 editing, remains challenging. Here, we developed HomoSelect-CT, an integrated workflow combining CRISPR-Cas12a nucleic acid detection with T7 Endonuclease I (T7EI) genotyping, designed to streamline the screening process for homozygous mutant cell lines. This method requires no specialized instrumentation, enhancing accessibility and efficiency. We validated HomoSelect-CT by successfully identifying homozygous mutants in CRISPR-Cas9-edited THP-1 cells, which was confirmed by Sanger sequencing and Western blot (WB). These findings demonstrate that HomoSelect-CT is a robust and efficient alternative for the rapid isolation of genome-edited cell lines. The entire screening workflow, from monoclonal cultures to confirmed homozygous mutants, is completed in under 4 h, requiring only standard PCR equipment and routine reagents. Thus, HomoSelect-CT represents a significant advancement in CRISPR screening methodology, offering remarkable simplicity and enabling high-throughput screening that is particularly suitable for mutants arising from low-efficiency editing events.},
}
@article {pmid40829585,
year = {2025},
author = {Xu, C and Niu, X and Sun, H and Yan, H and Tang, W and Ke, A},
title = {Conversion of IscB and Cas9 into RNA-guided RNA editors.},
journal = {Cell},
volume = {188},
number = {21},
pages = {5847-5861.e11},
doi = {10.1016/j.cell.2025.07.032},
pmid = {40829585},
issn = {1097-4172},
mesh = {Humans ; *RNA Editing ; *RNA, Guide, CRISPR-Cas Systems/metabolism/genetics ; *CRISPR-Cas Systems ; *CRISPR-Associated Protein 9/metabolism/genetics ; HEK293 Cells ; Adenosine Deaminase/metabolism/genetics ; RNA, Messenger/metabolism/genetics ; Gene Editing/methods ; RNA-Binding Proteins/metabolism/genetics ; DNA, Single-Stranded/metabolism ; Trans-Splicing ; },
abstract = {RNA-guided RNA editing represents an attractive alternative to DNA editing. However, the prevailing tool, CRISPR-Cas13, has collateral RNA cleavage activity that causes undesirable cytotoxicity in human cells. Here, we report an ultracompact RNA-editing platform engineered from IscB, which has comparable or higher activity than Cas13 but without cytotoxicity concerns. We show that IscB, the evolutionary ancestor of Cas9, has an intrinsic affinity for complementary single-stranded (ss)DNA and RNA. This activity becomes dominant when its double-stranded DNA binding activity is switched off through the deletion of its target-adjacent motif domain. The resulting R-IscB is comparable to or better than Cas13, can efficiently alter splicing outcomes in human cells, and can further mediate trans-splicing to correct any mutation at the mRNA level. R-IscB also drives efficient A-to-I editing on mRNA when fused to adenosine deaminase acting on RNA 2 (ADAR2) and mediates cleavage-based mRNA knockdown upon HNH engineering. Finally, we show that the same approach converts some Cas9s to RNA-targeting tools.},
}
@article {pmid40774253,
year = {2025},
author = {Zhou, Z and Zhu, S and Hong, Y and Jin, G and Ma, R and Lin, F and Zhang, Y and Lee, HY and Liu, N},
title = {Composite transposons with bivalent histone marks function as RNA-dependent enhancers in cell fate regulation.},
journal = {Cell},
volume = {188},
number = {21},
pages = {5878-5894.e18},
doi = {10.1016/j.cell.2025.07.014},
pmid = {40774253},
issn = {1097-4172},
mesh = {Humans ; *DNA Transposable Elements/genetics ; Erythropoiesis/genetics ; *Histone Code ; CRISPR-Cas Systems ; *Enhancer Elements, Genetic ; *Histones/metabolism ; Animals ; *RNA/metabolism ; Myelopoiesis/genetics ; },
abstract = {Discrete genomic units can recombine into composite transposons that transcribe and transpose as single units, but their regulation and function are not fully understood. We report that composite transposons harbor bivalent histone marks, with activating and repressive marks in distinct regions. Genome-wide CRISPR-Cas9 screening, using a reporter driven by the hominid-specific composite transposon SVA (SINE [short interspersed nuclear element]-VNTR [variable number of tandem repeats]-Alu) in human cells, identified diverse genes that modify bivalent histone marks to regulate SVA transcription. SVA transcripts are critical for SVA's cis-regulatory function in selectively contacting and activating long-range gene expression. Remarkably, a subset of bivalent SVAs is activated during erythropoiesis to boost multiple erythroid gene expression, and knocking down these SVAs leads to deficient erythropoiesis. The RNA-dependent cis-regulatory function of SVA activates genes for myelopoiesis and can contribute to aging-associated myeloid-biased hematopoiesis. These results reveal that the cis-regulatory functions of composite transposons are bivalently regulated to control cell fate transitions in development and aging.},
}
@article {pmid40769155,
year = {2025},
author = {Park, JC and Uhm, H and Kim, YW and Oh, YE and Lee, JH and Yang, J and Kim, K and Bae, S},
title = {AI-generated MLH1 small binder improves prime editing efficiency.},
journal = {Cell},
volume = {188},
number = {21},
pages = {5831-5846.e21},
doi = {10.1016/j.cell.2025.07.010},
pmid = {40769155},
issn = {1097-4172},
mesh = {Humans ; *Gene Editing/methods ; Animals ; *MutL Protein Homolog 1/metabolism/genetics/chemistry ; HeLa Cells ; Mice ; Mismatch Repair Endonuclease PMS2/metabolism ; CRISPR-Cas Systems ; DNA Mismatch Repair ; Protein Binding ; RNA, Guide, CRISPR-Cas Systems/metabolism ; },
abstract = {The prime editing (PE) system consists of a Cas9 nickase fused to a reverse transcriptase, which introduces precise edits into the target genomic region guided by a PE guide RNA. However, PE efficiency is limited by mismatch repair. To overcome this limitation, transient expression of a dominant-negative MLH1 (MLH1dn) has been used to inhibit key components of mismatch repair. Here, we designed a de novo MLH1 small binder (MLH1-SB) that binds to the dimeric interface of MLH1 and PMS2 using RFdiffusion and AlphaFold 3. The compact size of MLH1-SB enabled its integration into existing PE architectures via 2A systems, creating a PE-SB platform. The PE7-SB2 system significantly improved PE efficiency, achieving an 18.8-fold increase over PEmax and a 2.5-fold increase over PE7 in HeLa cells, as well as a 3.4-fold increase over PE7 in mice. This study highlights the potential of generative AI in advancing genome editing technology.},
}
@article {pmid40706591,
year = {2025},
author = {Marzook, NB and Song, OR and Baumgärtel, L and Bernitz, N and Mkandawire, TT and Watson, LC and Nunes, V and Warchal, S and MacRae, JI and Howell, M and Sateriale, A},
title = {The essential host genome for Cryptosporidium survival exposes metabolic dependencies that can be leveraged for treatment.},
journal = {Cell},
volume = {188},
number = {21},
pages = {5947-5961.e15},
doi = {10.1016/j.cell.2025.07.001},
pmid = {40706591},
issn = {1097-4172},
mesh = {Animals ; *Cryptosporidiosis/parasitology/drug therapy/genetics/metabolism ; Humans ; *Cryptosporidium/genetics/metabolism/drug effects ; Glutathione/metabolism ; CRISPR-Cas Systems ; Mice ; Epithelial Cells/metabolism/parasitology ; Farnesyl-Diphosphate Farnesyltransferase/antagonists &amp; inhibitors/metabolism ; Cholesterol/biosynthesis ; Host-Parasite Interactions/genetics ; },
abstract = {Cryptosporidium is a leading cause of diarrheal disease, yet little is known regarding the infection cell biology of this intracellular intestinal parasite. To this end, we implemented an arrayed genome-wide CRISPR-Cas9 knockout screen to microscopically analyze multiple phenotypic features of a Cryptosporidium infection following individual host gene ablation. We discovered parasite survival within the host epithelial cell hinges on squalene, an intermediate metabolite in the host cholesterol biosynthesis pathway. A buildup of squalene within intestinal epithelial cells creates a reducing environment, making more reduced glutathione available for parasite uptake. Remarkably, the Cryptosporidium parasite has lost the ability to synthesize glutathione and has become dependent on this host import. This dependency can be leveraged for treatment with the abandoned drug lapaquistat, an inhibitor of host squalene synthase that shifts the redox environment, blocking Cryptosporidium growth in vitro and in vivo.},
}
@article {pmid40685751,
year = {2025},
author = {Qin, W and Lin, SJ and Zhang, Y and Huang, K and Petree, C and Boyd, K and Varshney, P and Varshney, GK},
title = {Rationally Designed TadA-Derived Cytosine Editors Enable Context-Independent Zebrafish Genome Editing.},
journal = {Advanced science (Weinheim, Baden-Wurttemberg, Germany)},
volume = {12},
number = {39},
pages = {e09800},
doi = {10.1002/advs.202509800},
pmid = {40685751},
issn = {2198-3844},
support = {R24 OD034438/OD/NIH HHS/United States ; R24OD034438/RI/ORIP NIH HHS/United States ; /NH/NIH HHS/United States ; R24OD034438/RI/ORIP NIH HHS/United States ; },
mesh = {Animals ; *Zebrafish/genetics ; *Gene Editing/methods ; *Cytosine/metabolism ; CRISPR-Cas Systems/genetics ; Genome/genetics ; },
abstract = {CRISPR base editors are crucial for precise genome manipulation. Existing APOBEC-based cytosine base editors (CBEs), while powerful, exhibit indels and sequence context limitations, and editing CC and GC motifs is challenging and inefficient. To address these challenges, existing tRNA adenine deaminase (TadA)-derived CBEs are evaluated in zebrafish, and a series of zTadCBE variants is developed that demonstrate high editing efficiency, minimized off-target effects, and an expanded targeting range compared to existing tools. The approach integrates beneficial mutations from TadA-based adenine base editors (ABEs) with SpRYCas9n-enhanced protospacer-adjacent motif (PAM) compatibility. The expanded window zTadCBE variants enable the targeting of cytosines at a broader range of nucleotide positions relative to the PAM sequence, further enhancing the versatility of this tool. Using zTadCBEs, four zebrafish disease models affecting the auditory, nervous, metabolic, and muscular systems are generated directly in the F0 generation-models that cannot be efficiently produced using earlier CBE tools. Together, zTadCBE variants provide a robust and flexible toolkit for efficient and precise C-to-T base editing in zebrafish, facilitating rapid in vivo functional assessment of genetic variants.},
}
@article {pmid40683408,
year = {2025},
author = {Zhou, J and Zhou, C and Jiang, G and Wang, Y and Pan, L and Jia, X and Tu, X and Li, W and Wang, C},
title = {Engineering an Escherichia coli with performance-enhanced switch utilizing CRISPR-Cas9 system as living quorum quencher for biofilm formation inhibition.},
journal = {Environmental research},
volume = {285},
number = {Pt 2},
pages = {122383},
doi = {10.1016/j.envres.2025.122383},
pmid = {40683408},
issn = {1096-0953},
mesh = {*Biofilms/growth &amp; development ; *Escherichia coli/genetics/physiology ; *Quorum Sensing/genetics ; *CRISPR-Cas Systems ; Genetic Engineering ; },
abstract = {Quorum quenching (QQ) of signal molecules plays a critical role in disrupting bacterial communication, thereby suppressing biofilm formation. However, the wild-type QQ bacteria lacks the regulatory capacity to modulate gene expression levels. In this study, the QQ gene aiiO and reporter gene GFP were chromosomally integrated into Escherichia coli BW25113 using the clustered regularly interspaced short palindromic repeats-CRISPR associated protein 9 (CRISPR-Cas9) system. The performance-enhanced switch in the engineering bacteria (EB) allowed it to express aiiO weakly without the inducer isopropyl-beta-D-thiogalactopyranoside (IPTG) and express aiiO strongly with IPTG, and 1.00 mM IPTG induction enhanced EB's QQ activity by 2.34-fold. In activated sludge systems, the inoculation of EB reduced biofilm formation by 18.56 % versus controls after 168 h, with the performance-enhanced switch enhancing inhibition to 24.72 %. EB reduced biofilm thickness by 22.96 %, total microbial biomass by 57.68 %, and significantly decreased extracellular polymeric substances secretion and adhesion strength of the biofilm (maximum reductions: 29.88 % and 34.31 %, respectively) across all sampling points versus controls. 1.00 mM IPTG addition sustainedly intensified these biofilm-inhibitory effects by EB, demonstrating the genetic switch's persistent functionality under environmentally relevant conditions. Furthermore, the genetically modified strain exhibited minimal environmental impact according to standardized assessments. Therefore, this study successfully constructed an implementable strategy for engineering bacteria-mediated biofilm control, with demonstrated applicability in complex environmental systems.},
}
@article {pmid41097540,
year = {2025},
author = {Chen, C and Wu, T and Liu, J and Gao, J},
title = {Threat and Control of tet(X)-Mediated Tigecycline-Resistant Acinetobacter sp. Bacteria.},
journal = {Foods (Basel, Switzerland)},
volume = {14},
number = {19},
pages = {},
doi = {10.3390/foods14193374},
pmid = {41097540},
issn = {2304-8158},
support = {32402890//National Natural Science Foundation of China/ ; 2023M732993//China Postdoctoral Science Foundation/ ; },
abstract = {Tigecycline is regarded as one of the last-resort antibiotics against multidrug-resistant (MDR) Acinetobacter sp. bacteria. Recently, the tigecycline-resistant Acinetobacter sp. isolates mediated by tet(X) genes have emerged as a class of global pathogens for humans and food-producing animals. However, the genetic diversities and treatment options were not systematically discussed in the era of One Health. In this review, we provide a detailed illustration of the evolution route, distribution characteristics, horizontal transmission, and rapid detection of tet(X) genes in diverse Acinetobacter species. We also detail the application of chemical drugs, plant extracts, phages, antimicrobial peptides (AMPs), and CRISPR-Cas technologies for controlling tet(X)-positive Acinetobacter sp. pathogens. Despite excellent activities, the antibacterial spectrum and application safety need further evaluation and resolution. It is noted that deep learning is a promising approach to identify more potent antimicrobial compounds.},
}
@article {pmid41097079,
year = {2025},
author = {Wu, KC and Chang, YH and Chiang, RY and Ding, DC},
title = {CAP-LAMP2b-Modified Stem Cells' Extracellular Vesicles Hybrid with CRISPR-Cas9 Targeting ADAMTS4 to Reverse IL-1β-Induced Aggrecan Loss in Chondrocytes.},
journal = {International journal of molecular sciences},
volume = {26},
number = {19},
pages = {},
doi = {10.3390/ijms26199812},
pmid = {41097079},
issn = {1422-0067},
support = {TCRD 113-065//Hualien Tzu Chi Hospital/ ; },
mesh = {*Extracellular Vesicles/metabolism ; Humans ; *Chondrocytes/metabolism/cytology ; *ADAMTS4 Protein/genetics/metabolism ; *CRISPR-Cas Systems ; *Mesenchymal Stem Cells/metabolism/cytology ; *Aggrecans/metabolism ; *Interleukin-1beta/metabolism ; Cell Differentiation ; Cells, Cultured ; Gene Editing ; },
abstract = {Extracellular vesicles (EVs) from mesenchymal stem cells hold therapeutic promise for inflammatory and degenerative diseases; however, limited delivery and targeting capabilities hinder their clinical use. In this study, we sought to enhance the anti-inflammatory and chondroprotective effects of EVs through CAP-LAMP2b (chondrocyte affinity peptide fused to an EV membrane protein) engineering and ADAMTS4 gene editing hybrid vesicle formation. Human umbilical cord MSCs (hUCMSCs) were characterized via morphology, immunophenotyping, and trilineage differentiation. EVs from control and CAP-LAMP2b-transfected hUCMSCs were fused with liposomes carrying CRISPR-Cas9 ADAMTS4 gRNA. DiI-labeled EV uptake was assessed via fluorescence imaging. CAP-LAMP2b was expressed in hUCMSCs and their EVs. EVs exhibited the expected size (~120 nm), morphology, and exosomal markers (CD9, CD63, CD81, HSP70). CAP-modified hybrid EVs significantly enhanced chondrocyte uptake compared to control EVs and liposomes. IL-1β increased ADAMTS4 expression, whereas CAP-LAMP2b-ADAMTS4 EVs, particularly clone SG3, reversed these effects by reducing ADAMTS4 and restoring aggrecan. Western blotting confirmed suppressed ADAMTS4 and elevated aggrecan protein. CAP-LAMP2b-ADAMTS4 EVs, therefore, showed superior uptake and therapeutic efficacy in inflamed chondrocytes, attenuating inflammatory gene expression and preserving matrix integrity. These results support engineered EVs as a promising cell-free approach for cartilage repair and osteoarthritis treatment.},
}
@article {pmid41097026,
year = {2025},
author = {Jeong, SK and Park, JR and Kim, EG and Kim, KM},
title = {Development of Resistance to Damping-Off in Rice, Oryza sativa L., Using CRISPR/Cas9.},
journal = {International journal of molecular sciences},
volume = {26},
number = {19},
pages = {},
doi = {10.3390/ijms26199761},
pmid = {41097026},
issn = {1422-0067},
mesh = {*Oryza/genetics/microbiology/growth &amp; development ; *CRISPR-Cas Systems ; *Disease Resistance/genetics ; *Plant Diseases/genetics/microbiology ; Gene Editing/methods ; Plants, Genetically Modified/genetics ; Rhizoctonia/pathogenicity ; Plant Proteins/genetics ; },
abstract = {Damping-off disease hinders rice seedling growth and reduces yield. Current control methods, such as seed or soil sterilization, rely on chemicals that cause environmental pollution and promote pathogen resistance. As a sustainable alternative, we targeted the damping-off resistance-related gene OsDGTq1 using CRISPR/Cas9. Field experiments first verified OsDGTq1's significance in resistance. The CRISPR/Cas9 system, delivered via Agrobacterium-mediated transformation, was used to edit OsDGTq1 in rice cultivar Ilmi. Lesions from major damping-off pathogens, Rhizoctonia solani and Pythium graminicola, were observed on G0 plants. All 37 regenerated plants contained T-DNA insertions. Among them, edits generated by sgRNA1-1, sgRNA1-2, and sgRNA1-3 resulted in the insertion of two thymine bases as target mutations. Edited lines were assigned names and evaluated for agronomic traits, seed-setting rates, and pathogen responses. Several lines with edited target genes showed distinct disease responses and altered gene expression compared to Ilmi, likely due to CRISPR/Cas9-induced sequence changes. Further studies in subsequent generations are needed to confirm the stability of these edits and their association with resistance. These results confirm that genome editing of OsDGTq1 alters resistance to damping-off. The approach demonstrates that gene-editing technology can accelerate rice breeding, offering an environmentally friendly strategy to develop resistant varieties. Such varieties can reduce chemical inputs, prevent pollution, and minimize seedling loss, ultimately enhancing food self-sufficiency and stabilizing rice supply.},
}
@article {pmid41097019,
year = {2025},
author = {Kapitonova, MA and Shabalina, AV and Dedkov, VG and Dolgova, AS},
title = {CRISPR-Cas12a-Based Isothermal Detection of Mammarenavirus machupoense Virus: Optimization and Evaluation of Multiplex Capability.},
journal = {International journal of molecular sciences},
volume = {26},
number = {19},
pages = {},
doi = {10.3390/ijms26199754},
pmid = {41097019},
issn = {1422-0067},
support = {Ensuring chemical and biological safety of the Russian Federation//state program/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; *Nucleic Acid Amplification Techniques/methods ; Humans ; *Endodeoxyribonucleases/genetics ; *CRISPR-Associated Proteins/genetics ; *Molecular Diagnostic Techniques/methods ; *Arenaviridae/genetics/isolation &amp; purification ; Animals ; Bacterial Proteins ; },
abstract = {Bolivian hemorrhagic fever (BHF) is a zoonotic disease caused by Mammarenavirus machupoense (MACV) featuring severe neurological and hemorrhagic symptoms and a high mortality rate. BHF is usually diagnosed by serological tests or real-time polymerase chain reaction (RT-PCR); these methods are often inaccessible in endemic regions due to a lack of laboratory infrastructure, creating a demand for sensitive and rapid equipment-free alternatives. Here, we present an isothermal method for MACV nucleic acid detection based on the Cas12a-based DETECTR system combined with recombinase polymerase amplification (RPA) in a single tube: the RT-RPA/DETECTR assay. We demonstrate the possibility of using more than one primer set for the simultaneous detection of MACV genetic variants containing multiple point mutations. The method was optimized and tested using specially developed virus-like armored particles containing the target sequence. The multiplex RT-RPA/DETECTR method achieved a limit of detection of approximately 5 × 10[4] copies/ mL (80 aM) of armored particles. The method was validated using clinical samples spiked with virus-like particles. The assay proved to be selective and reliable in detecting certain nucleotide substitutions simultaneously.},
}
@article {pmid41096922,
year = {2025},
author = {Xu, J and Pan, M and Zhu, Y and Wang, P and Jiang, L and Xu, D and Wang, X and Chen, L and Guo, W and Yang, H and Cao, D},
title = {CRISPR/Cas9-Mediated Targeted Mutagenesis of GmAS1/2 Genes Alters Leaf Shape in Soybean.},
journal = {International journal of molecular sciences},
volume = {26},
number = {19},
pages = {},
doi = {10.3390/ijms26199657},
pmid = {41096922},
issn = {1422-0067},
support = {YBXM2529//National Nanfan Research Institute of CAAS/ ; },
mesh = {*Glycine max/genetics/anatomy &amp; histology/growth &amp; development ; *CRISPR-Cas Systems ; *Plant Leaves/genetics/anatomy &amp; histology/growth &amp; development ; Gene Expression Regulation, Plant ; Phenotype ; *Plant Proteins/genetics/metabolism ; *Mutagenesis ; *Genes, Plant ; Gene Editing ; Transcription Factors/genetics ; Gene Expression Profiling ; Mutation ; },
abstract = {ASYMMETRIC LEAVES1 (AS1) and AS2 play essential roles in regulating leaf development in plants. However, their functional roles in soybean remain poorly understood. Here, we identified two members of the soybean AS1 gene family, GmAS1a and GmAS1c, which exhibit high expression levels in stem and leaf tissues. Using the CRISPR/Cas9 system, we targeted four GmAS1 and three GmAS2 genes, generating mutant lines with distinct leaf development phenotypes, including wrinkling (refers to fine lines and creases on the leaf surface, like aged skin texture), curling (describes the inward or outward rolling of leaf edges, deviating from the typical flat shape), and narrow. We found that functional redundancy exists among the four GmAS1 genes in soybean. GmAS1 and GmAS2 cooperatively regulate leaf curling, leaf crinkling phenotypes, and leaf width in soybean, with functional redundancy also observed between these two genes. Transcriptome sequencing analysis of w3 mutant (as1b as1c as1d as2a as2b as2c) identified 1801 differentially expressed genes (DEGs), including 192 transcription factors (TFs). Gene ontology enrichment analysis revealed significant enrichment of DEGs in pathways associated with plant hormone biosynthesis and signal transduction. A detailed examination of the DEGs showed several genes involved in the development of leaf lateral organs, such as KNOX (SHOOT MERISTEMLESS (STM), KNAT1, KNAT2, and KNAT6), LOB (LBD25, LBD30), and ARP5, were down-regulated in w3/WT (wild-type) comparison. CRISPR/Cas9-mediated targeted mutagenesis of the GmAS1/2 genes significantly impairs leaf development and polarity establishment in soybean, providing valuable germplasm resources and a theoretical framework for future studies on leaf morphogenesis.},
}
@article {pmid41096782,
year = {2025},
author = {Huang, C and Liu, M and Kok, J},
title = {Chromosomal and Plasmid-Based CRISPRi Platforms for Conditional Gene Silencing in Lactococcus lactis.},
journal = {International journal of molecular sciences},
volume = {26},
number = {19},
pages = {},
doi = {10.3390/ijms26199516},
pmid = {41096782},
issn = {1422-0067},
support = {201706350298//China Scholarship Council/ ; 201505990303//China Scholarship Council/ ; },
mesh = {*Lactococcus lactis/genetics ; *Plasmids/genetics ; *CRISPR-Cas Systems ; *Gene Silencing ; Streptococcus pyogenes/genetics ; Bacterial Proteins/genetics ; Gene Expression Regulation, Bacterial ; *Chromosomes, Bacterial/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; Gene Editing ; Promoter Regions, Genetic ; },
abstract = {Inducible CRISPR interference (CRISPRi) systems were established in Lactococcus lactis using both plasmid and chromosomal approaches. Expression of nuclease-deficient Cas9 (dCas9) from Streptococcus pyogenes was placed under the control of the nisin-inducible promoter PnisA, while sgRNAs were transcribed from the constitutive Pusp45 promoter. To monitor expression, dCas9 was fused with superfolder GFP. Plasmid-based constructs successfully repressed a luciferase reporter gene and silenced the gene of the major autolysin, AcmA, leading to the expected morphological phenotype. However, plasmid systems showed leaky expression, producing mutant phenotypes even without induction. Chromosomal integration of dCas9 reduced its expression level by approximately 20-fold compared with plasmid-based expression, thereby preventing leaky activity and ensuring tight regulation. This chromosome-based (cbCRISPRi) platform enabled controlled repression of the essential gene ybeY, which resulted in severe growth defects. Restoration of wild-type phenotypes was achieved by introducing a synonymous codon substitution in the sgRNA target region. Transcriptome analysis of ybeY-silenced cells revealed downregulation of ribosomal protein genes and widespread effects on membrane-associated proteins, ATP synthase subunits, and various transporters. These inducible CRISPRi platforms provide robust and tunable tools for functional genomics in L. lactis, particularly for studying essential genes that cannot be deleted.},
}
@article {pmid41096720,
year = {2025},
author = {Peng, H and Li, J and Sun, K and Tang, H and Huang, W and Li, X and Wang, S and Ding, K and Han, Z and Li, Z and Xu, L and Wang, K},
title = {Advances and Applications of Plant Base Editing Technologies.},
journal = {International journal of molecular sciences},
volume = {26},
number = {19},
pages = {},
doi = {10.3390/ijms26199452},
pmid = {41096720},
issn = {1422-0067},
support = {32472182//National Natural Science Foundation of China/ ; 2024ZD0407704//Biological Breeding-National Science and Technology Major Project/ ; 2022BBF02039//Science and Technology Department of Ningxia in China/ ; },
mesh = {*Gene Editing/methods ; CRISPR-Cas Systems ; *Crops, Agricultural/genetics ; Genome, Plant ; Plant Breeding/methods ; Plants, Genetically Modified/genetics ; },
abstract = {Base editing represents a major breakthrough in the field of genome editing in recent years. By fusing deaminases with the CRISPR/Cas system, it enables precise single-base modifications of DNA. This review systematically summarizes the development of base editing technologies, including cytosine base editors (CBEs), adenine base editors (ABEs), and glycosylase base editors (GBEs), with a particular focus on their applications in crop improvement as well as future trends and prospects. We highlight advances in the creation of novel germplasm with enhanced stress resistance and desirable agronomic traits through base editing in rice, wheat, maize, potato, and other crops, particularly for improving herbicide resistance, disease resistance, and grain quality. Furthermore, we analyze factors that influence base editing efficiency, noting that challenges remain, such as PAM sequence constraints, limited base conversion types, off-target effects, narrow editing windows, and efficiency variation. Future efforts should aim to optimize deaminase activity, expand PAM compatibility, and develop versatile tools to facilitate the broad application of base editing in molecular breeding. This review provides a timely reference for researchers and breeders, offering theoretical guidance and practical insights into harnessing base editing for crop genetic improvement.},
}
@article {pmid41096693,
year = {2025},
author = {He, J and Shi, N and Yao, H and Li, J and Wang, Y and Zhang, J},
title = {Genome Editing in the Chicken: From PGC-Mediated Germline Transmission to Advanced Applications.},
journal = {International journal of molecular sciences},
volume = {26},
number = {19},
pages = {},
doi = {10.3390/ijms26199426},
pmid = {41096693},
issn = {1422-0067},
support = {2023ZD04053//the Biological Breeding-National Science and Technology Major Project/ ; 2025YFHZ0042//the Natural Science Foundation of Sichuan Province/ ; SCU2025D003//the fundamental research funds for the central universitie/ ; },
mesh = {Animals ; *Gene Editing/methods ; *Chickens/genetics ; *Germ Cells/metabolism ; CRISPR-Cas Systems ; },
abstract = {Avian genome editing has historically lagged behind mammalian research. This disparity is primarily due to a unique reproductive biology that precludes standard techniques like pronuclear injection. A pivotal breakthrough, however, came from the development of efficient in vitro culture systems for primordial germ cells (PGCs). This has established the chicken as a tractable and powerful model for genetic engineering. Our review chronicles the technological evolution this has enabled, from early untargeted methods to the precision of modern CRISPR-based systems. We then analyze the broad applications of these tools, which are now used to engineer disease resistance, enhance agricultural traits, and develop novel platforms such as surrogate hosts and oviduct bioreactors. Collectively, these advances have established PGC-based genome editing as a robust and versatile platform. Looking forward, emerging precision editors and the expansion of these techniques to other avian species are poised to drive the next wave of innovation in poultry science and biotechnology.},
}
@article {pmid41096689,
year = {2025},
author = {Haldrup, SB and McClements, ME and Cehajic-Kapetanovic, J and Corydon, TJ and MacLaren, RE},
title = {Gene Therapy Strategies for the Treatment of Bestrophinopathies.},
journal = {International journal of molecular sciences},
volume = {26},
number = {19},
pages = {},
doi = {10.3390/ijms26199421},
pmid = {41096689},
issn = {1422-0067},
support = {NA//Aarhus University/ ; NA//Fight for Sight Denmark/ ; NA//Synoptik Fonden/ ; NA//Maskinfabrikant Jochum Jensen og hustru Mette Marie Jensen, f. Poulsens Mindelegat (fond)/ ; NA//A.P. M&#xf8;ller Foundation/ ; NA//Mette Warburgs Fond/ ; NA//APT Holding/ ; 00038189//VELUX Foundation/ ; 24OC0088426//Novo Nordisk Foundation/ ; NA//NIHR Oxford Biomedical Research Centre/ ; 304408/Z/23/Z//Wellcome Discovery Award/ ; },
mesh = {Humans ; *Genetic Therapy/methods ; *Bestrophins/genetics/metabolism ; *Retinal Diseases/therapy/genetics ; *Eye Diseases, Hereditary/therapy/genetics ; Gene Editing/methods ; Mutation ; Animals ; CRISPR-Cas Systems ; Chloride Channels/genetics ; },
abstract = {The BEST1 gene encodes a transmembrane protein in the retinal pigment epithelium (RPE) in the eye, that functions as a calcium-dependent chloride channel (CaCC). Pathogenic variants in BEST1 are the underlying cause for bestrophinopathies, a group of inherited retinal disorders that vary in their pattern of inheritance, clinical appearance, and underlying molecular disease mechanisms. Currently, there are no treatments available for any of the bestrophinopathies, and gene therapy represents an attractive strategy due to the accessibility of the eye and slow disease progression. While gene augmentation may be effective for a subset of bestrophinopathies, others require allele-specific silencing or correction of the disease-causing variant to reconstitute expression of the BEST1 protein. This review aims to give an overview of the clinical diversity of bestrophinopathies and proposes the molecular disease mechanism of the pathogenic BEST1 variant as an important parameter for the choice of treatment strategy. Furthermore, we discuss the potential of different mutation-specific and mutation-independent CRISPR/Cas9-based gene editing strategies as a future treatment approach for bestrophinopathies.},
}
@article {pmid41096677,
year = {2025},
author = {Șerban, M and Toader, C and Covache-Busuioc, RA},
title = {CRISPR and Artificial Intelligence in Neuroregeneration: Closed-Loop Strategies for Precision Medicine, Spinal Cord Repair, and Adaptive Neuro-Oncology.},
journal = {International journal of molecular sciences},
volume = {26},
number = {19},
pages = {},
doi = {10.3390/ijms26199409},
pmid = {41096677},
issn = {1422-0067},
mesh = {Humans ; *Artificial Intelligence ; *Precision Medicine/methods ; Gene Editing/methods ; *CRISPR-Cas Systems ; Animals ; *Spinal Cord Regeneration ; *Clustered Regularly Interspaced Short Palindromic Repeats ; *Nerve Regeneration/genetics ; },
abstract = {Repairing the central nervous system (CNS) remains one of the most difficult obstacles to overcome in translational neurosciences. This is due to intrinsic growth inhibitors, extracellular matrix issues, the glial scar-form barrier, chronic neuroinflammation, and epigenetic silencing. The purpose of this review is to bring together findings from recent developments in genome editing and computational approaches, which center around the possible convergence of clustered regularly interspaced short palindromic repeats (CRISPR) platforms and artificial intelligence (AI), towards precision neuroregeneration. We wished to outline possible ways in which CRISPR-based systems, including but not limited to Cas9 and Cas12 nucleases, RNA-targeting Cas13, base and prime editors, and transcriptional regulators such as CRISPRa/i, can be applied to potentially reactivate axon-growth programs, alter inhibitory extracellular signaling, reprogram or lineage transform glia to functional neurons, and block oncogenic pathways in glioblastoma. In addition, we wanted to highlight how AI approaches, such as single-cell multi-omics, radiogenomic prediction, development of digital twins, and design of adaptive clinical trials, will increasingly be positioned to act as system-level architects that allow translation of complex datasets into predictive and actionable therapeutic approaches. We examine convergence consumers in spinal cord injury and adaptive neuro-oncology and discuss expanse consumers in ischemic stroke, Alzheimer's disease, Parkinson's disease, and rare neurogenetic syndromes. Finally, we discuss the ethical and regulatory landscape around beyond off-target editing and genomic stability of CRISPR, algorithmic bias, explainability, and equitable access to advanced neurotherapies. Our intent was not to provide a comprehensive inventory of possibilities but rather to provide a conceptual tool where CRISPR acts as a molecular manipulator and AI as a computational integrator, converging to create pathways towards precision neuroregeneration, personalized medicine, and adaptive neurotherapeutics that are ethically sound.},
}
@article {pmid41096563,
year = {2025},
author = {Simoni, S and Fambrini, M and Pugliesi, C and Rogo, U},
title = {Genome Editing by Grafting.},
journal = {International journal of molecular sciences},
volume = {26},
number = {19},
pages = {},
doi = {10.3390/ijms26199294},
pmid = {41096563},
issn = {1422-0067},
mesh = {*Gene Editing/methods ; CRISPR-Cas Systems ; *Genome, Plant ; *Plants, Genetically Modified/genetics ; },
abstract = {Grafting is the process of joining parts of two plants, allowing the exchange of molecules such as small RNAs (including microRNAs and small interfering RNAs), messenger RNAs, and proteins between the rootstock and the scion. Genome editing by grafting exploits RNAs, such as tRNA-like sequences (TLS motifs), to deliver the components (RNA) of the clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (Cas9) system from transgenic rootstock to wild-type scion. The complex Cas9 protein and sgRNA-TLS produced in the scion perform the desired modification without the integration of foreign DNA in the plant genome, resulting in heritable transgene-free genome editing. In this review, we examine the current state of the art of this innovation and how it helps address regulatory problems, improves crop recovery and selection, exceeds the usage of viral vectors, and may reduce potential off-target effects. We also discuss the promise of genome editing by grafting for plants recalcitrant to in vitro culture and for agamic-propagated species that must maintain heterozygosity for plant productivity, fruit quality, and adaptation. Furthermore, we explore the limitations of this technique, including variable efficiency, graft incompatibility among genotypes, and challenges in large-scale application, while highlighting its considerable potential for further improvement and future broader applications for crop breeding.},
}
@article {pmid41093884,
year = {2025},
author = {Moon, J and Zhang, J and Guan, X and Yang, R and Guo, C and Schalper, KT and Avery, L and Banach, D and LaSala, R and Warrier, R and Liu, C},
title = {CRISPR anti-tag-mediated room-temperature RNA detection using CRISPR/Cas13a.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {9142},
pmid = {41093884},
issn = {2041-1723},
mesh = {*CRISPR-Cas Systems/genetics ; Humans ; Hepacivirus/genetics/isolation &amp; purification ; *RNA, Viral/genetics/blood/analysis ; Temperature ; *CRISPR-Associated Proteins/metabolism/genetics ; HIV Infections/virology/diagnosis/blood ; Hepatitis C/diagnosis/virology ; HIV/genetics/isolation &amp; purification ; HIV-1/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; },
abstract = {The CRISPR/Cas13a enzyme serves as a powerful tool for RNA detection due to its RNA-targeting capabilities. However, simple and highly sensitive detection using Cas13a faces challenges, such as the need for pre-amplification and elevated reaction temperatures. In this study, we investigate the allosteric regulation mechanism of Cas13a activation by target RNAs with various structures containing the CRISPR anti-tag sequence. We discover that the target RNA secondary structure and anti-tag sequences inhibit the trans-cleavage reaction of Cas13a. By designing and introducing a specific CRISPR anti-tag hairpin, we develop CRISPR Anti-tag Mediated Room-temperature RNA Detection (CARRD) using a single CRISPR/Cas13a enzyme. This method enables one-step cascade signal amplification for RNA detection without the need for pre-amplification. We apply the CARRD method to detect human immunodeficiency virus (HIV) and hepatitis C virus (HCV), achieving a detection sensitivity of 10 aM. Furthermore, we validate its clinical feasibility by detecting HIV clinical plasma samples, demonstrating a simple, affordable, and efficient approach for viral RNA detection. Due to its simplicity, sensitivity, and flexible reaction temperature, the CARRD method is expected to have broad applicability, paving the way for the development of field-deployable diagnostic tools.},
}
@article {pmid41093851,
year = {2025},
author = {Zhou, SK and Luo, JT and Chen, YF and Lu, ZD and Jian, QH and Jiang, SQ and Li, J and Zhang, XQ and Tan, XY and Yang, XZ and Xu, CF and Wang, J},
title = {Muscle-specific gene editing therapy via mammalian fusogen-directed virus-like particles.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {9145},
pmid = {41093851},
issn = {2041-1723},
support = {32430059//National Natural Science Foundation of China (National Science Foundation of China)/ ; 32471434//National Natural Science Foundation of China (National Science Foundation of China)/ ; 32271442//National Natural Science Foundation of China (National Science Foundation of China)/ ; },
mesh = {Animals ; *Gene Editing/methods ; *Muscle, Skeletal/metabolism ; Mice ; *Muscular Dystrophy, Duchenne/therapy/genetics ; CRISPR-Cas Systems ; Dystrophin/genetics/metabolism ; *Genetic Therapy/methods ; Humans ; Disease Models, Animal ; CRISPR-Associated Protein 9/genetics/metabolism ; Male ; Ribonucleoproteins/genetics/metabolism ; Exons ; Mice, Inbred C57BL ; },
abstract = {Muscle genetic defects can lead to impaired movement, respiratory failure, and other severe symptoms. The development of curative therapies is challenging due to the need for the delivery of gene-editing tools into skeletal muscle cells throughout the body. Here, we use muscular fusogens (Myomaker and Myomerger) to engineer muscle-specific virus-like particles (MuVLPs) for the systemic delivery of gene-editing tools. We demonstrate that MuVLPs can be loaded with diverse payloads, including EGFP, Cre and Cas9/sgRNA ribonucleoproteins (Cas9 RNPs), and can be delivered into skeletal muscle cells via targeted membrane fusion. Systemic administration of MuVLPs carrying Cas9 RNPs enables skeletal muscle-specific gene editing, which excised the exon containing a premature terminator codon mutation in a mouse model for Duchenne muscular dystrophy (DMD). This treatment restores dystrophin expression in various skeletal muscle tissues, including the diaphragm, quadriceps, tibialis anterior, gastrocnemius, and triceps. As a result, the treated mice exhibit a significantly increased capacity for exercise and endurance. This study established a platform for precise gene editing in skeletal muscle tissues.},
}
@article {pmid41093849,
year = {2025},
author = {Shang, M and Li, Y and Cao, Q and Ren, J and Zeng, Y and Wang, J and Gonzalez, RVL and Zhang, X},
title = {A motif preferred adenine base editor with minimal bystander and off-targets editing.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {9153},
pmid = {41093849},
issn = {2041-1723},
mesh = {*Gene Editing/methods ; Animals ; Humans ; Mice ; *Adenine/metabolism/chemistry ; *Nucleotide Motifs/genetics ; HEK293 Cells ; CRISPR-Cas Systems ; },
abstract = {47% of hereditable diseases are caused by single C•G-to-T•A base conversions, which means efficient A-to-G base editing tools (ABEs) have great potential for the treatment of these diseases. However, the existing efficient ABEs, while catalyzing targeted A-to-G conversion, cause high A or C bystander editing and off-target events, which poses safety concerns for their clinical applications. To overcome this shortcoming, we have developed ABE8e-YA (ABE8e with TadA-8e A48E) for efficient and accurate editing of As in YA motifs with YAY > YAR (Y = T or C, R = A or G) hierarchy through structure-oriented rational design. Compared with ABE3.1, which is currently the only ABE version with a YAC motif preference, ABE8e-YA exhibits an average A-to-G editing efficiency improvement of an up to 3.1-fold increase in the indicated YA motif while maintaining reduced bystander C editing and minimized DNA or RNA off-targets. Additionally, we demonstrate that ABE8e-YA efficiently and precisely corrects pathogenic mutations in human cells, suggesting its high suitability for addressing 9.3% of pathogenic point mutations, higher than that of ABE8e and ABE9. Moreover, by using ABE8e-YA, we efficiently and precisely generate hypocholesterolemia and tail-loss mouse models mimicking human-associated disease, as well as performed in vivo mouse proprotein convertase subtilisin/kexin type 9 (Pcsk9) base editing for hypercholesterolemia gene therapy. Together these data indicate its great potential in broad applications for disease modeling and gene therapy.},
}
@article {pmid41093834,
year = {2025},
author = {Shibue, K and Kahraman, S and Castillo-Quan, JI and De Jesus, DF and Hu, J and Morita, H and Blackwell, TK and Yi, P and Kulkarni, RN},
title = {Genome-wide CRISPR Screen Identifies Sec31A as a Key Regulator of Alpha Cell Survival.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {9159},
pmid = {41093834},
issn = {2041-1723},
support = {DK067536//U.S. Department of Health &amp; Human Services | National Institutes of Health (NIH)/ ; R35GM122610//U.S. Department of Health &amp; Human Services | National Institutes of Health (NIH)/ ; R01AG054215//U.S. Department of Health &amp; Human Services | National Institutes of Health (NIH)/ ; JP22K16404//MEXT | Japan Society for the Promotion of Science (JSPS)/ ; },
mesh = {Animals ; Humans ; Mice ; Caenorhabditis elegans/metabolism/genetics ; *Vesicular Transport Proteins/genetics/metabolism ; *Glucagon-Secreting Cells/metabolism/cytology ; Cell Survival/genetics ; Endoplasmic Reticulum Stress/genetics ; CRISPR-Cas Systems ; Receptor, Insulin/metabolism ; Endoplasmic Reticulum/metabolism ; Signal Transduction ; Insulin/metabolism ; Clustered Regularly Interspaced Short Palindromic Repeats ; Insulin-Secreting Cells/metabolism ; Glucagon/metabolism ; },
abstract = {Glucagon, secreted by pancreatic alpha cells, is essential for maintaining normal blood glucose levels. In type 1 and advanced type 2 diabetes, alpha cells often fail to respond to low glucose, yet the mechanisms underlying their stress resistance remain unclear. To investigate this, we performed a genome-wide CRISPR screen and identify Sec31A, a gene involved in transporting proteins from the endoplasmic reticulum (ER), as a key regulator of alpha cell survival under stress. We show that loss of Sec31A enhances survival in stressed mouse alpha cells and in C. elegans. In human islets, SEC31A expression increases in alpha cells under inflammatory stress, and this upregulation is reversed by reducing ER stress. Functional studies in lab-grown human islet clusters reveal distinct responses in alpha versus beta cells following Sec31A suppression. We also find that Sec31A interacts with the insulin receptor, suggesting a link between stress adaptation and insulin signaling in alpha cells.},
}
@article {pmid41093525,
year = {2025},
author = {Wei, Z and Luo, H and Huang, D and Wei, P and Zhang, J and Wei, J and Tang, Q and Huang, L and Zhang, K and Liao, X},
title = {Structure-specific electrochemiluminescent biosensor for FEN1 detection via dumbbell probe-mediated transcription and CRISPR/Cas13a-induced G-quadruplexes cleavage.},
journal = {Analytica chimica acta},
volume = {1377},
number = {},
pages = {344662},
doi = {10.1016/j.aca.2025.344662},
pmid = {41093525},
issn = {1873-4324},
mesh = {*Biosensing Techniques/methods ; *G-Quadruplexes ; *Flap Endonucleases/analysis/blood/metabolism ; *Electrochemical Techniques/methods ; Humans ; *CRISPR-Cas Systems ; *Luminescent Measurements/methods ; *DNA Probes/chemistry/genetics ; Limit of Detection ; Transcription, Genetic ; *CRISPR-Associated Proteins/metabolism ; },
abstract = {Flap endonuclease 1 (FEN1) is crucial for DNA replication, repair, and telomere maintenance. Its dysregulation is linked to various cancers and diseases. Accurate detection of FEN1 is essential for early diagnosis and therapeutic monitoring. Thus, a novel electrochemiluminescent (ECL) biosensor has been developed for the structure-specific and detection of FEN1. The strategy integrates dumbbell DNA probe-mediated transcription and CRISPR/Cas13a-induced trans-cleavage of RNA G-quadruplexes. In the presence of FEN1, the 5'-flap structure of the probe was selectively cleaved and subsequently ligated by T4 DNA ligase to form a closed circular template. This enabled T7 RNA polymerase to transcribe crRNA-encoded RNA strands, which activated Cas13a to cleave surface-tethered G-quadruplexes/hemin complexes on a Ru(II)/Ti3C2/AuNPs-modified electrode, thereby restoring the quenched ECL signal. The biosensor exhibited an ultralow detection limit of 4.82 × 10[-9] U μL[-1] and a wide dynamic range (1 × 10[-8] to 1 × 10[-5] U μL[-1]), along with excellent specificity and stability. Successful application in human serum validated its reliability for complex biological samples. This work presents a powerful platform for sensitive FEN1 monitoring, holding potential for clinical diagnostics and enzymatic analysis.},
}
@article {pmid41093508,
year = {2025},
author = {Wei, Z and Huang, D and Luo, H and Zhang, J and Wei, J and Tang, Q and Huang, L and Zhang, K and Liao, X},
title = {A multi-level signal conversion architecture for enzyme sensing: Integrating MXene nanoplatforms with CRISPR-driven electrochemiluminescence.},
journal = {Analytica chimica acta},
volume = {1377},
number = {},
pages = {344636},
doi = {10.1016/j.aca.2025.344636},
pmid = {41093508},
issn = {1873-4324},
mesh = {*Biosensing Techniques/methods ; *Electrochemical Techniques/methods ; *Luminescent Measurements/methods ; *CRISPR-Cas Systems ; Humans ; *Flap Endonucleases/analysis/metabolism ; Nanocomposites/chemistry ; Gold/chemistry ; Limit of Detection ; DNA/chemistry ; Nitrites ; Titanium ; Transition Elements ; },
abstract = {Precise and ultrasensitive detection of flap endonuclease 1 (FEN1), a key DNA repair enzyme implicated in cancer diagnostics, remains challenging due to its subtle structural cleavage activity. Herein, we present a cascade-amplified electrochemiluminescence (ECL) biosensor based on a Ti3C2 MXene-supported Ru (bpy)3[2+]/Au nanocomposite integrated with a CRISPR-Cas13a system and DNA walker circuitry. Upon specific recognition and cleavage of a 5'-flap substrate by FEN1, a nicked DNA product is circularized and transcribed via T7 RNA polymerase, yielding RNA activators that trigger Cas13a-mediated collateral cleavage. This event releases a blocked DNA walker, which reorganizes Fc-labeled DNA on the electrode surface and restores the ECL signal suppressed by resonance energy transfer. The system achieves a detection limit as low as 1.48 fU/mL and exhibits a dynamic range spanning five orders of magnitude. Compared to fluorescence-based CRISPR detection systems, the ECL-based platform offers low background, high signal-to-noise ratios, and operational simplicity using standard electrochemical instrumentation, supporting practical deployment in clinical diagnostics. Furthermore, the platform demonstrates high selectivity against other nucleases and proteins, along with excellent performance in spiked human serum samples. This work presents a robust and modular strategy for accurate enzyme activity profiling with promising applications in early-stage disease diagnostics.},
}
@article {pmid41092254,
year = {2025},
author = {Yin, K and Tsai, CJ},
title = {Turbo-charging crop improvement: harnessing multiplex editing for polygenic trait engineering and beyond.},
journal = {The Plant journal : for cell and molecular biology},
volume = {124},
number = {1},
pages = {e70527},
pmid = {41092254},
issn = {1365-313X},
support = {//Georgia Research Alliance/ ; DE-SC0023166//U.S. Department of Energy/ ; DE-SC0023338//U.S. Department of Energy/ ; ERKP886//U.S. Department of Energy/ ; },
mesh = {*Gene Editing/methods ; *Crops, Agricultural/genetics ; CRISPR-Cas Systems/genetics ; Genome, Plant/genetics ; *Multifactorial Inheritance/genetics ; Plants, Genetically Modified/genetics ; *Genetic Engineering/methods ; },
abstract = {Multiplex CRISPR editing has emerged as a transformative platform for plant genome engineering, enabling the simultaneous targeting of multiple genes, regulatory elements, or chromosomal regions. This approach is effective for dissecting gene family functions, addressing genetic redundancy, engineering polygenic traits, and accelerating trait stacking and de novo domestication. Its applications now extend beyond standard gene knockouts to include epigenetic and transcriptional regulation, chromosomal engineering, and transgene-free editing. These capabilities are advancing crop improvement not only in annual species but also in more complex systems such as polyploids, undomesticated wild relatives, and species with long generation times. At the same time, multiplex editing presents technical challenges, including complex construct design and the need for robust, scalable mutation detection. We discuss current toolkits and recent innovations in vector architecture, such as promoter and scaffold engineering, that streamline workflows and enhance editing efficiency. High-throughput sequencing technologies, including long-read platforms, are improving the resolution of complex editing outcomes such as structural rearrangements-often missed by standard genotyping-when targeting repetitive or tandemly spaced loci. To fully realize the potential of multiplex genome engineering, there is growing demand for user-friendly, synthetic biology-compatible, and scalable computational workflows for gRNA design, construct assembly, and mutation analysis. Experimentally validated inducible or tissue-specific promoters are also highly desirable for achieving spatiotemporal control. As these tools continue to evolve, multiplex CRISPR editing is poised to become a foundational technology of next-generation crop improvement to address challenges in agriculture, sustainability, and climate resilience.},
}
@article {pmid41091748,
year = {2025},
author = {Yew, WN and Dean, CJ and Chan, DKH},
title = {STAG2 mutations in the normal colon induce upregulation of oncogenic pathways in neighbouring wildtype cells.},
journal = {PloS one},
volume = {20},
number = {10},
pages = {pone.0332499.exml},
pmid = {41091748},
issn = {1932-6203},
mesh = {Humans ; Organoids/metabolism ; *Colon/metabolism/pathology/cytology ; *Mutation ; Up-Regulation ; Signal Transduction/genetics ; CRISPR-Cas Systems ; Coculture Techniques ; Gene Editing ; Proto-Oncogene Proteins p21(ras)/metabolism/genetics ; *Carcinogenesis/genetics ; *Colorectal Neoplasms/genetics/pathology ; Tumor Necrosis Factor-alpha/metabolism ; },
abstract = {While driver mutations in the normal colon have been described, characterizing the role and function of these driver mutations in relation to colorectal oncogenesis remains incomplete. Here, we investigated the role of STAG2 mutants in the normal colon using patient-derived wildtype organoids. Using CRISPR-Cas9 gene editing, we generated STAG2 mutants, and co-cultured these mutants with wildtype organoids, mimicking the presence of such STAG2 mutants in the normal colon. We sought to determine the transcriptional impact of co-culture using scRNAseq. Surprisingly, we uncovered a possible cell-cell interaction between STAG2 mutants and wildtype organoids, in which wildtype organoids in co-culture with STAG2 mutants upregulated known oncogenic pathways. This included the upregulation of TNFα-signaling, as well as KRAS-signaling in wildtype organoids. These results suggested that STAG2 mutant cells exert a pro-oncogenic effect in a cell interactive manner, instead of via a cell autonomous approach. In conclusion, our findings demonstrate a novel mechanism of colorectal oncogenesis which can support further investigation.},
}
@article {pmid41091245,
year = {2025},
author = {Yang, S and Jiao, X and Liu, J and Liu, Y and Wang, M and Li, S and Qiao, J},
title = {CRISPR-Cas opens a new era of antimicrobial therapy as a powerful gene editing tool.},
journal = {World journal of microbiology &amp; biotechnology},
volume = {41},
number = {10},
pages = {388},
pmid = {41091245},
issn = {1573-0972},
support = {X2024739//College Students' Innovation and Entrepreneurship Training Program of Shandong Second Medical University/ ; ZR2020MH305//Natural Science Foundation of Shandong Province, China/ ; },
}
@article {pmid41090767,
year = {2025},
author = {Slattery, JR and Naung, NY and Kalinna, BH and Pal, M},
title = {CRISPR-Powered Liquid Biopsies in Cancer Diagnostics.},
journal = {Cells},
volume = {14},
number = {19},
pages = {},
pmid = {41090767},
issn = {2073-4409},
mesh = {Humans ; Liquid Biopsy/methods ; *Neoplasms/diagnosis/genetics ; Biomarkers, Tumor/genetics ; *CRISPR-Cas Systems/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Gene Editing/methods ; },
abstract = {Liquid biopsies promise major advantages for cancer screening and diagnosis. By detecting biomarkers in peripheral blood samples, liquid biopsies reduce the need for invasive techniques and provide important genetic information integral to the emerging molecular classification of cancers. Unfortunately, the concentrations of most biomarkers, particularly circulating tumour nucleic acids, are vanishingly small-beyond the sensitivity and specificity of most assays. Clustered Regularly Interspaced Short Palindromic Repeats diagnostics (herein labelled 'CRISPR-Dx') use gene editing tools to detect, rather than modify, nucleic acids with extremely high specificity. These tools are commonly combined with isothermal nucleic acid amplification to also achieve sensitivities comparable to high-performance laboratory-based techniques, such as digital PCR. CRISPR assays, however, are inherently well suited to adaptation for point-of-care (POC) use, and unlike antigen-based POC assays, are significantly easier and faster to develop. In this review, we summarise current CRISPR-Dx platforms and their analytical potential for cancer biomarker discovery, with an emphasis on enhancing early diagnosis, disease monitoring, point-of-care testing, and supporting cancer therapy.},
}
@article {pmid41090360,
year = {2025},
author = {Wei, W and Zhu, W and Silver, S and Armstrong, AM and Robbins, FS and Rameshbabu, AP and Walz, K and Quan, Y and Du, W and Kim, Y and Indzhykulian, AA and Shu, Y and Liu, XZ and Chen, ZY},
title = {Single-dose genome editing therapy rescues auditory and vestibular functions in adult mice with DFNA41 deafness.},
journal = {The Journal of clinical investigation},
volume = {135},
number = {20},
pages = {},
pmid = {41090360},
issn = {1558-8238},
mesh = {Animals ; *Gene Editing ; Mice ; *Genetic Therapy ; Humans ; Dependovirus/genetics ; *Deafness/therapy/genetics ; Disease Models, Animal ; *Vestibule, Labyrinth/physiopathology ; Mutation, Missense ; CRISPR-Cas Systems ; },
abstract = {Genome editing has the potential to treat genetic hearing loss. However, current editing therapies for genetic hearing loss have shown efficacy only in hearing rescue. In this study, we evaluated a rescue strategy using adeno-associated virus (AAV) type 2-mediated delivery of Staphylococcus aureus Cas9-sgRNA in the mature inner ear of the P2rx2V61L/+ mouse model of autosomal dominant deafness-41 (DFNA41), a dominant, delayed-onset, and progressive hearing loss in humans. We demonstrate that local injection in adult mice results in efficient and specific editing that abolishes the mutation without notable off-target effects or AAV genome integration. Editing effectively restores long-term auditory and vestibular function. Editing further protects P2rx2V61L/+ mice from hypersensitivity to noise-induced hearing loss, a phenotype also observed in patients with DFNA41. Intervention in mice at a juvenile stage broadens the frequency range rescued, highlighting the importance of early intervention. An effective and specific gRNA for the human P2RX2 V60L mutation has been identified. Our study establishes the feasibility of editing to treat DFNA41 caused by P2RX2 V60L mutation in humans and opens an avenue for using editing to rescue hearing and vestibular function while mitigating noise-induced hearing loss.},
}
@article {pmid40921807,
year = {2025},
author = {Zhang, Y and Zhao, Z and Liu, M and Yang, J and Yang, C and Su, N and Sun, J and Fang, Y and Wang, Y and Li, X and Chen, W and Wu, J and Bai, J},
title = {Asymmetric volume-mediated buffer control overcomes sensitivity limits in one-pot RAA-CRISPR/Cas12a visual detection.},
journal = {Analytical and bioanalytical chemistry},
volume = {417},
number = {26},
pages = {5971-5981},
pmid = {40921807},
issn = {1618-2650},
mesh = {*CRISPR-Cas Systems ; Limit of Detection ; *Nucleic Acid Amplification Techniques/methods ; Buffers ; *Recombinases/metabolism/chemistry ; Drug Resistance, Bacterial/genetics ; Colistin/pharmacology ; Animals ; *Endodeoxyribonucleases/genetics ; Bacterial Proteins ; CRISPR-Associated Proteins ; },
abstract = {Rapid, low-cost, and visual nucleic acid detection methods are highly attractive for curbing colistin resistance spread through the food chain. CRISPR/Cas12a combined with recombinase-aided amplification (RAA) offers a one-pot, aerosol-free approach for visual detection. However, traditional one-pot systems often run Cas12a trans-cleavage in a buffer suitable for RAA, thus limiting Cas12a cleavage efficiency. This study proposes an asymmetric volume-optimized RAA-CRISPR/Cas12a assay for ultrasensitive visual detection of mobile colistin resistance gene mcr-1. Unlike conventional one-pot systems constrained by buffer incompatibility, our design spatially segregates a minimal-volume RAA-MIX (lid) from a CRISPR-dominant buffer microenvironment (tube bottom). This architecture leverages RAA's exponential amplification power to ensure sufficient product yield from minimal reaction volumes, while enabling subsequent enhancement of Cas12a trans-cleavage through automatic buffer assimilation upon mixing. The results were able to be visually observed under UV light, achieving 63.1% cost reduction compared to standard one-pot methods. The sensitivity of the proposed method for the mcr-1 gene was 2.5 copies/reaction, with anti-interference against other plasmids or bacteria. This method was applied to the detection of mcr-1 in animal-derived foods, showing satisfactory practical performance. By fundamentally reengineering buffer microenvironments through volume asymmetry, this work provides a general strategy for one-pot molecular diagnostics, achieving dual optimization of amplification and cleavage without trade-offs.},
}
@article {pmid40914368,
year = {2025},
author = {Liu, X and Fu, Y and Li, M and Xiong, S and Huang, L and Zhang, S and Zhang, W and Liang, X and Wang, W and Tang, K and Shen, Q},
title = {Biofortification of tomatoes with beta-carotene through targeted gene editing.},
journal = {International journal of biological macromolecules},
volume = {327},
number = {Pt 2},
pages = {147396},
doi = {10.1016/j.ijbiomac.2025.147396},
pmid = {40914368},
issn = {1879-0003},
mesh = {*Solanum lycopersicum/genetics/metabolism/chemistry ; *beta Carotene/biosynthesis/genetics/metabolism ; *Biofortification/methods ; *Gene Editing/methods ; Fruit/genetics/chemistry ; Lycopene/metabolism ; Nutritive Value ; Carotenoids/metabolism ; CRISPR-Cas Systems ; },
abstract = {Vitamin A deficiency is one of the most severe micronutrient-related health issues worldwide. Tomatoes, a widely cultivated crop for their adaptability, nutritional value, and lycopene content (a beta-carotene precursor), are ideal candidates for biofortification. In this study, CRISPR-mediated knockout mutants (cr-SlLCYe and cr-SlBCH) were generated to enhance the precursor supply to the β-carotene biosynthetic pathway and reduce its degradation. Carotenoids profiling showed that β-carotene levels in the mutants were 1.7 to 2.5-fold higher than in the wild-type, whereas lycopene levels remained unaltered without altering lycopene content. To evaluate potential trade-offs, the characteristics of the mutant fruits were comprehensively assessed, including appearance quality (color, firmness), nutritional quality (sugars, organic acids, vitamin C), and postharvest traits (shelf life, resistance to Botrytis cinerea). These results provide a new strategy for elevating β-carotene without compromising fruit quality and offer new insights into combating vitamin A deficiency through targeted tomato breeding programs.},
}
@article {pmid40830415,
year = {2025},
author = {Bi, J and Mo, W and Liu, M and Song, Y and Xiao, Q and Fan, S and Wang, W and Shi, T and Zheng, Y and Lian, J and Liu, R and Chen, B and Huang, X and Li, P and Zhao, Z and Shi, J and Zhang, L and Su, G and Zhang, N and Lu, W},
title = {Systematic decoding of functional enhancer connectomes and risk variants in human glioma.},
journal = {Nature cell biology},
volume = {27},
number = {10},
pages = {1838-1847},
pmid = {40830415},
issn = {1476-4679},
support = {32130018//National Natural Science Foundation of China (National Science Foundation of China)/ ; },
mesh = {Humans ; *Glioma/genetics/pathology/metabolism ; *Enhancer Elements, Genetic/genetics ; *Polymorphism, Single Nucleotide/genetics ; *Brain Neoplasms/genetics/pathology/metabolism ; Genetic Predisposition to Disease ; Gene Expression Regulation, Neoplastic ; Cell Line, Tumor ; Myeloid Ecotropic Viral Integration Site 1 Protein/genetics/metabolism ; *Connectome/methods ; CRISPR-Cas Systems ; Disease Progression ; Risk Factors ; },
abstract = {Genetic and epigenetic variations contribute to the progression of glioma, but the mechanisms underlying these effects, particularly for enhancer-associated genetic variations in non-coding regions, still remain unclear. Here we performed high-throughput CRISPR interference screening to identify pro-tumour enhancers in glioma cells. By integrating genome-wide H3K27ac HiChIP data, we identified the target genes of these pro-tumour enhancers and revealed the essential role of enhancer connectomes in promoting glioma progression. Through systematic analysis of enhancers carrying glioma risk-associated single-nucleotide polymorphisms (SNPs), we found that these SNPs can promote glioma progression through the enhancer connectome. Using CRISPR-Cas9-mediated enhancer interference and SNP editing, we demonstrated that glioma-specific enhancer carrying the risk SNP rs2297440 regulates SOX18 expression by specifically recruiting transcription factor MEIS1 binding, thereby contributing to glioma progression. Our study sheds light on the molecular mechanisms underlying glioma susceptibility and provides potential therapeutic targets to treat glioma.},
}
@article {pmid40646310,
year = {2025},
author = {Rodschinka, G and Forcelloni, S and Kühner, FM and Wani, S and Riemenschneider, H and Edbauer, D and Behrens, A and Nedialkova, DD},
title = {Comparative CRISPRi screens reveal a human stem cell dependence on mRNA translation-coupled quality control.},
journal = {Nature structural &amp; molecular biology},
volume = {32},
number = {10},
pages = {1932-1946},
pmid = {40646310},
issn = {1545-9985},
mesh = {Humans ; *RNA, Messenger/genetics/metabolism ; *Protein Biosynthesis ; *Induced Pluripotent Stem Cells/metabolism/cytology ; *CRISPR-Cas Systems ; Ribosomes/metabolism ; Cell Differentiation ; Myocytes, Cardiac/metabolism/cytology ; },
abstract = {The translation of mRNA into proteins in multicellular organisms needs to be carefully tuned to changing proteome demands in development and differentiation, while defects in translation often have a disproportionate impact in distinct cell types. Here we used inducible CRISPR interference screens to compare the essentiality of genes with functions in mRNA translation in human induced pluripotent stem cells (hiPS cells) and hiPS cell-derived neural and cardiac cells. We find that core components of the mRNA translation machinery are broadly essential but the consequences of perturbing translation-coupled quality control factors are cell type dependent. Human stem cells critically depend on pathways that detect and rescue slow or stalled ribosomes and on the E3 ligase ZNF598 to resolve a distinct type of ribosome collision at translation start sites on endogenous mRNAs with highly efficient initiation. Our findings underscore the importance of cell identity for deciphering the molecular mechanisms of translational control in metazoans.},
}
@article {pmid41090337,
year = {2025},
author = {Cherdantsev, AI and Kulagin, KA and Polyakova, AN and Karpov, VL and Sosnovtseva, AO and Karpov, DS},
title = {[DNA Double-Strand Break Repair System by a Mechanism of Non-Homologous End Joining Provides Resistance to DNA-Damaging and Oxidizing Stresses in the Yeast Debaryomyces hansenii].},
journal = {Molekuliarnaia biologiia},
volume = {59},
number = {4},
pages = {616-628},
doi = {10.31857/S0026898425040083},
pmid = {41090337},
issn = {0026-8984},
mesh = {*DNA End-Joining Repair ; *DNA Breaks, Double-Stranded ; *Oxidative Stress/genetics ; CRISPR-Cas Systems ; *Debaryomyces/genetics/metabolism ; *Fungal Proteins/genetics/metabolism ; Osmotic Pressure ; },
abstract = {The unconventional halotolerant yeast Debaryomyces hansenii is of great importance in biotechnology and the food industry, and in basic research it serves as a model for studying the molecular mechanisms of resistance to increased salinity and osmotic stress. We have previously established an efficient method for editing the D. hansenii genome using the CRISPR/Cas9 system. In turn, this has stimulated further investigation of the structure and physiological role of DNA double-strand break repair pathways in D. hansenii. The aim of the present work was to evaluate the involvement of key components of the DNA double-stranded break repair system by the non-homologous end joining (NHEJ) mechanism in the resistance of D. hansenii to DNA-damaging compounds and compounds that induce oxidative, high salinity, and osmotic stress. Using the CRISPR/Cas9 system, mutant strains with knockout of the DEHA2F10208g (DhKU70), DEHA2B01584g (DhKU80) , and DEHA2G04224g (DhLIG4) genes encoding key components of NHEJ were obtained. It was found that mutant strains, unlike the wild-type strain, are sensitive to chemical compounds that damage DNA, as well as to compounds that cause oxidative stress. Osmotic and high salinity stresses and vanillin do not cause significant changes in the rate of colony formation of mutant strains. Unexpectedly, mutant strains exhibit increased resistance to caffeine compared to the wild-type strain. The data indicate that the NHEJ systems of D. hansenii play a significant role in the response to DNA-damaging and oxidative types of stress. The importance of the NHEJ system in the processes of maintaining yeast cell homeostasis should be taken into account when creating strains producing valuable substances.},
}
@article {pmid41090155,
year = {2025},
author = {Li, JM and Huang, J and Liao, Y and Hu, T and Wang, CL and Zhang, WZ and Huang, CW},
title = {Gene and RNA Editing: Revolutionary Approaches to Treating Diseases.},
journal = {MedComm},
volume = {6},
number = {10},
pages = {e70389},
pmid = {41090155},
issn = {2688-2663},
abstract = {Gene editing and RNA editing technologies are advancing modern medicine by enabling precise manipulation of genetic information at the DNA and RNA levels, respectively. The third-generation gene editing tools, particularly Clustered regularly interspaced shortpalindromic repeats (CRISPR)/CRISPR-associated (Cas) system, have transformed genetic disease treatment with high efficiency, precision, and cost effectiveness, while RNA editing, via adenosine deaminase acting on RNA (ADAR) enzymes and CRISPR-Cas13, offers reversible regulation to avoid genomic integration risks. Despite advancements, challenges persist in delivery efficiency, tissue specificity, and long-term safety, limiting their clinical translation. This review systematically discusses the molecular mechanisms and technological evolution of these tools, focusing on their promising applications in treating nervous system disorders (e.g., Alzheimer's, Parkinson's), immune diseases (e.g., severe combined immunodeficiency, lupus), and cancers. It compares their technical attributes, analyzes ethical and regulatory issues, and highlights synergies between the two technologies. By bridging basic research and clinical translation, this review provides critical insights for advancing precision medicine, reshaping disease diagnosis, prevention, and treatment paradigms.},
}
@article {pmid41088816,
year = {2025},
author = {Mo, T and Ren, HY and Zhang, XX and Lu, YW and Teng, ZQ and Zhang, X and Dai, LP and Hou, L and Zhao, N and He, J and Qin, T},
title = {Phenotypic Function of Legionella pneumophila Type I-F CRISPR-Cas.},
journal = {Biomedical and environmental sciences : BES},
volume = {38},
number = {9},
pages = {1105-1119},
doi = {10.3967/bes2025.107},
pmid = {41088816},
issn = {2214-0190},
mesh = {*Legionella pneumophila/genetics/physiology/pathogenicity ; *CRISPR-Cas Systems ; Biofilms/growth &amp; development ; Phenotype ; Bacterial Proteins/genetics/metabolism ; Gene Deletion ; },
abstract = {OBJECTIVE: CRISPR-Cas protects bacteria from exogenous DNA invasion and is associated with bacterial biofilm formation and pathogenicity.<br><br>METHODS: We analyzed the type I-F CRISPR-Cas system of Legionella pneumophila WX48, including Cas1, Cas2-Cas3, Csy1, Csy2, Csy3, and Cas6f, along with downstream CRISPR arrays. We explored the effects of the CRISPR-Cas system on the in vitro growth, biofilm-forming ability, and pathogenicity of L. pneumophila through constructing gene deletion mutants.<br><br>RESULTS: The type I-F CRISPR-Cas system did not affect the in vitro growth of wild-type or mutant strains. The biofilm formation and intracellular proliferation of the mutant strains were weaker than those of the wild type owing to the regulation of type IV pili and Dot/Icm type IV secretion systems. In particular, Cas6f deletion strongly inhibited these processes.<br><br>CONCLUSION: The type I-F CRISPR-Cas system may reduce biofilm formation and intracellular proliferation in L. pneumophila.},
}
@article {pmid41086252,
year = {2025},
author = {Libri, AB and Wang, J and Marton, T and Yu, W and Dossin, F and Balmus, G and Reina-San-Martin, B and Frock, R and Lescale, C and Deriano, L},
title = {Senataxin promotes recombination fidelity during antigen receptor gene diversification.},
journal = {Science signaling},
volume = {18},
number = {908},
pages = {eadv8801},
doi = {10.1126/scisignal.adv8801},
pmid = {41086252},
issn = {1937-9145},
mesh = {Animals ; *V(D)J Recombination/genetics ; Mice ; DNA End-Joining Repair ; *RNA Helicases/genetics/metabolism ; Ataxia Telangiectasia Mutated Proteins/genetics/metabolism ; DNA Breaks, Double-Stranded ; Multifunctional Enzymes ; CRISPR-Cas Systems ; Mice, Knockout ; *DNA Helicases/genetics/metabolism ; Humans ; *Receptors, Antigen, T-Cell/genetics ; Precursor Cells, B-Lymphoid/metabolism/immunology ; },
abstract = {Antigen receptor diversity depends on the assembly of variable (V), diverse (D), and joining (J) exons in genes encoding immunoglobulins (Igs) and T cell receptors (TCRs). During V(D)J recombination, DNA double-strand breaks (DSBs) introduced by the RAG1/2 nuclease complex are repaired by the process of nonhomologous end-joining (NHEJ). We hypothesized that functional redundancies between NHEJ and the chromatin DSB response, which depends on the kinase ATM, potentially masked the activity of additional factors that regulate V(D)J recombination. We performed targeted CRISPR-Cas9 knockout screens for genes implicated in V(D)J recombination in pro-B cells that were either untreated or treated with an ATM inhibitor. We found that loss of the RNA/DNA helicase senataxin (SETX) impaired V(D)J recombination and led to the formation of aberrant hybrid joints between coding ends and signal ends, both in vitro and in mice. The loss of SETX in a background deficient in the NHEJ factor XLF or in which ATM was inhibited led to substantial impairment of V(D)J recombination and to the presence of unsealed coding ends. SETX limited aberrant activation-induced cytidine deaminase (AID)-induced DNA end-joining between Igh-containing alleles during the process of class-switch recombination. Together, our findings reveal a previously uncharacterized role for SETX in promoting recombination fidelity during antigen receptor gene diversification.},
}
@article {pmid41085803,
year = {2025},
author = {de Souza, HCA and Panzenhagen, P and Portes, AB and Dos Santos, AMP and Fidelis, J and Junior, CAC},
title = {CRISPR-Cas systems in combating antimicrobial resistance: which system to choose? A systematic review.},
journal = {World journal of microbiology &amp; biotechnology},
volume = {41},
number = {10},
pages = {381},
pmid = {41085803},
issn = {1573-0972},
support = {FinanceCode001//Coordena&#xe7;&#xe3;o de Aperfei&#xe7;oamento de Pessoal de N&#xed;vel Superior/ ; 313119/2020-1//Conselho Nacional de Desenvolvimento Cient&#xed;fico e Tecnol&#xf3;gico/ ; E26/202.227/2018//Funda&#xe7;&#xe3;o Carlos Chagas Filho de Amparo &#xe0; Pesquisa do Estado do Rio de Janeiro/ ; },
mesh = {*CRISPR-Cas Systems ; *Gene Editing/methods ; *Bacteria/genetics/drug effects ; *Drug Resistance, Bacterial/genetics ; Humans ; Anti-Bacterial Agents/pharmacology ; },
abstract = {Antimicrobial resistance (AMR) poses a growing threat to global public health, progressively compromising the efficacy of available antimicrobials. Technologies based on Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) have emerged as promising tools for controlling resistant pathogens, offering high specificity and versatility. However, a comprehensive and systematic synthesis of CRISPR strategies applied to AMR remains limited. From February 12, 2025, we conducted a systematic review of the PubMed, Embase, and Scopus databases, using the following search strategy: Population (resistant bacteria or plasmid-mediated resistance), Intervention (CRISPR, including variants such as CRISPR-Cas9, Cas3, Cas12, Cas13, and CRISPR interference [CRISPRi]), and Outcomes (bacterial resensitization or plasmid curing). The CRISPR-Cas9 system was the most frequently employed (75.7%), with conjugation identified as the primary delivery method. We identified the advantages and limitations of each system, highlighting CRISPRi and CRISPR-Cas13a as alternatives to overcome the constraints of direct genome editing. Delivery efficiency remains a central challenge, although nanocarrier- and bacteriophage-based methodologies show promising potential. We also propose a decision map that guides the selection of the most appropriate CRISPR-Cas system and delivery strategy, considering factors such as therapeutic objective, gene location, methodology efficiency, application environment, and clinical feasibility. This review provides an updated and structured synthesis of CRISPR strategies applied to AMR, emphasizing their potential translational and clinical applications. The findings can inform the development of CRISPR-based therapeutics, guide the design of preclinical studies, and support future strategies for combating multidrug-resistant infections in clinical settings.},
}
@article {pmid41085033,
year = {2025},
author = {Levassor, L and Whitford, CM and Petersen, SD and Blin, K and Weber, T and Frandsen, RJN},
title = {StreptoCAD: An Open-Source Software Toolbox Automating Genome Engineering Workflows in Streptomycetes.},
journal = {ACS synthetic biology},
volume = {},
number = {},
pages = {},
doi = {10.1021/acssynbio.5c00261},
pmid = {41085033},
issn = {2161-5063},
abstract = {Streptomycetes hold immense potential for discovering novel bioactive molecules for applications in medicine or sustainable agriculture. However, high-throughput exploration is hampered by the current Streptomyces genetic engineering methods that involve the manual design of complex experimental molecular biological engineering strategies for each targeted gene. Here, we introduce StreptoCAD, an open-source software toolbox that automates and streamlines the design of genome engineering strategies in Streptomyces, supporting various CRISPR-based and gene overexpression methods. Once initiated, StreptoCAD designs all necessary DNA primers and CRISPR guide sequences, simulates plasmid assemblies (cloning) and the resulting modification of the genomic target(s), and further summarizes the information needed for laboratory implementation and documentation. StreptoCAD currently offers six design workflows, including the construction of overexpression libraries, base-editing, including multiplexed CRISPR-BEST plasmid generation, and genome engineering using CRISPR-Cas9, CRISPR-Cas3, and CRISPRi systems. In addition to automating the design process, StreptoCAD further secures compliance with the FAIR principles, ensuring reproducibility and ease of data management via standardized output files. To experimentally demonstrate the design process and output of StreptoCAD, we designed and constructed a series of gene overexpression strains, and performed CRISPRi knockdowns in Streptomyces Gö40/10, underscoring the tool's efficiency and user-friendliness.. This tool simplifies complex genetic engineering tasks and promotes collaboration through standardized workflows and design parameters. StreptoCAD is set to transform genome engineering in Streptomyces, making sophisticated genetic manipulations accessible for all and accelerating natural product discovery.},
}
@article {pmid41084992,
year = {2025},
author = {Han, J and Ganguly, R and Yi, JY and Yun, H and Jung, SY and Sung, C and Lee, CS},
title = {Osmotically Tunable Microdroplets Enable Amplification-Free CRISPR Detection of Gene Doping.},
journal = {Advanced science (Weinheim, Baden-Wurttemberg, Germany)},
volume = {},
number = {},
pages = {e15861},
doi = {10.1002/advs.202515861},
pmid = {41084992},
issn = {2198-3844},
abstract = {Gene doping is an increasing challenge in sports, demanding highly sensitive and specific detection tools beyond the limitations of the current amplification-dependent methods. Here, an innovative amplification-free clustered regularly interspaced short palindromic repeats/CRISPR-associated protein (CRISPR/Cas) 12a assay integrated with osmotically tunable double emulsion (DE) droplets is reported for rapid and ultrasensitive gene doping detection. Target DNA and CRISPR/Cas12a complexes are encapsulated within DE droplets, where osmotic shrinkage rapidly concentrates the reaction components, thereby enhancing the fluorescent signal intensity without nucleic acid amplification. This platform enables the detection of the human erythropoietin (hEPO) gene at unprecedented attomolar levels within 30 min, achieving a 25-fold improvement in sensitivity compared with that of nonshrinkable formats. Notably, the assay demonstrated a robust and specific performance in complex serum samples with minimal matrix interference. This novel approach offers a rapid, reliable, and inherently contamination-free solution for gene doping surveillance with broad potential for versatile amplification-free nucleic acid diagnostics.},
}
@article {pmid41083939,
year = {2025},
author = {Menelih, A and Girma, A and Aemiro, A},
title = {Advancing nutritional quality in oilseed crops through genome editing: a comprehensive review.},
journal = {GM crops &amp; food},
volume = {16},
number = {1},
pages = {709-732},
pmid = {41083939},
issn = {2164-5701},
mesh = {*Gene Editing/methods ; *Crops, Agricultural/genetics/metabolism ; *Nutritive Value ; CRISPR-Cas Systems ; Plants, Genetically Modified/genetics ; *Plant Oils/metabolism ; Seeds/genetics ; Fatty Acid Desaturases/genetics/metabolism ; Genome, Plant ; },
abstract = {Genome editing has emerged as a powerful approach to enhancing the nutritional quality of oilseed crops. Clustered regularly interspaced short palindromic repeats case9 (CRISPR/Cas9) is the predominant editing tool, while transcription activator-like effector nucleases (TALENs) and base editors are used less commonly. Key fatty acid desaturase genes such as FAD2 and FAD3 are prime targets because of their critical functions in fatty acid desaturation. This review summarizes recent progress in editing genes associated with oil composition and related traits across oilseed species. Visual data representations including, Sankey diagrams, heat maps, and crop-trait matrices illustrate shared editing priorities and emerging trait targets across crops. Despite its promise, genome editing still faces challenges in transformation efficiency, field-level validation, and regulatory acceptance. This review underscores the increasing impact of target gene editing on nutritional trait improvement and its potential to accelerate the development of healthier, more sustainable oilseed varieties.},
}
@article {pmid41002041,
year = {2025},
author = {Clark, MB and Funk, AT and Paporakis, A and Brown, GP and Beach, SJ and Tay, A and Deering, S and Cooper, C and Tizard, M and Jolly, CJ and Ward-Fear, G and Waddle, AW and Shine, R and Maselko, M},
title = {Efficient CRISPR-Cas9-Mediated Genome Editing of the Cane Toad (Rhinella marina).},
journal = {The CRISPR journal},
volume = {8},
number = {5},
pages = {321-332},
doi = {10.1177/25731599251382427},
pmid = {41002041},
issn = {2573-1602},
mesh = {Animals ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; Monophenol Monooxygenase/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; *Bufo marinus/genetics ; Albinism/genetics ; Introduced Species ; },
abstract = {Invasive species inflict major ecological, economic, and cultural harm worldwide, highlighting the urgent need for innovative control strategies. Genome editing offers exciting possibilities for targeted control methods for invasive species. Here, we demonstrate CRISPR-Cas9 genome editing in the cane toad (Rhinella marina), one of Australia's most notorious invasive species, by targeting the tyrosinase gene to produce albino phenotypes as visual markers for assessing editing efficiency. Microinjection of Cas9 protein and guide RNAs into one-cell zygotes resulted in 87.6% of mosaic larvae displaying nearly complete albinism, with 2.3% exhibiting complete albinism. For completely albino individuals, genomic analysis confirmed predominantly frameshift mutations or large deletions at the target site, with no wild-type alleles detected. Germline transmission rates reflected the extent of albinism in the mosaic adult, with maternal transmission approaching 100%. This first application of CRISPR-Cas9 in the Bufonidae family opens possibilities for exploring basic research questions and population control strategies.},
}
@article {pmid40875132,
year = {2025},
author = {Skipper, TS and Dickson, KA and Denes, CE and Waller, MA and Du, TY and Neely, GG and Bowden, NA and Faiz, A and Marsh, DJ},
title = {Revealing genetic drivers of ovarian cancer and chemoresistance: insights from whole-genome CRISPR-knockout library screens.},
journal = {Cellular oncology (Dordrecht, Netherlands)},
volume = {48},
number = {5},
pages = {1245-1265},
pmid = {40875132},
issn = {2211-3436},
mesh = {Humans ; *Ovarian Neoplasms/genetics/drug therapy ; Female ; *Drug Resistance, Neoplasm/genetics ; *Gene Knockout Techniques ; *CRISPR-Cas Systems/genetics ; *Gene Library ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; },
abstract = {Understanding genetic dependencies in cancer is key to identifying novel actionable drug targets to advance precision medicine. Whole-genome CRISPR-knockout library screening methods have facilitated this goal. Pooled libraries of single guide RNAs (sgRNAs) targeting over 90% of the annotated protein coding genome are used to induce gene knockouts in pre-clinical cancer models. Novel genes of interest are identified by evaluating sgRNA dropout or enrichment following selection pressure application. This method is particularly beneficial for researching cancers where effective treatment strategies are limited. One example of a commonly chemoresistant cancer, particularly at relapse, is the low survival malignancy epithelial ovarian cancer (EOC), made up of multiple histotypes with distinct molecular profiles. CRISPR-knockout library screens in pre-clinical EOC models have demonstrated the ability to predict biomarkers of treatment response, identify targets synergistic with standard-of-care chemotherapy, and determine novel actionable targets which are synthetic lethal with cancer-associated mutations. Robust experimental design of CRISPR-knockout library screens, including the selection of strong pre-clinical cell line models, allows for meaningful conclusions to be made. We discuss essential design criteria for the use of CRISPR-knockout library screens to discover genetic dependencies in cancer and draw attention to discoveries with translational potential for EOC.},
}
@article {pmid40789680,
year = {2025},
author = {Sun, X and Li, M and Wang, H and Yang, Y and Kang, Y and Sun, P and Dong, J and Jin, M and Jin, W},
title = {Possible Reversion of CRISPR-Cas9-Edited Sequences in Octoploid Strawberry.},
journal = {The CRISPR journal},
volume = {8},
number = {5},
pages = {375-389},
doi = {10.1177/25731599251361374},
pmid = {40789680},
issn = {2573-1602},
mesh = {*Fragaria/genetics ; *Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; Plant Leaves/genetics ; Polyploidy ; Gene Expression Regulation, Plant ; Plants, Genetically Modified/genetics ; Seedlings/genetics ; Plant Proteins/genetics ; },
abstract = {Gene editing is more challenging in octoploids due to the presence of multiple copies of each gene. However, the ability to edit genes in these plants would allow editing in commercial varieties. Here, we delivered sequences targeting FaMYB9 into octoploid strawberry "Honeoye" and identified several gene-edited lines. Among them, the heterozygous gene-edited line FaMYB9[CR]-15 had curved and wrinkled leaves at 3 months, whereas leaves of 3-month-old wild-type (WT) strawberry seedlings were elliptical with a smooth surface. At that stage, FaMYB9[CR]-15 leaves also had large patches of wax. We identified 11,402 differentially expressed genes, divided into four clusters, between WT and FaMYB9[CR]-15 seedlings at 3 months. Notably, cluster 4 genes-related to nonhomologous end joining, microhomology-mediated end joining repairs, homologous recombination, nucleotide excision repair, and mismatch repair-were more highly expressed in the gene-edited line than in the WT. Surprisingly, by 6 months of age, FaMYB9[CR]-15 leaves had become smooth with small patches of wax, and expression levels of cluster 4 genes were significantly lower than at 3 months. Over the same period, the percentage of FaMYB9 loci harboring the mutant allele decreased from 70.2% to 43.7%. These findings lead us to conclude that there could be reversion of mutated sequences in octoploid strawberry, emphasizing the challenges of gene editing high-ploidy materials.},
}
@article {pmid40675779,
year = {2025},
author = {Sherkow, JS},
title = {A "Bare Hope of A Result": The Second CRISPR Patent Appeal.},
journal = {The CRISPR journal},
volume = {8},
number = {5},
pages = {317-320},
doi = {10.1177/25731599251361362},
pmid = {40675779},
issn = {2573-1602},
mesh = {*Patents as Topic/legislation &amp; jurisprudence ; Humans ; *Gene Editing/legislation &amp; jurisprudence ; *CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; United States ; Dissent and Disputes/legislation &amp; jurisprudence ; },
abstract = {On May 12, 2025, the US Court of Appeals for the Federal Circuit issued its second decision in the long-running CRISPR patent dispute between the Regents of the University of California and related institutions (CVC) and the Broad Institute. This Perspective recounts the principal dispute to date, reviews the Federal Circuit's recent opinion, and provides a critique of its analysis. In particular, this Perspective highlights how the decision is self-contradictory and in tension with patent law's conception doctrine-when an inventor has formed a "definite and permanent" idea of an invention in the mind or whether the invention was little more than a "bare hope" of a result. This Perspective briefly concludes with the implications of this recent decision and where the underlying dispute is likely headed.},
}
@article {pmid40659333,
year = {2025},
author = {Manuvera, VA and Bobrovsky, PA and Kharlampieva, DD and Grafskaia, EN and Brovina, KA and Serebrennikova, MY and Lazarev, VN},
title = {Bacterial Expression System with Deep Repression and Activation via CRISPR-Cas9.},
journal = {The CRISPR journal},
volume = {8},
number = {5},
pages = {353-365},
doi = {10.1177/25731599251358852},
pmid = {40659333},
issn = {2573-1602},
mesh = {*CRISPR-Cas Systems/genetics ; *Escherichia coli/genetics ; Plasmids/genetics ; *Gene Editing/methods ; Promoter Regions, Genetic ; *Gene Expression Regulation, Bacterial ; Green Fluorescent Proteins/genetics ; 5' Untranslated Regions ; Terminator Regions, Genetic ; },
abstract = {Incomplete repression of recombinant genes encoding toxic polypeptides can suppress cell growth even in the absence of a transcription inducer. To address this issue, we developed a CRISPR-Cas9-based genome editing approach that directly modifies the plasmid encoding the toxic peptide during Escherichia coli cultivation. The constructed plasmids contained a transcription terminator between the promoter and coding region, preventing full gene expression through abortive transcription. Upon CRISPR-Cas9 activation, this region is excised, thus restoring the functional gene. To implement this approach, we modified widely used pET-series expression plasmids by adding extra terminators in the 5'-untranslated region of the recombinant gene. Four antimicrobial peptides with strong bactericidal properties served as toxic gene products, while green fluorescent protein was used to assess the efficiency of expression repression. As a result, we developed an expression system with strong repression, which is activated by CRISPR-Cas9-mediated excision of a DNA fragment from the plasmids.},
}
@article {pmid40637801,
year = {2025},
author = {Han, J and Yu, B and Jing, J and He, X and Hua, Y and Xu, G},
title = {EGFR blockade confers sensitivity to pan-RAS inhibitors in KRAS-mutated cancers.},
journal = {Cellular oncology (Dordrecht, Netherlands)},
volume = {48},
number = {5},
pages = {1317-1335},
pmid = {40637801},
issn = {2211-3436},
mesh = {Humans ; *ErbB Receptors/antagonists &amp; inhibitors/metabolism ; Animals ; *Proto-Oncogene Proteins p21(ras)/genetics/antagonists &amp; inhibitors ; Cell Line, Tumor ; *Mutation/genetics ; *Protein Kinase Inhibitors/pharmacology/therapeutic use ; Mice ; *Neoplasms/genetics/drug therapy/pathology ; CRISPR-Cas Systems/genetics ; Cell Proliferation/drug effects ; Drug Resistance, Neoplasm/drug effects ; *ras Proteins/antagonists &amp; inhibitors ; },
abstract = {INTRODUCTION: KRAS is one of the most commonly occurring mutated oncogene in human cancers. Development of KRAS G12C or G12D inhibitors exhibit promising clinical activities, but patients harboring other hotspot KRAS mutations cannot benefit from those strategies. Recent development in pan-RAS inhibitors have broad therapeutic implications and merit clinical investigation. However, intrinsic and acquired drug resistance caused by tumor heterogeneity greatly limit the clinical application, posing a significant challenge in this field.<br><br>RESULTS: In this study, through CRISPR/Cas9 sgRNA screening using a human kinome sgRNA library, EGFR was discovered to correlate with the sensitivity of KRAS-mutated tumors to pan-RAS inhibitor RMC-7977. Through multiple in vitro cell proliferation or viability assays, EGFR loss or pharmacological EGFR inhibition significantly enhances the effectiveness of pan-RAS inhibitors in multiple KRAS[G12C] or KRAS[G12D] cancer cell lines, disregarding their cellular origins. Mechanistically, co-inhibition of EGFR and pan-RAS may further dampen the RTK-RAS-RAF-MEK-ERK pathway activation than either alone, thereby enhancing the anti-tumor activity of pan-RAS inhibitors. Strikingly, with the LL/2 syngeneic mice tumor model, the combination of pan-RAS inhibitors and EGFR inhibitors demonstrated more significant in vivo therapeutic efficacy compared to either single agent.<br><br>CONCLUSION: In conclusion, this study employed high-throughput CRISPR/Cas9 sgRNA screening to identify the enhanced anti-cancer effects when combining EGFR inhibitors with pan-RAS inhibitors in multiple human KRAS-mutated cancer cell lines as well as a mouse syngeneic tumor model. This synergy underscores the potential for a combinational therapy strategy, leveraging EGFR and pan-RAS inhibitors to improve treatment outcomes for patients with KRAS-driven cancers.},
}
@article {pmid40637619,
year = {2025},
author = {Guerra, I and Jensen, K and Perez-Pinera, P},
title = {Implementation of an Undergraduate Laboratory-Based Mammalian Genome Editing Course.},
journal = {The CRISPR journal},
volume = {8},
number = {5},
pages = {366-374},
doi = {10.1089/crispr.2025.0017},
pmid = {40637619},
issn = {2573-1602},
mesh = {*Gene Editing/methods ; Humans ; Curriculum ; Animals ; Genetic Engineering/methods ; Laboratories ; Students ; Mammals/genetics ; CRISPR-Cas Systems ; Universities ; },
abstract = {Genome engineering methods can be utilized to perform complex genetic manipulations in living cells with remarkable efficiency and precision. Given the transformative potential of these enabling technologies, their applications are steadily expanding into most biology and biomedical fields where they play a central role in many experimental frameworks. For these reasons, in order to effectively prepare future generations of biologists and bioengineers for successful careers, there is a high need to incorporate courses teaching genome editing fundamentals into existing curricula. To accomplish this objective, lecture-based courses are rapidly integrating genome editing concepts; however, there are few laboratory courses that teach the practical skills needed to successfully perform genome editing experiments. Here, we describe the development and implementation of a semester-long laboratory course that teaches students not only the techniques needed to perform gene knockout, gene activation, gene repression, and base editing in mammalian cells but also prepares them to design and troubleshoot experiments, write scientific manuscripts, as well as prepare and deliver scientific presentations. Course evaluations demonstrate that this class effectively equips students with the knowledge and hands-on experience needed to succeed in careers related to genome engineering, cell and tissue engineering, and, more broadly, biology.},
}
@article {pmid41083470,
year = {2025},
author = {McCallion, O and Du, W and Glaser, V and Milward, K and Short, S and Bilici, M and Cross, A and Stark, H and Franke, C and Kath, J and Valkov, M and Yang, M and Amini, L and Künkele, A and Polansky, JK and Schmueck-Henneresse, M and Volk, HD and Reinke, P and Wagner, DL and Hester, J and Issa, F},
title = {HLA matching or CRISPR editing of HLA class I/II enables engraftment and effective function of allogeneic human regulatory T cell therapy in a humanized mouse transplantation model.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {9090},
pmid = {41083470},
issn = {2041-1723},
support = {825392//EC | Horizon 2020 Framework Programme (EU Framework Programme for Research and Innovation H2020)/ ; 825392//EC | Horizon 2020 Framework Programme (EU Framework Programme for Research and Innovation H2020)/ ; 825392//EC | Horizon 2020 Framework Programme (EU Framework Programme for Research and Innovation H2020)/ ; 825392//EC | Horizon 2020 Framework Programme (EU Framework Programme for Research and Innovation H2020)/ ; 825392//EC | Horizon 2020 Framework Programme (EU Framework Programme for Research and Innovation H2020)/ ; 211122/Z/18//Wellcome Trust (Wellcome)/ ; FS/ 12/72/29754//British Heart Foundation (BHF)/ ; },
mesh = {Animals ; Humans ; *T-Lymphocytes, Regulatory/immunology/transplantation ; Mice ; Skin Transplantation ; Gene Editing/methods ; Graft Survival/immunology ; CRISPR-Cas Systems ; Transplantation, Homologous ; *Histocompatibility Antigens Class I/genetics/immunology ; Adoptive Transfer ; Forkhead Transcription Factors/metabolism/genetics ; CD8-Positive T-Lymphocytes/immunology ; },
abstract = {Regulatory T cells (Tregs) hold promise for treating autoimmune disease and transplant rejection, yet generation of autologous products for adoptive transfer can suffer donor variability and slow turnaround, limiting their use in urgent indications. We therefore examine whether allogeneic, pre-manufactured ('off-the-shelf') Tregs could overcome these barriers. In a human skin-xenograft model, HLA-mismatched Tregs are swiftly eliminated by recipient CD8[+] T cells and fail to protect grafts. Stringent matching of HLA class I and II restores efficacy but is clinically impractical. Using non-viral CRISPR editing we disrupt B2M and CIITA while inserting an HLA-E-B2M fusion, generating hypo-immunogenic Tregs that evade both T and NK cell attack. Engineered cells retain FOXP3 stability and potent in vitro suppression, and after a single low-dose infusion, prolong human skin graft survival in a humanized mouse model comparably to autologous Tregs. Histology and spatial transcriptomics reveal minimal cytotoxic infiltration and enrichment of immunoregulatory and tissue-repair programmes. Multiplex HLA engineering thus enables ready-to-use allogeneic Tregs that withstand host immune attack for adoptive transfer.},
}
@article {pmid41082405,
year = {2025},
author = {Yang, L and Fang, Y and Lian, Y and Kong, Z and Miao, J and Chen, Y and Li, W and Chen, F and Zhang, B and Chen, Y and Bian, Y},
title = {Aloe-Emodin Targeting FOXC2 Disrupts NETs Formation and EMT-Driven Postoperative Peritoneal Adhesion Through TGF-β1-Smad2/3 Pathway.},
journal = {Advanced science (Weinheim, Baden-Wurttemberg, Germany)},
volume = {},
number = {},
pages = {e11013},
doi = {10.1002/advs.202511013},
pmid = {41082405},
issn = {2198-3844},
support = {81704084//National Natural Science Foundation of China/ ; 0121/2022/A3//Science and Technology Development Fund, Macau SAR/ ; 0127/2023/RIA2//Science and Technology Development Fund, Macau SAR/ ; SJCX24_1123//Postgraduate Research &amp; Practice Innovation Program of Jiangsu Province/ ; SJCX25_0992//Postgraduate Research &amp; Practice Innovation Program of Jiangsu Province/ ; //333 High-Level Talents Cultivation Project of Jiangsu Province/ ; },
abstract = {Postoperative peritoneal adhesion (PPA) develops through TGF-β1-driven fibrotic remodeling, characterized by neutrophil extracellular trap (NETs)-induced aberrant epithelial-to-mesenchymal transition (EMT) deposition. Although aloe-emodin (AE) exhibits anti-fibrosis potential, its molecular mechanisms remain elusive. Forkhead box protein C2 (FOXC2) is a critical regulator of fibrotic tissue formation, yet its role in PPA is unknown. Here, it is demonstrated that FOXC2 expression is elevated in human ileostomy tissue, PPA rodent model, and TGF-β1-exposed peritoneal mesothelial cells (PMCs), where it orchestrates NETs formation and extracellular matrix (ECM) remodeling. Mechanically, CRISPR/Cas-based knockdown and overexpression of FOXC2 alter EMT changes in PMCs, which is achieved via TGF-β1-Smad2/3 signaling. FOXC2 functions as a dual mediator and amplifier through the TGF-β1-Smad2/3 pathway feedback loop to drive EMT alterations. Its overexpression further induces neutrophil recruitment and NETs formation, exacerbating EMT in PMCs. Notably, AE ameliorates FOXC2-driven peritoneal fibrosis by impeding NETs formation and EMT changes through the TGF-β1-Smad2/3 pathway. Moreover, AE binds directly to FOXC2, and the Ser125 residue is critical for the binding of FOXC2 to AE. These findings identify FOXC2 as a pivotal effector in fibrotic responses during PPA formation and reveal that AE targeting the Ser125 residue of FOXC2 may be a promising therapeutic approach to attenuate PPA.},
}
@article {pmid41082121,
year = {2026},
author = {Kim, WD and Huber, RJ},
title = {Modeling Lysosomal Disease in Dictyostelium discoideum: Examining the Trafficking and Secretion of Lysosomal Enzymes.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2976},
number = {},
pages = {189-207},
pmid = {41082121},
issn = {1940-6029},
mesh = {*Dictyostelium/genetics/metabolism/enzymology ; *Lysosomes/enzymology/metabolism ; Protein Transport ; *Lysosomal Storage Diseases/genetics/metabolism/enzymology ; Gene Editing/methods ; CRISPR-Cas Systems ; Gene Knockout Techniques ; Protozoan Proteins/metabolism/genetics ; },
abstract = {Non-mammalian models are powerful systems for enhancing our understanding of lysosomal function and lysosomal storage diseases. The social amoeba Dictyostelium discoideum is an excellent model organism for studying lysosomal function, as its genome encodes many proteins associated with lysosomal disease. Methods for gene knockout are straightforward in D. discoideum and include restriction enzyme-mediated integration (REMI) mutagenesis, homologous recombination via the Cre-loxP system, and CRISPR/Cas9-mediated gene editing, which collectively allow researchers to study protein function (e.g., lysosomal enzymes) in a genetically tractable biomedical model system. Additionally, activity assays for conserved lysosomal enzymes are well-established in D. discoideum. In this chapter, we outline methods for studying the intracellular localization and secretion of conserved lysosomal proteins in D. discoideum.},
}
@article {pmid41082119,
year = {2026},
author = {Chear, S and Chiam, A and Talbot, J and Thorne, BN and Wilkinson, EJ and Hewitt, AW and Cook, AL},
title = {Generation of Donor-Specific iPSC for Modelling Lysosomal Storage Disorders.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2976},
number = {},
pages = {151-173},
pmid = {41082119},
issn = {1940-6029},
mesh = {Humans ; *Lysosomal Storage Diseases/pathology/genetics/metabolism ; *Induced Pluripotent Stem Cells/metabolism/cytology ; Cell Differentiation ; Fibroblasts/metabolism/cytology ; *Cell Culture Techniques/methods ; CRISPR-Cas Systems ; Cells, Cultured ; Tissue Donors ; },
abstract = {iPSC technology has enabled the generation of human cell-based models of lysosomal storage disorders and has provided disease-relevant systems to undertake drug discovery or pre-clinical testing of gene- or cell-based therapies. Here, we provide a protocol to generate iPSCs derived from people with lysosomal storage disorders and illustrate expected results using a CLN2 disease donor-specific skin fibroblast culture. Protocol steps include lipofection of episomal plasmids, picking of putative iPSC colonies following live cell TRA-1-60 immunofluorescence, and quality control steps such as immunofluorescence for expression of undifferentiated cell markers, germ layer differentiation, and confirmation of pathological variant genotype. The iPSC generated by this protocol can be differentiated to several cell lineages and can be used with CRISPR/Cas technology to generate isogenic disease models.},
}
@article {pmid41082037,
year = {2025},
author = {Kim, HJ and Kwon, MY and Song, S and Cheon, SW and Kim, HJ},
title = {Engineered Lactiplantibacillus plantarum and Levilactobacillus brevis utilizing ribonucleoprotein-mediated editing for inactivation of hemolysin gene.},
journal = {World journal of microbiology &amp; biotechnology},
volume = {41},
number = {10},
pages = {373},
pmid = {41082037},
issn = {1573-0972},
mesh = {*Gene Editing/methods ; CRISPR-Cas Systems ; *Ribonucleoproteins/genetics/metabolism ; *Hemolysin Proteins/genetics ; Probiotics ; Bacterial Proteins/genetics ; Plasmids/genetics ; *Lactobacillus plantarum/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; *Lactobacillus/genetics ; },
abstract = {Lactiplantibacillus plantarum and Levilactobacillus brevis are widely used probiotics with significant potential as chassis organisms for probiotic engineering. However, their bioengineering remains underdeveloped compared to that of other probiotic bacteria due to the limited availability of genetic tools. Although CRISPR-Cas systems have shown promise for genome editing in Lactobacillus species, strain- or site-specific targeting challenges must be overcome to enhance their broader applicability. This study aimed to develop a novel editing system with reduced dependency on plasmids and antibiotics in L. plantarum WCFS1, L. plantarum SPC 72 - 1 and L. brevis SPC-SNU 70 - 2 using a Cas9-gRNA ribonucleoprotein (RNP) complex. Although the hlyIII gene has been annotated as a hemolysin-related gene in several Lactobacillus genomes, no functional hemolytic activity has been definitively demonstrated to date. In this study, hlyIII was selected as a target to evaluate genome editing efficiency and to assess its potential relevance to strain safety. To construct ΔhlyIII strains, the RNP complex targeting hlyIII was separately transformed with recombinase RecE/T and double-stranded donor DNA. As a result, ΔhlyIII mutants were obtained under optimized electroporation conditions. Sequencing analysis revealed a 50 bp deletion and the introduction of a stop codon in hlyIII across all mutant strains. The hemolytic activity test showed a reduction in free hemoglobin levels in the ΔhlyIII strains compared to the wild type: 27.0%, 74.3%, and 5.0% in L. plantarum WCFS1, L. plantarum SPC 72 - 1, and L. brevis SPC-SNU 70 - 2, respectively. These results suggest strain-dependent differences in hemolytic activity and indicate that inactivation of hlyIII may contribute to reduced hemolysis, although further validation is needed to clarify its functional role. In conclusion, the hlyIII gene was successfully edited in L. plantarum and L. brevis using Cas9-gRNA ribonucleoprotein-mediated editing, demonstrating the feasibility of this genome editing platform for application in probiotic strains.},
}
@article {pmid41039923,
year = {2025},
author = {Xiao, J and Hu, X and Chen, H and Diao, B and Huang, X and Liu, L},
title = {CRISPR-Programmed CuO Nanocatalyst Release for Ultrasensitive Detection of Pathogens in Sterile Body Fluids.},
journal = {Analytical chemistry},
volume = {97},
number = {40},
pages = {22427-22435},
doi = {10.1021/acs.analchem.5c05043},
pmid = {41039923},
issn = {1520-6882},
mesh = {*Copper/chemistry ; Humans ; Catalysis ; *CRISPR-Associated Proteins/metabolism/chemistry ; Benzidines/chemistry ; *Endodeoxyribonucleases/metabolism/chemistry ; Limit of Detection ; RNA, Ribosomal, 16S/genetics/analysis ; *Body Fluids/microbiology ; *Metal Nanoparticles/chemistry ; *CRISPR-Cas Systems ; Bacterial Proteins/metabolism/chemistry ; Colorimetry ; },
abstract = {Treatment of sterile body fluids (SBFs) infections is delayed by conventional methods that require up to 72 h to detect pathogens. Here, we present a CRISPR-associated protein 12a (Cas12a)-programmed nanocatalyst release (CNR) method for culture-free diagnostics. To enhance both sensitivity and coverage, three starter DNA (sDNA)-complementary DNA (cDNA) probe pairs were designed for conserved regions and additional three pairs for variable regions of bacterial 16S or fungal 18S rRNA. Upon target recognition, cDNA undergoes strand displacement, releasing sDNA to activate Cas12a. The activated Cas12a cleaves copper oxide nanoparticle (CuONPs)-loaded magnetic probes, releasing tandem CuONPs. Upon acid dissolution, each CuONP generates Cu[2+] ions that catalyze the oxidation of 3,3',5,5'-tetramethylbenzidine (TMB), producing a visible colorimetric signal. This quadruple signal amplification strategy integrates high-copy rRNA targets, multi-cDNA recognition, Cas12a-mediated continuous release of tandem CuONPs, and Cu[2+]-driven chromogenic amplification. This nucleic acid amplification-free assay detects pathogens at 0.69 CFU mL[-1] in original SBFs samples (after 10-fold centrifugation) within 70 min. In 64 clinical samples, it achieved 100% sensitivity and 100% specificity versus culture. Notably, one culture-negative but clinically confirmed case was correctly identified. Overall, the CNR method offers a rapid, ultrasensitive, and accessible diagnostic solution for resource-limited settings.},
}
@article {pmid40980924,
year = {2025},
author = {Deng, L and He, X and Zhou, S and Gu, T and Dong, J and Zhu, S and Luo, X and Huo, D and Hou, C},
title = {A sticky end-driven PAM-free RPA-CRISPR/Cas12a dual amplification system for ultrasensitive detection of KRAS G12C.},
journal = {Chemical communications (Cambridge, England)},
volume = {61},
number = {83},
pages = {16282-16285},
doi = {10.1039/d5cc04401d},
pmid = {40980924},
issn = {1364-548X},
mesh = {*Proto-Oncogene Proteins p21(ras)/genetics ; *CRISPR-Cas Systems ; Humans ; *Biosensing Techniques/methods ; Limit of Detection ; DNA/chemistry/genetics ; *Nucleic Acid Amplification Techniques ; *Endodeoxyribonucleases/metabolism ; Bacterial Proteins ; CRISPR-Associated Proteins ; },
abstract = {Herein, a fluorescent biosensing platform was constructed for KRAS G12C single base mutation detection by CRISPR/Cas12a-coupled RPA without the PAM site. The KRAS G12C gene sequence was cleaved into double-stranded DNA containing a sticky end using HindIII enzyme cleavage site specificity. Sticky end dsDNA activated the trans-cleavage activity of Cas12a and generates an intense fluorescent signal. This strategy detected KRAS G12C targets in a linear range of 10 aM-10 pM with a detection limit of 1.5 aM. What's more, the method was able to distinguish 0.1% KRAS G12C mutation in a total of 10 pM gene concentration and demonstrated excellent detection performance in real samples.},
}
@article {pmid40721863,
year = {2025},
author = {Rybarikova, M and Rey, M and Hasanovic, E and Sipion, M and Rambousek, L and Déglon, N},
title = {Gene editing for Spinocerebellar ataxia type 3 taking advantage of the human ATXN3L paralog as replacement gene.},
journal = {Gene therapy},
volume = {32},
number = {5},
pages = {462-474},
pmid = {40721863},
issn = {1476-5462},
support = {FN 310030_184761/1//Schweizerischer Nationalfonds zur F&#xf6;rderung der Wissenschaftlichen Forschung (Swiss National Science Foundation)/ ; No. 31ER30_179594//EC | EU Framework Programme for Research and Innovation H2020 | H2020 European Institute of Innovation and Technology (H2020 The European Institute of Innovation and Technology)/ ; },
mesh = {*Machado-Joseph Disease/therapy/genetics ; Animals ; *Ataxin-3/genetics ; Humans ; *Gene Editing/methods ; Mice, Transgenic ; Mice ; *Genetic Therapy/methods ; CRISPR-Cas Systems ; Exons ; Cerebellum/metabolism ; Disease Models, Animal ; Repressor Proteins ; },
abstract = {Spinocerebellar ataxia type 3 (SCA3) is a rare neurodegenerative disease caused by a CAG expansion of the ataxin-3 gene (ATXN3). SCA3 patients suffer from ataxia, spasticity and dystonia in mid-adulthood, with spinocerebellar dysfunction and degeneration. As a monogenic disease for which only symptomatic treatment is available, ATXN3 is an attractive target for gene editing. We used the KamiCas9, a self-inactivating gene editing system, to explore gene editing strategies suitable for all SCA3 patients. We first tested the deletion of exon 10 or the introduction of a premature stop codon into exon 9. High editing events were observed in vitro, but efficiency was very low in SCA3 transgenic mice. We then evaluated an ablate-and-replace strategy. The ablate experiments resulted in 55 ± 18% cerebellar editing of the ATXN3 gene. A human ATXN3L paralog, expressed in the brains of SCA3 patients, may act as a natural, CRISPR-resistant replacement gene. In a proof-of-principle study, ablate and ablate-and-replace strategies were evaluated in SCA3 transgenic mice. Two months after injection, similar editing efficiencies were obtained in the ablate and ablate-and-replace groups. Immunofluorescence and RT-qPCR analyses of cerebellar markers support the development of this strategy for SCA3 treatment.},
}
@article {pmid40581821,
year = {2025},
author = {Luo, Y and Zhan, X and Zhang, Y and Wang, B and Wang, G and Zhang, Y and Li, G and Liu, Q and Shen, X and Chen, D and Hong, Y and Wu, W and Ye, G and Cheng, S and Pan, G and Cao, L},
title = {CRISPR-Cas9-mediated knockup of OsDREB1C enhances rice yield without compromising grain quality.},
journal = {Plant communications},
volume = {6},
number = {10},
pages = {101433},
doi = {10.1016/j.xplc.2025.101433},
pmid = {40581821},
issn = {2590-3462},
mesh = {*Oryza/genetics/growth &amp; development ; *CRISPR-Cas Systems/genetics ; *Edible Grain/genetics ; *Plant Proteins/genetics/metabolism ; Plants, Genetically Modified/genetics ; Gene Editing ; Gene Knock-In Techniques ; },
abstract = {This study presents a CRISPR-Cas9-based strategy for engineering structural variations in the OsDREB1C gene in rice, leading to a yield increase of over 20% without compromising grain quality. The resulting homozygous plants are transgene-free, highlighting the potential of this approach for precise and effective crop improvement.},
}
@article {pmid39572737,
year = {2025},
author = {Yan, RE and Corman, A and Katgara, L and Wang, X and Xue, X and Gajic, ZZ and Sam, R and Farid, M and Friedman, SM and Choo, J and Raimondi, I and Ganesan, S and Katsevich, E and Greenfield, JP and Dahmane, N and Sanjana, NE},
title = {Pooled CRISPR screens with joint single-nucleus chromatin accessibility and transcriptome profiling.},
journal = {Nature biotechnology},
volume = {43},
number = {10},
pages = {1628-1634},
pmid = {39572737},
issn = {1546-1696},
support = {DP2HG010099//U.S. Department of Health &amp; Human Services | NIH | National Human Genome Research Institute (NHGRI)/ ; GM136573//U.S. Department of Health &amp; Human Services | NIH | National Institute of General Medical Sciences (NIGMS)/ ; R03OD034499//U.S. Department of Health &amp; Human Services | NIH | NIH Office of the Director (OD)/ ; },
mesh = {Humans ; *Chromatin/genetics/metabolism ; *Gene Expression Profiling/methods ; Single-Cell Analysis/methods ; *CRISPR-Cas Systems/genetics ; Transcriptome/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; *Cell Nucleus/genetics ; },
abstract = {Pooled single-cell CRISPR screens have profiled either gene expression or chromatin accessibility but not both modalities. Here we develop MultiPerturb-seq, a high-throughput CRISPR screening platform with joint single-nucleus chromatin accessibility, transcriptome and guide RNA capture using combinatorial indexing combined with droplet microfluidics to scale throughput and integrate all three modalities. We identify key differentiation genes in a rare pediatric cancer and establish ZNHIT1 as a potential target for cancer reprogramming therapy.},
}
@article {pmid39482449,
year = {2025},
author = {Jabalera, Y and Tascón, I and Samperio, S and López-Alonso, JP and Gonzalez-Lopez, M and Aransay, AM and Abascal-Palacios, G and Beisel, CL and Ubarretxena-Belandia, I and Perez-Jimenez, R},
title = {A resurrected ancestor of Cas12a expands target access and substrate recognition for nucleic acid editing and detection.},
journal = {Nature biotechnology},
volume = {43},
number = {10},
pages = {1663-1672},
pmid = {39482449},
issn = {1546-1696},
mesh = {*Gene Editing/methods ; *CRISPR-Associated Proteins/genetics/metabolism/chemistry ; Humans ; *CRISPR-Cas Systems/genetics ; *Endodeoxyribonucleases/genetics/metabolism/chemistry ; DNA/metabolism/genetics ; *Bacterial Proteins/genetics/metabolism ; Substrate Specificity ; },
abstract = {The properties of Cas12a nucleases constrict the range of accessible targets and their applications. In this study, we applied ancestral sequence reconstruction (ASR) to a set of Cas12a orthologs from hydrobacteria to reconstruct a common ancestor, ReChb, characterized by near-PAMless targeting and the recognition of diverse nucleic acid activators and collateral substrates. ReChb shares 53% sequence identity with the closest Cas12a ortholog but no longer requires a T-rich PAM and can achieve genome editing in human cells at sites inaccessible to the natural FnCas12a or the engineered and PAM-flexible enAsCas12a. Furthermore, ReChb can be triggered not only by double-stranded DNA but also by single-stranded RNA and DNA targets, leading to non-specific collateral cleavage of all three nucleic acid substrates with similar efficiencies. Finally, tertiary and quaternary structures of ReChb obtained by cryogenic electron microscopy reveal the molecular details underlying its expanded biophysical activities. Overall, ReChb expands the application space of Cas12a nucleases and underscores the potential of ASR for enhancing CRISPR technologies.},
}
@article {pmid38472508,
year = {2025},
author = {Gould, SI and Wuest, AN and Dong, K and Johnson, GA and Hsu, A and Narendra, VK and Atwa, O and Levine, SS and Liu, DR and Sánchez Rivera, FJ},
title = {High-throughput evaluation of genetic variants with prime editing sensor libraries.},
journal = {Nature biotechnology},
volume = {43},
number = {10},
pages = {1648-1662},
pmid = {38472508},
issn = {1546-1696},
support = {V2022-028//V Foundation for Cancer Research (V Foundation)/ ; P30-CA14051//U.S. Department of Health &amp; Human Services | NIH | National Cancer Institute (NCI)/ ; U01AI142756//U.S. Department of Health &amp; Human Services | National Institutes of Health (NIH)/ ; RM1HG009490//U.S. Department of Health &amp; Human Services | National Institutes of Health (NIH)/ ; },
mesh = {Humans ; *Genetic Variation/genetics ; *Gene Editing/methods ; *Tumor Suppressor Protein p53/genetics ; *High-Throughput Screening Assays/methods ; RNA, Guide, CRISPR-Cas Systems/genetics ; CRISPR-Cas Systems/genetics ; Neoplasms/genetics ; },
abstract = {Tumor genomes often harbor a complex spectrum of single nucleotide alterations and chromosomal rearrangements that can perturb protein function. Prime editing has been applied to install and evaluate genetic variants, but previous approaches have been limited by the variable efficiency of prime editing guide RNAs. Here we present a high-throughput prime editing sensor strategy that couples prime editing guide RNAs with synthetic versions of their cognate target sites to quantitatively assess the functional impact of endogenous genetic variants. We screen over 1,000 endogenous cancer-associated variants of TP53-the most frequently mutated gene in cancer-to identify alleles that impact p53 function in mechanistically diverse ways. We find that certain endogenous TP53 variants, particularly those in the p53 oligomerization domain, display opposite phenotypes in exogenous overexpression systems. Our results emphasize the physiological importance of gene dosage in shaping native protein stoichiometry and protein-protein interactions, and establish a framework for studying genetic variants in their endogenous sequence context at scale.},
}
@article {pmid41081710,
year = {2025},
author = {Serreze, DV and Tousey-Pfarrer, M and Racine, JJ},
title = {Humanized Mouse Models for Type 1 Diabetes.},
journal = {Current protocols},
volume = {5},
number = {10},
pages = {e70224},
doi = {10.1002/cpz1.70224},
pmid = {41081710},
issn = {2691-1299},
mesh = {*Diabetes Mellitus, Type 1/immunology/genetics ; Animals ; *Disease Models, Animal ; Mice, Inbred NOD ; Humans ; Mice ; CRISPR-Cas Systems ; },
abstract = {T cell-mediated autoimmune type 1 diabetes (T1D) is under complex polygenic control in both humans and the NOD mouse model. However, in both species, particular major histocompatibility complex (MHC; designated HLA in humans) haplotypes provide the primary T1D risk factor. Both MHC/HLA class I and II variants interactively contribute to T1D by respectively driving autoreactive CD8 and CD4 T cell responses that cooperatively destroy insulin-producing pancreatic β cells. While NOD mice have provided important insights to the pathogenic basis of T1D, the model has so far provided only a limited means to identify possible clinically translatable disease intervention approaches. This highlights a need to humanize NOD mice in ways that their pathogenic basis of T1D development becomes more similar to that characterizing the disease course in patients. In this review, we discuss the use of CRISPR/Cas9-generated murine-MHC-deficient NOD mice as a platform for introduction of patient-relevant HLA and T cell receptor molecules. These mice provide ever-improving models for development of clinically applicable interventions for T1D and other autoimmune diseases. © 2025 The Author(s) Current Protocols published by Wiley Periodicals LLC.},
}
@article {pmid41081480,
year = {2025},
author = {Ma, F and Zheng, Q and Sun, Y and Zhang, N and Xu, W},
title = {Toward Tissue-Free Plant Engineering: Emerging Platforms for Sustainable Horticultural Transformation.},
journal = {Journal of agricultural and food chemistry},
volume = {},
number = {},
pages = {},
doi = {10.1021/acs.jafc.5c06803},
pmid = {41081480},
issn = {1520-5118},
abstract = {Genetic transformation in horticultural crops is being reshaped by the emergence of nontissue culture technologies that bypass entrenched barriers of genotype dependence, regeneration inefficiency, and sterile culture requirements. This review surveys recent in planta methods, including regenerative activity-dependent in Planta injection delivery (RAPID), cut-dip-budding (CDB), virus-based delivery, nanoparticle-mediated transformation, and Agrobacterium rhizogenes-induced regeneration, and evaluates their operational versatility across species. We further examine their integration with developmental regulators (BABY BOOM [BBM], WUSCHEL [WUS]), visual markers (RUBY), and CRISPR/Cas systems to enhance transformation efficiency and precision. Case studies across fruit, vegetable, and ornamental crops illustrate broad applicability and growing technical maturity. Despite these advances, unresolved challenges in biosafety, reproducibility, and regulatory alignment remain. We advocate a new transformation paradigm that is rapid, genotype-independent, and environmentally compatible, enabling scalable and more accessible broadly applicable crop improvement in horticultural biotechnology.},
}
@article {pmid41080519,
year = {2025},
author = {Chaturvedi, A and Ranjan, R},
title = {Strategies for plant-virus disease management from gene editing to nanotechnology.},
journal = {Physiology and molecular biology of plants : an international journal of functional plant biology},
volume = {31},
number = {8},
pages = {1293-1308},
pmid = {41080519},
issn = {0971-5894},
abstract = {Plant viruses are a global agricultural threat and can result in large financial losses. The globalization of agriculture and its international trading are the major causes of viruses and their vectors expanding to new environmental niches. Conventional methods are not effective in managing virus infection. To mitigate the virus spread, one of the cutting-edge biotechnological approaches, CRISPR/Cas is a robust tool. CRISPR/Cas is a powerful genome editing technology, and provides a highly specific viral genome targeting. Additionally, nanotechnology is a cutting-edge method for mitigating plant viruses. Nanoparticles in biosensors aid in the early identification of plant viruses, hence preventing the spread of disease in the future. Moreover, nanoparticles can also be used as a flexible delivery system. Nanoparticle-mediated delivery of dsRNA ensures minimal off-target while maintaining biosafety. This review explores the genome editing approach and nanotechnological strategies for ensuring sustainable agriculture practices for virus disease management, focusing on biosafety, efficacy, and practical applicability. It also aims to provide a clear insight into the limitations and strengths of each approach.},
}
@article {pmid41076478,
year = {2025},
author = {Wu, M and Wang, F and Wang, Y and Wu, YX and Tian, BY and Ou, XY and Xu, QC and Wu, XY and Han, C and Liu, WL and Xing, S},
title = {Mismatch-introduced crRNA guided PCR-CRISPR/Cas12a platform improves EGFR point mutation detection in single tumor cell.},
journal = {Mikrochimica acta},
volume = {192},
number = {11},
pages = {727},
pmid = {41076478},
issn = {1436-5073},
support = {2021B1515230006//the Guangdong Basic and Applied Basic Research Foundation/ ; },
mesh = {Humans ; ErbB Receptors/genetics ; *Point Mutation ; *CRISPR-Cas Systems ; *Polymerase Chain Reaction/methods ; *Single-Cell Analysis/methods ; Cell Line, Tumor ; Neoplastic Cells, Circulating ; DNA Mutational Analysis/methods ; Base Pair Mismatch ; },
abstract = {Dynamic monitoring of epidermal growth factor receptor (EGFR) mutations is essential for the early identification of resistance and treatment adaptation. Single-cell heterogeneity analysis is crucial for precision cancer medicine, yet sensitive and specific detection methods for individual tumor cells remain challenging. Here, we develop a PCR-CRISPR/Cas12a platform enhanced by the incorporation of mismatched base in crRNA at specific site for single-cell point mutation detection. This platform demonstrated high specificity and sensitivity, detecting point mutation at a frequency of 0.1% and in as low as 1.02 ng of genomic DNA, which represents an improvement over the amplification-refractory mutation system PCR (ARMS-PCR). Notably, the accuracy of the platform is highly consistent with next-generation sequencing (NGS), as evidenced by Kappa test values surpassing 0.9. By utilizing a conical-pore membrane with optimized porosity for single circulating tumor cell (CTC) enrichment, our platform enables point mutations detection in individual tumor cells, offering potential enhancements in precision and reliability for EGFR mutation analysis. This novel methodology holds potential for more accurate and personalized cancer treatment strategies.},
}
@article {pmid41075940,
year = {2025},
author = {Zhang, J and Dai, P and Weng, Z and Xu, R and Li, Y and Liu, X and Lei, J},
title = {Efficient CRISPR/Cas-based gene editing in cotton induced by cotton leaf crumple virus.},
journal = {Journal of biotechnology},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.jbiotec.2025.10.001},
pmid = {41075940},
issn = {1873-4863},
abstract = {Plant viral vectors can replicate autonomously and spread within host cells, making them an ideal tool for the delivery of CRISPR/Cas gene-editing elements. Here, we constructed a cotton CRISPR/Cas system mediated by cotton leaf crumple virus (CLCrV) as a delivery vector. We first inoculated Pro35s::Cas9 and ProUbi::Cas9 cotton with sgRNAs designed to knock out GhAGL16, GhPDS, and GhCLA1 target genes via the CLCrV vector and then compared the effects of these two transformation receptors on the editing efficiency of the same target genes. We next explored the feasibility of simultaneous multi-target editing in cotton via pooled virus inoculation. Finally, we used a cotton line overexpressing nCas9-TadA7.10 as the transformation receptor to explore the feasibility of CLCrV-mediated adenine base editing and verify the specificity of gene editing in this system. Mutation detection and deep sequencing revealed that the Pro35s::Cas9 and ProUbi::Cas9 cotton lines did not differ significantly in editing efficiency, and both could be used as successful receptors for the CLCrV-mediated Cas9 system. Pooled inoculation with CLCrV-sgRNAs enabled the simultaneous editing of multiple target genes in Pro35s::Cas9 and ProUbi::Cas9 cotton, although this approach had somewhat lower editing efficiency than inoculation with single sgRNAs. The CLCrV-mediated adenine base-editing system enabled A-to-G conversion at target sites in cotton GhPEBP and showed high gene-editing specificity. In summary, this study establishes an efficient CLCrV-mediated CRISPR system in cotton, providing a powerful technical tool for editing of multiple target genes and base editing.},
}
@article {pmid41074985,
year = {2025},
author = {Gupta, I and Sharma, JG and Kaul, T},
title = {Nanoparticle-driven CRISPR-Cas9 genome editing: a new frontier in crop improvement.},
journal = {Molecular biology reports},
volume = {52},
number = {1},
pages = {1015},
pmid = {41074985},
issn = {1573-4978},
mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; *Crops, Agricultural/genetics ; *Nanoparticles/chemistry ; Genome, Plant/genetics ; Plants, Genetically Modified/genetics ; Nanotechnology/methods ; Genetic Engineering/methods ; Agriculture/methods ; },
abstract = {The agricultural sector has experienced unpredictable and extreme climatic aberrations, which have severely hampered food production. However, applying advanced nanotechnological approaches in agriculture will be crucial for ensuring more secure and sustainable food production. The revolutionizing phyto-nanotechnology enables the precise delivery of biomolecules i.e., nucleotides and proteins, and the modulated release of agrochemicals, including fungicides and pesticides. In addition, CRISPR-Cas-based genetic engineering holds great promise for food security, agriculture, and environmental sustainability. However, its application in plants faces challenges, including cargo delivery, germline transformation, species independence, HDR efficiency, and overall editing effectiveness. Nanomaterials offer innovative and effective solutions to overcome these challenges by enhancing genome-editing tools precision, efficiency, and delivery mechanisms. This review examines the key limitations of CRISPR-mediated plant genome editing and how nanoparticle technologies can overcome them. We highlight essential nanotech innovations that enhance genome modification, paving the way for a faster, more versatile genomic toolbox in plant biotechnology.},
}
@article {pmid41074026,
year = {2025},
author = {Frey, T and Kandolf-Zumpf, C and Kaempf, A and Schaffer, K and Hollenstein, M and Lampl, A and Kovarik, JJ and Strobl, J and Stary, G and Eckl-Dorna, J and Schmidt, R and Schmetterer, KG},
title = {T cell receptor associated transmembrane adaptor 1 (TRAT1) modulates human Th17 and Treg responses via PI3-kinase and STAT dependent mechanisms.},
journal = {Cell communication and signaling : CCS},
volume = {23},
number = {1},
pages = {431},
pmid = {41074026},
issn = {1478-811X},
support = {P34728-B//Austrian Science Funds/ ; P34728-B//Austrian Science Funds/ ; P34728-B//Austrian Science Funds/ ; },
mesh = {Humans ; *T-Lymphocytes, Regulatory/metabolism/immunology/cytology ; *Th17 Cells/metabolism/immunology/cytology ; Signal Transduction ; *Phosphatidylinositol 3-Kinases/metabolism ; *STAT Transcription Factors/metabolism ; *Adaptor Proteins, Signal Transducing/metabolism ; CRISPR-Cas Systems ; },
abstract = {BACKGROUND: Adaptor proteins associated with the T cell receptor (TCR) play critical roles in regulating immune responses by Translating receptor engagement into intracellular signals. T cell Receptor Associated Transmembrane Adaptor 1 (TRAT1) has been implicated in modulating TCR complex stability, but its functional role in human effector and regulatory CD4[+] T cell subsets remains poorly understood. This study aimed to elucidate the role of TRAT1 in regulating T cell activation and differentiation, particularly in helper T cells function and regulatory T cells.<br><br>METHODS: Primary human CD4&#x207a; T cells, including thymus-derived and induced regulatory T cells (Treg), were genetically modified by CRISPR/Cas9-mediated gene deletion or retro-/lentiviral overexpression of TRAT1. Functional assays, flow cytometry, cytokine quantification, and RNA sequencing were performed to evaluate modulation of T cell functions. Mechanistic studies included pathway inhibition using small molecules and phospho-protein analysis. The influence of TRAT1 on Treg function was further assessed in a CAR Treg context in an immune organoid model of allo-rejection.<br><br>RESULTS: Thymus-derived, TGFb-induced and FOXP3-transgenic Treg displayed reduced expression of TRAT1 compared to effector T cells, which showed pronounced up-regulation of TRAT1 following activation. In effector T cells, deletion of TRAT1 led to increased signaling through the phosphoinositide 3-kinase pathway resulting in enhanced proliferation and increased expression of activation markers. However, this was accompanied by reduced production of interleukin-17, which was linked to elevated activity of STAT6 as shown by inhibition experiments using small molecule inhibitors. Overexpression and CRISPR/Cas9-mediated knockout of TRAT1 in Treg enhanced suppression of CD4&#x207a; target cells via up-regulation of LAP/GARP but reduced suppression of CD8&#x207a; target cells, an effect confirmed in HLA-A2-specific CAR Treg in a human organoid model of allo-rejection.<br><br>CONCLUSIONS: TRAT1 acts as a dual regulator of human CD4&#x207a; T cell function, limiting effector activation through modulation of intracellular signaling and supporting regulatory T cell-mediated suppression. These findings reveal a novel mechanism of immune regulation with potential implications for the development of cell-based immunotherapies.},
}
@article {pmid41073589,
year = {2025},
author = {Das, A and Debnath, S and Pramanik, S and Monshi, FI and Rahimi, M},
title = {Bio-digital feedback loop systems: a synergistic integration of predictive genomics, genome editing, and AI-driven phenomic synthesis for next-generation edible and medicinal mushroom breeding.},
journal = {Antonie van Leeuwenhoek},
volume = {118},
number = {11},
pages = {168},
pmid = {41073589},
issn = {1572-9699},
mesh = {*Gene Editing/methods ; *Genomics/methods ; *Agaricales/genetics ; *Artificial Intelligence ; CRISPR-Cas Systems ; *Phenomics/methods ; *Breeding/methods ; },
abstract = {Edible mushrooms face persistent challenges in yield optimization, bioactive compound production, and climate resilience that conventional breeding methods struggle to address. Traditional approaches such as cross-breeding, protoplast fusion, and mutagenesis are limited by genetic noise, laborious screening, and unstable trait inheritance. This review proposes a transformative paradigm built upon converging advances in molecular biology and data science: the bio-digital feedback loop (BDFL) framework, integrating multi-omics, CRISPR-engineered chassis strains, and predictive phenomics for precision mushroom breeding. Our framework employs multi-omics to decipher gene networks governing critical traits, such as substrate degradation enzymes, developmental synchrony regulators, and secondary metabolite pathways. CRISPR-Cas9 and synthetic biology tools then deploy these insights to verify and design modular gene circuits in pre-engineered "plug-and-play" chassis strains, enabling conflict-free stacking of desirable traits. Artificial intelligence serves as the linchpin, not only automating high-throughput phenotyping through advanced imaging but also accelerating the entire breeding cycle by predicting trait heritability from omics data and optimizing the design of CRISPR guide RNAs and genetic constructs for efficient editing. The BDFL we describe iteratively refines strains by feeding phenomics data back into AI algorithms, enabling rapid trait optimization cycles. This transcends the trial-and-error limitations of classical methods, accelerating development of climate-smart mushrooms for circular bioeconomies including strains engineered to thrive on agricultural waste, overproduce immunomodulatory compounds, or resist emerging pathogens. The integration of predictive genomics, AI-driven phenomics, and CRISPR-edited chassis strains heralds a new era of precision mycology, where mushrooms are computationally designed as sustainable solutions for global food security, pharmaceutical innovation, and ecological resilience, ultimately transforming fungi into programmable biological factories tailored to address pressing agricultural and ecological challenges.},
}
@article {pmid40959957,
year = {2025},
author = {Chen, T and Hu, G and Fu, J and Tu, J},
title = {Structural Basis of PAM-Induced Conformational Changes in SpCas9: A Molecular Dynamics Study.},
journal = {Journal of chemical information and modeling},
volume = {65},
number = {19},
pages = {10624-10633},
doi = {10.1021/acs.jcim.5c01626},
pmid = {40959957},
issn = {1549-960X},
mesh = {*Molecular Dynamics Simulation ; *CRISPR-Associated Protein 9/chemistry/metabolism ; Protein Conformation ; DNA/metabolism/chemistry ; RNA, Guide, CRISPR-Cas Systems/metabolism/chemistry ; CRISPR-Cas Systems ; },
abstract = {As the most widely utilized CRISPR gene-editing enzyme, SpCas9 has been extensively studied and applied. However, its strict dependence on the canonical NGG PAM sequence significantly restricts its targeting scope. Although recent research has successfully engineered SpCas9 variants capable of recognizing noncanonical (non-NGG) PAMs, these variants still exhibit limitations when binding noncanonical PAMs, including substantially reduced cleavage efficiency. To elucidate the molecular mechanisms underlying noncanonical PAM recognition by SpCas9, we employed molecular dynamics simulations to compare the structural differences within the Cas9-gRNA-DNA ternary complex when bound to various PAM sequences. Our analysis revealed significant conformational changes within SpCas9 upon engagement with noncanonical PAMs and uncovered the regulatory mechanisms underpinning these changes. We further identified key dynamic determinants governing the extensive conformational transitions occurring during the noncanonical PAM binding process. These findings provide insights into the dynamic landscape of noncanonical PAM recognition, offering crucial mechanistic guidance for designing efficient, PAM-compatible Cas9 variants.},
}
@article {pmid40925496,
year = {2025},
author = {Yamazaki, M and Ueta, A and Nakanishi, T and Tachikawa, K and Kawai, M and Ozono, K and Michigami, T},
title = {Involvement of impaired phosphate production and aberrant extracellular ATP signaling in the pathogenesis of hypophosphatasia: Analysis of ALPL-Knockout human iPS cell models.},
journal = {Bone},
volume = {201},
number = {},
pages = {117629},
doi = {10.1016/j.bone.2025.117629},
pmid = {40925496},
issn = {1873-2763},
mesh = {Humans ; *Induced Pluripotent Stem Cells/metabolism/pathology ; *Hypophosphatasia/metabolism/pathology/genetics ; *Alkaline Phosphatase/metabolism/genetics/deficiency ; *Phosphates/metabolism ; *Adenosine Triphosphate/metabolism ; *Signal Transduction ; Gene Knockout Techniques ; Osteogenesis ; Cell Differentiation ; Osteoblasts/metabolism/pathology ; CRISPR-Cas Systems/genetics ; *Models, Biological ; *Extracellular Space/metabolism ; },
abstract = {Hypophosphatasia (HPP) is caused by inactivating variants of ALPL, the gene encoding tissue non-specific alkaline phosphatase (TNSALP). In order to deepen our understanding of the pathogenic mechanisms of HPP, we herein generated ALPL-knockout (KO) human induced pluripotent stem (iPS) cells by applying CRISPR/Cas9-mediated gene deletion to an iPS clone derived from a healthy subject. We analyzed two ALPL-KO clones, one ALPL-hetero KO clone, and a control clone isogenic except for ALPL. In an osteogenic culture using β-glycerophosphate, which generates inorganic phosphate (Pi) by TNSALP-mediated degradation, ALPL-KO clones showed impaired mineralization, elevated levels of extracellular pyrophosphate (PPi), and reduced levels of extracellular Pi. Osteogenic induction using 3 mM Pi instead of β-glycerophosphate rescued the decreased content of hydroxyapatite in ALPL-KO cells despite the still high levels of extracellular PPi; however, abnormal distribution of hydroxyapatite was noted. Osteoblast lineage cells differentiated from ALPL-KO iPS clones showed the up-regulation of SPP1 and the down-regulation of ANKH and the genes for type III sodium/phosphate co-transporters in the culture using β-glycerophosphate, but not when 3 mM Pi was used. Extracellular ATP levels were elevated in osteoblast lineage cells derived from ALPL-KO iPS clones in both culture conditions, which was associated with the down-regulation of P2X7 encoding a purinergic receptor. Moreover, osteoblast lineage cells differentiated from ALPL-KO iPS clones in the culture using β-glycerophosphate showed a change in cellular response to extracellular Pi. These results suggest that the reduced local production of extracellular Pi and aberrant ATP signaling play substantial roles in the pathogenesis of HPP.},
}
@article {pmid40884048,
year = {2025},
author = {Mikdar, M and Shabani, E and Grüring, C and Chaand, M and Kanjee, U and Goldberg, JM and Azouzi, S and Tennessen, JA and Elsworth, B and Keutcha, C and Barteneva, NS and Doench, JG and Duraisingh, MT},
title = {Sialyl-T Antigen: A Novel Red Blood Cell Determinant for Plasmodium falciparum Invasion.},
journal = {American journal of hematology},
volume = {100},
number = {11},
pages = {1952-1962},
doi = {10.1002/ajh.70037},
pmid = {40884048},
issn = {1096-8652},
support = {P300P3_151146//Schweizerischer Nationalfonds zur Frderung der Wissenschaftlichen Forschung/ ; PBSKP3_140144//Schweizerischer Nationalfonds zur Frderung der Wissenschaftlichen Forschung/ ; 5R01AI140751//National Institute of Allergy and Infectious Diseases/ ; 5R01HL139337//National Heart Lung and Blood Institute/ ; 23POST1017743//American Heart Association/ ; },
mesh = {*Plasmodium falciparum/pathogenicity/physiology ; Humans ; *Erythrocytes/parasitology/metabolism ; *Malaria, Falciparum/parasitology/blood/genetics ; *Galactosyltransferases/genetics/metabolism ; Animals ; N-Acetylneuraminic Acid/metabolism ; CRISPR-Cas Systems ; },
abstract = {Malaria continues to pose significant health challenges globally despite advances in control measures. Plasmodium falciparum, the parasite responsible for most severe malaria cases, uses multiple redundant invasion pathways to enter the red blood cell (RBC) during the blood stage of infection. Through a combination of RNA interference screening in erythroid cells and validation by CRISPR/Cas9-mediated knockout in primary human hematopoietic stem cells, we identified the glycosyltransferase Core 1 Synthase Glycoprotein-N-Acetylgalactosamine 3-Beta-Galactosyltransferase 1 (C1GALT1) as a novel host determinant for P. falciparum invasion. Analyses of C1GALT1-deficient cultured reticulocytes and RBCs with the glycophorin A/B-null MkMk blood group phenotype demonstrated that the C1GALT1-dependent α(2-3) sialic acid structures within mucin-type O-glycans are crucial for efficient invasion of both sialic acid-dependent and sialic acid-independent P. falciparum strains, but not the primate malaria parasite Plasmodium knowlesi. However, different P. falciparum parasite strains exhibit variable dependencies on distinct sialic acid configurations on the RBC surface. Overall, our findings highlight a key role for RBC glycans in malaria infection.},
}
@article {pmid40992249,
year = {2025},
author = {Greisle, T and Kunze, I and Wang, X and Malinowski, AR and Böttcher, A and Lickert, H and Burtscher, I},
title = {Generation of a Flattop-T2A-H2B-Venus x C-peptide-mCherry double reporter human iPSC line to monitor WNT/Planar cell polarity pathway activity.},
journal = {Stem cell research},
volume = {88},
number = {},
pages = {103838},
doi = {10.1016/j.scr.2025.103838},
pmid = {40992249},
issn = {1876-7753},
mesh = {Humans ; *Induced Pluripotent Stem Cells/metabolism/cytology ; *Cell Polarity ; *Wnt Signaling Pathway ; Cell Line ; CRISPR-Cas Systems ; Insulin-Secreting Cells/metabolism/cytology ; Genes, Reporter ; C-Peptide/metabolism/genetics ; Cell Differentiation ; },
abstract = {Deriving functional β-cells from human induced pluripotent stem cells (hiPSCs) holds potential for cell replacement therapy, disease modeling, and drug testing in diabetes research. Wnt/Planar cell polarity (PCP) signaling is crucial for endocrine cell development and β-cell maturation in murine models and can be tracked by the expression of the tissue-specific effector gene Flattop. Here, we report the generation of a human fluorescent FLTP/CFAP126 (Flattop-T2A-H2B-Venus) and FLTP-Insulin (Flattop-T2A-H2B-Venus x C-peptide-mCherry) double reporter by CRISPR/Cas9 gene editing. These hiPSC reporter lines allow monitoring of WNT/PCP signaling during endocrine cell formation and studying its role in β-cells in a human model system.},
}
@article {pmid40983220,
year = {2025},
author = {Madhusudhan, K and Padmanaban, A and Parvathi, VD},
title = {Early detection of Parkinson's disease via aptamer-CRISPR platform.},
journal = {Neuroscience},
volume = {586},
number = {},
pages = {163-195},
doi = {10.1016/j.neuroscience.2025.09.027},
pmid = {40983220},
issn = {1873-7544},
mesh = {Humans ; *Parkinson Disease/diagnosis/genetics ; *Aptamers, Nucleotide ; Early Diagnosis ; *CRISPR-Cas Systems ; Biomarkers ; Animals ; *Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {Parkinson's disease (PD) is a neurodegenerative disorder with a worldwide prevalence of around 9.4 million that is expected to double by 2040. It's extended prodromal phase allows irreversible neuronal loss to occur before manifestation of symptoms. Current diagnostic approaches, primarily based on clinical assessment and neuroimaging, are often delayed and lack sensitivity in the early stages, highlighting the need for an early, conclusive, and minimally invasive test. This review focuses on the integration of CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) diagnostics with aptamers to detect PD-associated biomarkers. CRISPR systems utilising Cas12 and Cas13 enzymes offer high specificity and collateral cleavage activity that can be harnessed for signal amplification. Aptamers are short, single-stranded oligonucleotides that can be designed to identify nucleic and non-nucleic acid targets. Their fusion with CRISPR may enable the sensitive detection of key PD biomarkers such as α-Syn, dopa decarboxylase, glial fibrillary acidic protein, and neurofilament light chain in biological fluids like blood, CSF, urine, saliva, and sweat. We explore various strategies for aptamer-CRISPR integration, detection, and multiplexing techniques for parallel biomarker detection. We also examine existing diagnostic platforms and discuss barriers to clinical translation. Ultimately, aptamer-CRISPR diagnostics could represent a powerful, next-generation approach for early PD detection.},
}
@article {pmid40953792,
year = {2025},
author = {Watts, JL and Willeke, L and Stottmann, RW},
title = {Mouse variants in Taf1c result in reduced survival to birth.},
journal = {Developmental biology},
volume = {528},
number = {},
pages = {143-151},
doi = {10.1016/j.ydbio.2025.09.011},
pmid = {40953792},
issn = {1095-564X},
mesh = {Animals ; Mice ; *TATA-Binding Protein Associated Factors/genetics/metabolism ; Humans ; *Transcription Factor TFIID/genetics ; Mutation, Missense ; Female ; Craniofacial Abnormalities/genetics ; Phenotype ; Ribosomes/metabolism/genetics ; Male ; CRISPR-Cas Systems ; },
abstract = {Ribosome biogenesis is a key cellular function and disruptions in this process can lead to congenital anomalies or "ribosomopathies" with varying phenotypes including craniofacial malformations and neurodevelopment symptoms. Classically, the mouse is a robust model to understand the molecular mechanisms underlying ribosomopathies to further elucidate human pathogenesis. We identified novel compound heterozygous missense variants in the TATA-box binding protein associated factor, RNA polymerase I subunit C (TAF1C) locus in a patient with some phenotypes consistent with ribosomopathies. TAF1C encodes a subunit of the SL1 complex which is critical for the RNA PolI complex to initiate ribosomal RNA transcription. We hypothesized that functional TAF1C is required at developmental stages critical for craniofacial and neurodevelopment. To test this hypothesis, we created mouse Taf1c variants orthologous to the human variants using CRISPR-CAS9 technology (Taf1c[R202Q] and Taf1c[S428A]). We also created an 11bp deletion to complement the missense variants (Taf1c[11bpdel]). We created multiple allelic combinations to determine the roles for Taf1c in survival and craniofacial development. Homozygous mice for any of these novel variants were underrepresented at organogenesis stages. We did not observe craniofacial anomalies in any surviving mice. Our results suggest that these specific TAF1C variants are not the cause of any human phenotype present in the patient motivating the study. However, we showed that Taf1c is required for embryonic survival and our studies contribute to knowledge about the role of ribosome biogenesis machinery throughout organogenesis.},
}
@article {pmid40929753,
year = {2025},
author = {Herbrich, S and Ramachandran, H and Seibt, A and Tolle, I and Zink, A and Prigione, A and Rossi, A and Distelmaier, F},
title = {CRISPR/Cas9-mediated editing of COQ4 in induced pluripotent stem cells: A model for investigating COQ4-associated human coenzyme Q10 deficiency.},
journal = {Stem cell research},
volume = {88},
number = {},
pages = {103825},
doi = {10.1016/j.scr.2025.103825},
pmid = {40929753},
issn = {1876-7753},
mesh = {*Induced Pluripotent Stem Cells/metabolism/cytology ; Humans ; *CRISPR-Cas Systems/genetics ; *Ubiquinone/deficiency/analogs &amp; derivatives/genetics/metabolism ; *Gene Editing/methods ; *Mitochondrial Diseases/genetics/pathology/metabolism ; *Ataxia/genetics/pathology/metabolism ; *Mitochondrial Proteins/genetics/metabolism ; *Muscle Weakness/genetics/pathology/metabolism ; Cell Line ; },
abstract = {Pathogenic variants in the gene COQ4 cause primary coenzyme Q10 deficiency, which is associated with symptoms ranging from early epileptic encephalopathy up to adult-onset ataxia-spasticity spectrum disease. We genetically modified commercially available wild-type iPS cells by using a CRISPR/Cas9 approach to create heterozygous and homozygous isogenic cell lines carrying the disease-causing COQ4 variants c.458C > T, p.Ala153Val and c.437T > G, p.Phe146Cys, respectively. All iPSCs lines exhibited a normal cell morphology, expression of pluripotency markers, and the ability to differentiate into the three primary germ layers. The COQ4-deficient cell lines will provide a helpful tool to investigate the disease mechanism and to develop therapeutic strategies.},
}
@article {pmid40925290,
year = {2025},
author = {Raabe, J and Lewandowski, V and Fuchs, S and Hammerschmidt, A and Piasecki, A and Orthey, E and Krämer, E and Ehler, E and Cuello, F},
title = {Generation of a biallelic NRAP-knockout mutant from a human iPSC line.},
journal = {Stem cell research},
volume = {88},
number = {},
pages = {103829},
doi = {10.1016/j.scr.2025.103829},
pmid = {40925290},
issn = {1876-7753},
mesh = {Humans ; *Induced Pluripotent Stem Cells/metabolism/cytology ; Cell Differentiation ; Cell Line ; CRISPR-Cas Systems ; Gene Knockout Techniques ; Alleles ; *Adaptor Proteins, Signal Transducing/genetics/metabolism ; Myocytes, Cardiac/metabolism/cytology ; Mutation ; },
abstract = {Cardiomyopathies, a leading cause of mortality, are associated with dysfunctional intercalated discs, which connect neighbouring cardiomyocytes and ensure proper contractility. In human cardiac diseases, loss-of-function mutations of the intercalated disc-associated protein Nebulin-Related Anchoring Protein (NRAP) have been reported. NRAP plays a crucial role in myofibril assembly and mechanotransduction, however, its regulatory functions remain unclear. To investigate the effects of NRAP loss-of-function in cardiac disease, a human induced pluripotent stem cell (hiPSC) line was generated carrying a biallelic NRAP-knockout (KO) using the CRISPR-Cas9 technology. Control and mutant cell lines were assessed for karyotype integrity, pluripotency, off-target effects, mycoplasma contamination, and differentiation into ectoderm, mesoderm, and endoderm. This hiPSC line provides a valuable tool to study how NRAP modulates cardiac function and contributes to disease progression.},
}
@article {pmid40902326,
year = {2025},
author = {Ran, Y and Ruan, J and Wang, Y and Feng, X and Tan, P and Guan, Y and Guo, X},
title = {Generation of a PHF19 knockout human embryonic stem cell line by CRISPR/Cas9 system.},
journal = {Stem cell research},
volume = {88},
number = {},
pages = {103824},
doi = {10.1016/j.scr.2025.103824},
pmid = {40902326},
issn = {1876-7753},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Human Embryonic Stem Cells/metabolism/cytology ; *Transcription Factors/genetics/metabolism/deficiency ; Cell Line ; *Gene Knockout Techniques ; *DNA-Binding Proteins/genetics/metabolism/deficiency ; },
abstract = {PHD finger protein 19 (PHF19) is a polycomb protein that promoted cardiac hypertrophy via epigenetic targeting SIRT2. To determine the role of PHF19 in myocardial hypertrophy, we established a large fragment knockout model of PHF19 gene in human embryonic stem cells (hESCs-H7) using the CRISPR/Cas9 system based on a vector. This PHF19-KO cell line has a normal karyotype, classical human pluripotent stem cell morphology, strong pluripotency, and significantly reduced PHF19 gene expression, which will become a useful tool for further in-depth research on the pathogenesis of PHF19 gene deficiency induced myocardial hypertrophy.},
}
@article {pmid40850232,
year = {2025},
author = {Kim, JW and Jo, S and Kang, EH and Ryu, JH and Noh, H and Park, HJ and Kim, H},
title = {Generation of human embryonic stem cell line expressing dCas9-TET1 fusion protein for epigenetic editing.},
journal = {Stem cell research},
volume = {88},
number = {},
pages = {103811},
doi = {10.1016/j.scr.2025.103811},
pmid = {40850232},
issn = {1876-7753},
mesh = {Humans ; *Human Embryonic Stem Cells/metabolism/cytology ; *Gene Editing/methods ; *Proto-Oncogene Proteins/genetics/metabolism ; *Mixed Function Oxygenases/genetics/metabolism ; *Epigenesis, Genetic ; CRISPR-Cas Systems ; Cell Line ; *CRISPR-Associated Protein 9/metabolism/genetics ; Epigenome Editing ; },
abstract = {CRISPR-based epigenome editing systems can induce site-specific transcriptional activation or repression of target genes. Ten-eleven translocation methylcytosine dioxygenase 1 (TET1) is a transcriptional activation effector involved in the cytosine demethylation of CpG dinucleotides in gene regulatory regions. In this study, we generated a human embryonic stem cell line that stably expresses catalytically dead Cas9 (dCas9) fused to the catalytic domain of TET1 via lentiviral transduction. This cell line can be used for locus-specific transcriptional activation in combination with single guide RNAs and serves as a valuable tool for epigenetic regulation in stem cell and organoid models.},
}
@article {pmid40848400,
year = {2025},
author = {Cota-Coronado, A and Manning, M and Kim, DH and Lee, J and Gibbons, A and Rosenbluh, J and Hill, RA and Sundram, S},
title = {Generation of two Betacellulin CRISPR-Cas9 knockout hiPSC lines to study the affected EGF system paradigm in schizophrenia.},
journal = {Stem cell research},
volume = {88},
number = {},
pages = {103808},
doi = {10.1016/j.scr.2025.103808},
pmid = {40848400},
issn = {1876-7753},
mesh = {*Schizophrenia/genetics/metabolism/pathology ; Humans ; *CRISPR-Cas Systems/genetics ; *Induced Pluripotent Stem Cells/metabolism/cytology ; *Betacellulin/genetics/metabolism ; *Epidermal Growth Factor/metabolism/genetics ; Gene Knockout Techniques ; Cell Differentiation ; Cell Line ; },
abstract = {Several members of the epidermal growth factor (EGF) family have been implicated in the biology of schizophrenia (Ketharanathan et al., 2024). The EGF-related ligand, Betacellulin (BTC), plays an important role in the proliferation and differentiation of neural stem cells and our group found markedly reduced BTC levels in patients with schizophrenia. Nevertheless, the interplay of affected BTC and its participation in neural specification and neurodevelopment remains elusive. We generated Knockout (KO) - BTC clones from an existing hiPSC line through CRISPR/Cas9-mediated modification. Furthermore, we validated BTC-KO through genotyping/sequencing, FACS and Western Blot. Finally, we demonstrated trilineage differentiation potential in vitro.},
}
@article {pmid41073543,
year = {2025},
author = {Shi, Z and Cheng, TL},
title = {UGI relocation inside Cas9 reduces Cas9 dependent off target effects in cytosine base editors.},
journal = {Scientific reports},
volume = {15},
number = {1},
pages = {35518},
pmid = {41073543},
issn = {2045-2322},
support = {20ZR1403100//Shanghai Natural Science Foundation/ ; },
mesh = {*Gene Editing/methods ; *Cytosine/metabolism ; *CRISPR-Associated Protein 9/metabolism/genetics/chemistry ; *CRISPR-Cas Systems ; *Uracil-DNA Glycosidase/antagonists &amp; inhibitors/metabolism ; Humans ; HEK293 Cells ; },
abstract = {Cytosine base editors (CBEs) achieve precise C-to-T conversions by addition of uracil DNA glycosylase inhibitor (UGI) with Cas9 nickase (nCas9) and cytidine deaminase, and the conventional fusion at the nCas9 carboxyl terminus effectively inhibits uracil excision repair to enhance editing efficiency. However, despite potent on-target activity, classical CBEs exhibit significant Cas9-dependent DNA off-target effects that necessitate optimization for future applications. Here we present a strategic UGI relocation through internal fusion within the nCas9 architecture. This spatial reorganization maintains comparable on-target editing efficiency while substantially reducing Cas9-dependent DNA off-target activity. Our findings establish an alternative engineering paradigm to develop high-fidelity CBEs, offering an improved platform for widespread genome editing applications.},
}
@article {pmid41073413,
year = {2025},
author = {Zhang, H and Li, M and Wang, G and Zhu, K and Guo, G and Fu, H and Hu, C and Chu, Z and Hu, J and Wu, Q and Chen, Y and Qiu, D and Xie, J and Li, D and Li, B and Li, W and Dong, L and Hou, Y and Cui, X and Huang, B and Liu, Y and Li, Y and Li, H and Yuan, C and Dong, L and Liu, Z and Lu, P},
title = {Paired NLRs originated from Triticum dicoccoides coordinately confer resistance to powdery mildew in wheat.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {9040},
pmid = {41073413},
issn = {2041-1723},
mesh = {*Triticum/genetics/microbiology/immunology ; *Disease Resistance/genetics ; *Plant Diseases/microbiology/genetics/immunology ; *Ascomycota/physiology/pathogenicity ; *Plant Proteins/genetics/metabolism ; *NLR Proteins/genetics/metabolism ; Haplotypes ; Gene Expression Regulation, Plant ; CRISPR-Cas Systems ; },
abstract = {Wheat has evolved diverse resistance genes against powdery mildew, typically controlled by single-gene-encoded proteins. Here, we report the map-based cloning of PmWR183, a resistance locus encoding two adjacent NLR proteins (PmWR183-NLR1 and PmWR183-NLR2) from wild emmer wheat. Stable transformation and CRISPR/Cas9 knockout experiments demonstrate that the two NLRs function cooperatively: neither gene alone confers resistance, but their co-expression restores immunity, while disruption of either gene abolishes resistance. PmWR183 mediates a developmental stage-dependent response, with susceptibility at the seedling stage and strong resistance at the adult stage. Protein interaction assays reveal constitutive association of PmWR183-NLR1 and PmWR183-NLR2, supporting their cooperative role. Geographical and haplotype analyses show the locus originates from wild emmer and is rare in cultivated wheat, exhibiting at least nine haplotypes. Together, our findings uncover a rare NLR gene pair conferring effective resistance to powdery mildew, providing valuable resources for wheat breeding.},
}
@article {pmid41073016,
year = {2025},
author = {Nie, Y and Wang, W and Wang, N and Yuan, M and Huang, L and Sun, Y and Li, K and Liu, Z and Mu, Y},
title = {PCR-CRISPR/Cas12a-based fluorescence and lateral flow dipstick platforms for efficient screening of CD71 biallelic mutants.},
journal = {Analytica chimica acta},
volume = {1376},
number = {},
pages = {344622},
doi = {10.1016/j.aca.2025.344622},
pmid = {41073016},
issn = {1873-4324},
mesh = {*CRISPR-Cas Systems/genetics ; Humans ; *Receptors, Transferrin/genetics ; *Polymerase Chain Reaction/methods ; *Mutation ; *Antigens, CD/genetics ; *Alleles ; Fluorescence ; *CRISPR-Associated Proteins/genetics/metabolism ; Bacterial Proteins ; Endodeoxyribonucleases ; },
abstract = {CRISPR/Cas9 technology plays a pivotal role in gene editing and has been extensively utilized in gene function studies, disease modeling, and gene therapy. However, efficient and accurate detection of CRISPR/Cas9-induced mutants remains a challenge due to the complexity, time-consuming nature, and high cost of existing detection methods. Meanwhile, CRISPR/Cas12a systems have been widely applied in molecular diagnostics due to the non-specific trans-cleavage activity of Cas12a, yet their application in detecting CRISPR/Cas9-induced mutations remains limited. In this study, we developed a PCR-CRISPR/Cas12a-based method to enable the rapid and accurate screening of CD71 biallelic mutants. The detection system was mainly composed of CRISPR RNA specific to the CD71 gene-editing site, Cas12a protein, target DNA, and ssDNA probes for fluorescence or lateral flow dipstick assays. The system demonstrated high specificity in distinguishing CD71 biallelic mutants, with validation through TA cloning confirming its accuracy. Additionally, the method exhibited high sensitivity, establishing it as an efficient tool for biallelic mutated cell clone screening. These findings underscore the potential of PCR-CRISPR/Cas12a as a rapid, sensitive, and cost-effective approach for the precise identification of biallelic mutants, contributing to advancements in gene-editing research and molecular diagnostics.},
}
@article {pmid41073014,
year = {2025},
author = {Cao, R and Wang, S and Guo, Q and Xie, H and Wang, Y and Li, T and Chang, F and Shi, H and Ding, S and Min, X and Duan, X},
title = {DNAzyme-driven SDA reaction regulates CRISPR/Cas12a for highly sensitive and selective analysis of underexpressed miRNA.},
journal = {Analytica chimica acta},
volume = {1376},
number = {},
pages = {344620},
doi = {10.1016/j.aca.2025.344620},
pmid = {41073014},
issn = {1873-4324},
mesh = {*MicroRNAs/analysis/genetics/metabolism ; *DNA, Catalytic/metabolism/chemistry ; *CRISPR-Cas Systems ; Humans ; *Biosensing Techniques/methods ; *Nucleic Acid Amplification Techniques/methods ; Limit of Detection ; Male ; Prostatic Neoplasms/genetics ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {Underexpressed microRNA (miRNA) exerts a pivotal influence across a spectrum of physiological and pathological processes, with their role being particularly pronounced in the incipient stages of tumorigenesis. However, there are several challenges to analyzing these underexpressed miRNAs for their lower abundance and relative decreases in some cancers. Here, we developed a novel fluorescence biosensor based on the DNAzyme-driven strand displacement amplification (SDA) regulating CRISPR/Cas12a for the sensitive and selective detection of underexpressed miRNA, using prostate cancer-associated miR-222 as a proof-of-concept. This study innovatively expanded the application of DNAzyme substrates, designed as templates to trigger SDA and CRISPR/Cas12a reaction, which could effectively generate a positive signal output for detecting underexpressed miRNA. In the absence of miR-222, DNAzyme formation was blocked, allowing the complete substrate to activate SDA, which generated ssDNA that triggered CRISPR/Cas12a trans-cleavage activity to produce a strong fluorescent signal. In contrast, intact DNAzymes (in the presence of miR-222) cleaved the substrates into short DNA fragments, preventing SDA and CRISPR/Cas12a activation, thereby maintaining the sensor in a low fluorescent state. The biosensor demonstrated a linear detection range spanning from 0.1 pmol/L to 1 nmol/L, accompanied by a detection limit of 33.5 fmol/L. Moreover, it exhibited excellent specificity and anti-interference capacity, enabling the successful detection of miR-222 in blood samples. This "DNAzyme-SDA-CRISPR" fluorescence strategy offers a effective, programmability and scalable solution for detecting underexpressed miRNAs in early cancer screening, which is expected to become a powerful tool in early tumor diagnostics and precision therapy.},
}
@article {pmid41043311,
year = {2026},
author = {Hu, M and Zhou, C and Li, M and Zhao, J},
title = {From 3D culture to clinical decision-making: Systematic innovations in breast cancer organoids.},
journal = {Biomaterials advances},
volume = {179},
number = {},
pages = {214528},
doi = {10.1016/j.bioadv.2025.214528},
pmid = {41043311},
issn = {2772-9508},
mesh = {Humans ; *Organoids/pathology ; *Breast Neoplasms/pathology/therapy/genetics ; Female ; *Clinical Decision-Making ; Animals ; CRISPR-Cas Systems ; *Cell Culture Techniques, Three Dimensional/methods ; Tumor Microenvironment ; Gene Editing ; Precision Medicine ; },
abstract = {Breast cancer is a malignant tumour with high heterogeneity. Traditional research models rely mainly on 2D cell culture and patient-derived tumour xenografts (PDXs). However, these models have limited use in clinical trials because of their shortcomings in mimicking the tumour microenvironment and preserving the genetic background. In recent years, organoids, emerging models capable of self-organizing to form 3D structures in vitro, have become key tools for overcoming the traditional dilemma and are promising alternatives for breast cancer research. This review integrates cutting-edge technologies such as organ-on-a-chip and CRISPR/Cas9 gene editing to summarize the multidimensional generation strategy of breast cancer organoids and discusses the clinical value of translation from diagnosis to therapy. Compared with existing studies, this review provides a systematic solution from "model generation" to "precision medicine" for breast cancer research, and the hope is that this review will pave the way for the further development of organoids.},
}
@article {pmid41015100,
year = {2025},
author = {Deng, D and Yi, X and Wen, W and He, L and Peng, W},
title = {The role of the transformer gene in sex determination and its employment in CRISPR/Cas9-based homing gene drive in the global fruit pest Drosophila suzukii.},
journal = {Insect biochemistry and molecular biology},
volume = {184},
number = {},
pages = {104406},
doi = {10.1016/j.ibmb.2025.104406},
pmid = {41015100},
issn = {1879-0240},
mesh = {Animals ; *Sex Determination Processes/genetics ; CRISPR-Cas Systems ; Female ; Male ; *Drosophila Proteins/genetics/metabolism ; *Drosophila/genetics/growth &amp; development ; *Gene Drive Technology ; Nuclear Proteins ; },
abstract = {Sex determination of Diptera is established by the cascade genes such as transformer (tra), though the primary signals for sex determination differ among different insects. Here, we report the isolation, expression and function of tra gene in an invasive pest, Drosophila suzukii, and study the potential use of the D. suzukii tra (Dstra) gene in CRISPR/Cas9-based homing gene drive for genetic-based pest management. The Dstra gene is highly conserved in structure and has a sex-specific transcript. To test the function of this gene in sex determination, Dstra dsRNA was injected into embryos. Almost all XX embryos developed into masculinized phenotypic male adults with intersex morphology. Abnormal ovaries were revealed in XX pseudomales upon dissection. Based on the necessary role of Dstra for female development, we developed and evaluated a homing gene drive that targets Dstra in D. suzukii. The drive component consisting of multiplex Dstra single guide RNAs and Cas9 with Dsvasa promoter was introduced into the Dstra locus. Abnormal development of both the external genitalia and gonads was observed in G0 and G1 chromosomal female adults that expressed the male-specific doublesex (dsx) transcript. Interestingly, knocking out Dstra led to significantly reduced fertility in adults of corresponding sex and moderate transmission rates of the DsRed gene (63.54 %) were observed. Our results not only confirm the conserved function of the Dstra gene in sex determination, but also highlight the potential of sex conversion-based suppression gene-drive strategy targeting the Dstra gene in controlling of D. suzukii populations.},
}
@article {pmid41072992,
year = {2025},
author = {Shi, S and Qin, F and Wu, J and Yang, J and Zhang, X and Wang, S and Wen, W and Wu, Z},
title = {Ultrasensitive single-particle collision electrochemical platform employing CRISPR/Cas12a for ctDNA biosensing.},
journal = {Analytica chimica acta},
volume = {1376},
number = {},
pages = {344590},
doi = {10.1016/j.aca.2025.344590},
pmid = {41072992},
issn = {1873-4324},
mesh = {*Biosensing Techniques/methods ; *Circulating Tumor DNA/blood/analysis/genetics ; *Electrochemical Techniques/methods ; Humans ; *CRISPR-Cas Systems ; Silver/chemistry ; Metal Nanoparticles/chemistry ; Limit of Detection ; },
abstract = {Circulating tumor DNA (ctDNA) is a characteristic tumor biomarker used for cancer diagnosis, treatment, and prognosis. However, the low concentration of ctDNA in peripheral blood and the interference of complex matrices with signals make the detection of ctDNA extremely challenging. Single-particle collision electrochemistry (SPCE) has been widely used in bioanalysis due to its advantages such as high throughput, simple operation, high sensitivity, and low detection limit. In this work, a novel SPCE biosensor for the rapid detection of ctDNA was developed by combining CRISPR/Cas12a with excellent cleavage activity and magnetic beads (MBs) with good separation and enrichment capabilities. The trans-cleavage ability of CRISPR/Cas12a can only be triggered in the presence of target ctDNA to effectively cleave the ssDNA2 on the surface of Ag NPs-ssDNA2 within 1 h, thereby activating the collision activity of silver nanoparticles (Ag NPs). ctDNA was quantified by the collision frequency of Ag NPs. The detection limit of the developed SPCE biosensor for ctDNA was as low as 4.2 fM, and the linear range was 10 fM-1 nM. In addition, MBs allow the biosensor to detect ctDNA in complex samples by directly sampling from complex matrices, with excellent sensitivity and specificity, demonstrating the great potential of the developed SPCE biosensor in the detection of patient samples.},
}
@article {pmid41072860,
year = {2025},
author = {Patel, RR and Arun, PP and Singh, SK and Singh, M},
title = {Overcoming Antimicrobial Resistance: Phage Therapy as a Promising Solution to Combat ESKAPE Pathogens.},
journal = {International journal of antimicrobial agents},
volume = {},
number = {},
pages = {107640},
doi = {10.1016/j.ijantimicag.2025.107640},
pmid = {41072860},
issn = {1872-7913},
abstract = {The global escalation of antimicrobial resistance (AMR) has intensified the search for alternative therapies, with bacteriophage (phage) therapy re-emerging as a promising solution. This review critically examines the therapeutic potential of phage therapy against multidrug-resistant (MDR) ESKAPE pathogens which are among the leading causes of hospital-acquired infections. The review discusses the distinct antibacterial strategies of phage namely, targeted lysis, enzymatic biofilm disruption, and synergy with antibiotics. It also explores the molecular regulation of phage life cycles, highlighting the therapeutic importance of the lytic-lysogenic switch. A central focus is the interplay between advanced delivery systems such as liposomes, hydrogels, nanofibers, and nanoemulsions, and specific administration routes including oral, topical, intravenous, intranasal, and intravesical approaches. These delivery strategies are essential for overcoming key physiological barriers such as gastric acidity, enzymatic degradation, and immune clearance, thereby enhancing phage stability, retention, and therapeutic efficacy. Recent innovations in phage engineering are also explored, particularly the use of CRISPR-Cas systems, synthetic biology, and continuous evolution platforms to broaden host range and optimize lytic function. The review further evaluates emerging clinical evidence, including outcomes from compassionate use cases and early-phase trials, which emphasize both the safety and therapeutic potential of phage therapy in real-world settings. Despite these advances, significant challenges persist, including bacterial resistance to phages, the need for regulatory clarity, and scalability of personalized treatments. With the integration of microbiology, nanotechnology, and clinical practice, phage therapy bridges the gap between ecological solutions and modern medicine, positioning itself as a versatile, sustainable pillar in the post-antibiotic era.},
}
@article {pmid41072406,
year = {2025},
author = {Henriques, WS and Bowman, J and Hall, LN and Gauvin, CC and Wei, H and Kuang, H and Zimanyi, CM and Eng, ET and Santiago-Frangos, A and Wiedenheft, B},
title = {Structures reveal how the Cas1-2/3 integrase captures, delivers, and integrates foreign DNA into CRISPR loci.},
journal = {Structure (London, England : 1993)},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.str.2025.09.007},
pmid = {41072406},
issn = {1878-4186},
abstract = {Cas1 and Cas2 are the hallmark proteins of prokaryotic adaptive immunity. However, these two proteins are often fused to other proteins and the functional association of these fusions often remain poorly understood. Here we purify and determine structures of Cas1 and the Cas2/3 fusion proteins from Pseudomonas aeruginosa at distinct stages of CRISPR adaptation. Collectively, these structures reveal a prominent, positively charged channel on one face of the integration complex that captures short fragments of foreign DNA. Foreign DNA binding triggers conformational changes in Cas2/3 that expose new DNA binding surfaces necessary for homing the DNA-bound integrase to specific CRISPR loci. The length of the foreign DNA substrate determines if Cas1-2/3 docks completely onto the CRISPR repeat to successfully catalyze two sequential transesterification reactions required for integration. Together, these structures clarify how the Cas1-2/3 proteins orchestrate foreign DNA capture, site-specific delivery, and integration of new DNA into the bacterial genome.},
}
@article {pmid41071884,
year = {2025},
author = {Guo, Y and Xu, M and Xue, H and Ding, X and Wong, AM and Lin, N and Pu, D and Wong, AM and Wang, X and Zhao, H and Wong, N},
title = {Genome-wide CRISPR screen identifies splicing factor SF3B4 in driving hepatocellular carcinoma.},
journal = {Science advances},
volume = {11},
number = {41},
pages = {eadw7181},
doi = {10.1126/sciadv.adw7181},
pmid = {41071884},
issn = {2375-2548},
mesh = {*Carcinoma, Hepatocellular/genetics/pathology/metabolism/drug therapy ; Humans ; *Liver Neoplasms/genetics/pathology/metabolism/drug therapy ; Animals ; Mice ; *RNA Splicing Factors/genetics/metabolism ; Ferroptosis/genetics ; Gene Expression Regulation, Neoplastic ; *CRISPR-Cas Systems ; Cell Line, Tumor ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Phenylurea Compounds/pharmacology ; Drug Resistance, Neoplasm/genetics ; T-Box Domain Proteins/genetics/metabolism ; Quinolines ; },
abstract = {Although genome sequencings have recognized many cancer-associated genes in hepatocellular carcinoma (HCC), distinguishing their functional effect remains challenging. Leveraging on a genome-wide CRISPR knockout (KO) screening, we uncovered spliceosome factors as major survival essential genes in HCC and up-regulations of ferroptosis suppressors [particularly glutamate-cysteine ligase catalytic subunit (GCLC)] in lenvatinib resistance. Our KO screen in patient-derived HCC organoid showed splicing factor 3b subunit 4 (SF3B4) to be top-ranked, conferring prosurvival signal in HCC organoid and driving tumorigenic potentials in both hepatic progenitor organoids and hydrodynamic tail vein injection HCC murine model. The combined RNA immunoprecipitation sequencing, long-read isoform sequencing, and transcriptome revealed characteristic splicing landscape regulated by SF3B4 and identified T-box transcription factor 3 (TBX3) variant TBX3+2a as a potent downstream effector. Our findings highlighted vital roles of SF3B4 in HCC cell survival and tumor progression, and the phenomenon of ferroptosis resistance in patients unresponsive to first-line agent lenvatinib.},
}
@article {pmid41070887,
year = {2025},
author = {Zhang, Y and Wu, Y and Guo, A and Liu, Y and Sun, Q and Zou, X and Sun, Z},
title = {Fluorescent biosensors for the detection of foodborne pathogenic bacteria in food: a comprehensive review.},
journal = {Analytical methods : advancing methods and applications},
volume = {},
number = {},
pages = {},
doi = {10.1039/d5ay01025j},
pmid = {41070887},
issn = {1759-9679},
abstract = {Foodborne pathogenic bacterial contamination poses a major challenge to global food safety and public health, making the development of rapid, sensitive, and specific detection technologies critically important. Conventional methods are limited by their long turnaround time, complex operations, and reliance on large-scale instruments, making them unsuitable for on-site rapid detection. Fluorescent biosensors, which combine highly specific biological recognition elements with highly sensitive fluorescent signal output, demonstrate significant advantages in detecting foodborne pathogens. This review systematically summarizes recent advances in fluorescent biosensors for the detection of common foodborne pathogenic bacteria, with a focus on the application of signal amplification strategies such as functional nanomaterials, amplification techniques, CRISPR/Cas systems, and Argonaute proteins. Furthermore, it analyzes performance metrics including multiplex pathogen detection, real-time quantification, anti-interference capability, and on-site applicability. Finally, future development trends and challenges are discussed, aiming to provide insights for the innovation of food safety monitoring technologies.},
}
@article {pmid41069986,
year = {2025},
author = {Pandey, V and Sharma, S and Pokharel, YR},
title = {Exploring CRISPR-Cas: The transformative impact of gene editing in molecular biology.},
journal = {Molecular therapy. Nucleic acids},
volume = {36},
number = {4},
pages = {102717},
pmid = {41069986},
issn = {2162-2531},
abstract = {This review traces the evolution of clustered regularly interspaced short palindromic repeats (CRISPR) technology from a prokaryotic immune mechanism to a versatile tool for precise genome engineering. We compare CRISPR with traditional gene-editing methods like RNA interference (RNAi), zinc finger nucleases (ZFNs), and transcription activator-like effector nucleases (TALENs), emphasizing its advantages in target specificity, multiplexing, and ease of design. We examine various Cas enzyme classes, engineered variants, and their applications in dissecting genetic alterations at the cellular level. The review further explores CRISPR's expanding role in developing disease models using tissues, organoids, and animal systems, enhancing our understanding of disease mechanisms. Finally, we discuss CRISPR's emerging applications in diagnostics and its transformative impact on immunotherapy and cell-based cancer treatments.},
}
@article {pmid41069170,
year = {2025},
author = {Zhu, Z and Xue, J and Cao, J and Zhang, Z and Gu, T and Sun, Y and Wang, H},
title = {One-pot assay for rapid detection of heterozygous herbicide resistance in Digitaria ciliaris var. chrysoblephara by combining CRISPR/Cas and LAMP.},
journal = {Pest management science},
volume = {},
number = {},
pages = {},
doi = {10.1002/ps.70279},
pmid = {41069170},
issn = {1526-4998},
support = {CX(22)5003//Jiangsu Agricultural Science and Technology Innovation Fund/ ; 2023YFD1401100//National Key R&amp;D Program of China/ ; },
abstract = {BACKGROUND: Resistance to the acetyl-CoA carboxylase (ACCase) inhibitor herbicide cyhalofop-butyl in Digitaria ciliaris var. chrysoblephara is mainly caused by a mutation at the W2027C or W2027S site; however, the existing methods for this mutation site have insufficient detection performance and are difficult to achieve integrated detection in the field.<br><br>RESULTS: In this work, we have developed and optimized a One-Pot single-nucleotide polymorphism (SNP) detection for herbicide resistance based on CRISPR/Cas recognition coupled with the loop-mediated isothermal amplification (LAMP), named OpCas-LAMP. By designing specific CRISPR/Cas guide RNAs and LAMP primers, the OpCas-LAMP can accurately identify with 1% heterozygous mutants of the W2027S or W2027C mutations ACCase gene in D.ciliaris var. chrysoblephara. The optimized reaction system exhibits optimal amplification efficiency at 65&#xb0;C, effectively distinguishing within 60 min (30-min LAMP detection after 30 min CRISPR/Cas pre-cleavage) between homozygous mutant (HM), heterozygous mutant (HT) and wild-type (WT).<br><br>CONCLUSION: This method enables real-time one-pot field detection by integrated with miniaturized detection devices, significantly enhancing its practicality and potential for widespread application. This work provides a novel technical approach for detecting herbicide resistance for global weed resistance monitoring and management. &#xa9; 2025 Society of Chemical Industry.},
}
@article {pmid41068986,
year = {2025},
author = {Wu, J and Jang, H and Kwak, H and Son, M and Jiang, W and Hwang, HY and Jo, DH and Kim, D and Kim, HH and Kim, JH},
title = {Engineered virus-like particle-assembled Vegfa-targeting Cas9 ribonucleoprotein treatment alleviates neovascularization in wet age-related macular degeneration.},
journal = {Genome biology},
volume = {26},
number = {1},
pages = {346},
pmid = {41068986},
issn = {1474-760X},
support = {2022M3A9F3017506//National Research Foundation of Korea/ ; 2022M3A9E4017127//National Research Foundation of Korea/ ; 202200004004//Kun-hee Lee Child Cancer &amp; Rare Disease Project/ ; 18-2023-0010//Seoul National University Hospital/ ; GTL24021-000//National Research Council of Science and Technology/ ; },
mesh = {Animals ; *Vascular Endothelial Growth Factor A/genetics/metabolism ; Mice ; Gene Editing/methods ; *Ribonucleoproteins/genetics ; *CRISPR-Associated Protein 9/genetics/metabolism ; *Choroidal Neovascularization/therapy/genetics ; CRISPR-Cas Systems ; NIH 3T3 Cells ; Disease Models, Animal ; Humans ; *Wet Macular Degeneration/therapy/genetics ; Genetic Therapy ; *Macular Degeneration/therapy/genetics ; },
abstract = {BACKGROUND: Age-related macular degeneration, particularly the wet form, is a leading cause of vision loss, characterized by vascular endothelial growth factor A (VEGFA) overproduction. Engineered virus-like particles (eVLPs) combine the efficiency of viral systems with the transient nature of non-viral platforms to offer a potential solution for delivering VEGFA-targeting genome editing enzymes in a safe and efficient manner. Here, we investigate the therapeutic efficacy of eVLPs for transient delivery of Vegfa-targeting Cas9 ribonucleoprotein in a laser-induced choroidal neovascularization mouse model of wet age-related macular degeneration.<br><br>RESULTS: We find that Cas9-eVLPs enables efficient intracellular delivery in vitro, achieving up to 99% insertion and deletion frequency at Vegfa target locus and significant VEGFA protein downregulation in NIH/3T3 cells. A single subretinal injection of Cas9-eVLPs into the mouse retinal pigment epithelium effectively disrupts Vegfa expression, achieving an average indel efficiency of 16.7%. Compared to control groups, the laser-induced choroidal neovascularization mouse model exhibits significantly reduced choroidal neovascularization formation following Cas9-eVLPs intervention, and decreased VEGFA protein levels are detected in the retinal pigment epithelium. Furthermore, the retinal anatomical and functional toxicity are not affected after treatment.<br><br>CONCLUSIONS: eVLPs exhibit the potential as a safe and efficient delivery platform for Cas9 ribonucleoproteins, achieving precise Vegfa downregulation and significant reduction in choroidal neovascularization in a mouse model of wet age-related macular degeneration. With transient delivery of gene editing enzymes, high editing efficiency, and minimal risk of genomic integration, eVLPs present a promising alternative to conventional delivery systems for advancing genome editing therapies in retinal diseases.},
}
@article {pmid41066575,
year = {2025},
author = {Groessl, S and Kalis, R and Snaebjornsson, MT and Wambach, L and Haider, J and Andersch, F and Schulze, A and Palm, W and Zuber, J},
title = {Acidosis orchestrates adaptations of energy metabolism in tumors.},
journal = {Science (New York, N.Y.)},
volume = {390},
number = {6769},
pages = {eadp7603},
doi = {10.1126/science.adp7603},
pmid = {41066575},
issn = {1095-9203},
mesh = {*Energy Metabolism/genetics ; *Acidosis/metabolism/genetics ; Humans ; *Pancreatic Neoplasms/metabolism/genetics ; Mitochondria/metabolism ; Cell Line, Tumor ; *Adaptation, Physiological/genetics ; Animals ; Mice ; Stress, Physiological ; CRISPR-Cas Systems ; MAP Kinase Signaling System ; Mitochondrial Dynamics ; },
abstract = {Malignant tumors are characterized by diverse metabolic stresses, including nutrient shortages, hypoxia, and buildup of metabolic by-products. To understand how cancer cells adapt to such challenges, we conducted sequential CRISPR screens to identify genes that affect cellular fitness under specific metabolic stress conditions in cell culture and to then probe their relevance in pancreatic tumors. Comparative analyses of hundreds of fitness genes revealed that cancer metabolism in vivo was shaped by bioenergetic adaptations to tumor acidosis. Mechanistically, acidosis suppressed cytoplasmic activity of extracellular signal-regulated kinase (ERK), thereby preventing oncogene-induced mitochondrial fragmentation and promoting fused mitochondria. The resulting boost in mitochondrial respiration supported cancer cell adaptations to various metabolic stresses. Thus, acidosis is an environmental factor that alters energy metabolism to promote stress resilience in cancer.},
}
@article {pmid41065057,
year = {2025},
author = {Anjali, and Punetha, M and Kumar, A and Tripathi, MK and Kishor Kumar, DG and Khanna, S and Nanda, R and Yadav, P and Sharma, S and Maurya, VP and Singh, G and Chouhan, VS},
title = {Application of CRISPR/Cas9 for GDF9 Gene Editing in Caprine Granulosa Cells: Effects on Receptor Signalling and FGF2 Response.},
journal = {Reproduction in domestic animals = Zuchthygiene},
volume = {60},
number = {10},
pages = {e70128},
doi = {10.1111/rda.70128},
pmid = {41065057},
issn = {1439-0531},
support = {NASF/GTR-8004/2019-20//ICAR - National Agricultural Science Fund/ ; },
mesh = {Animals ; Female ; *Granulosa Cells/metabolism ; *Growth Differentiation Factor 9/genetics/metabolism ; *CRISPR-Cas Systems ; *Gene Editing/veterinary ; *Goats/genetics ; *Fibroblast Growth Factor 2/pharmacology/metabolism ; Signal Transduction ; Receptors, FSH/metabolism/genetics ; },
abstract = {Fecundity-related genes, such as GDF9, play a critical role in regulating ovulation, fertilisation and early embryonic development. This study aimed to elucidate the functional role of GDF9 in caprine granulosa cells by employing CRISPR/Cas9-mediated gene editing. The CRISPR/Cas9 system, incorporating single guide RNA (sgRNA) and Cas9 endonuclease, was used to specifically disrupt the GDF9 gene. Successful GDF9 knockout was confirmed via the T7 Endonuclease I (T7E1) cleavage assay. Subsequent analyses assessed the impact of GDF9 disruption on the expression of GDF9 and its associated receptors-BMPR-1A, BMPR-1B and BMPR-II. Additionally, the study examined the modulatory effects of fibroblast growth factor 2 (FGF2) on receptor expression. FGF2 treatment led to increased mRNA expression of BMPR-1A, BMPR-1B and BMPR-II in wild-type granulosa cells. Furthermore, follicle-stimulating hormone receptor (FSHR) levels were significantly upregulated, whereas luteinising hormone receptor (LHR) expression decreased following FGF2 stimulation in wild-type cells. In contrast, GDF9-knockout cells showed elevated expression of both FSHR and LHR. The study also investigated the impact of GDF9 deletion on the expression of key steroidogenic genes, particularly StAR. The combined presence of GDF9 and FGF2 synergistically enhanced StAR expression. Cellular responses to FGF2 included a downregulation of CASPASE 3, indicating reduced apoptosis and an upregulation of PCNA, suggesting increased cell proliferation. In conclusion, this study provides novel insights into the regulatory role of GDF9 in ovarian granulosa cell function and highlights the utility of CRISPR/Cas9 technology for functional genomics in caprine species. The findings have significant implications for enhancing reproductive performance through targeted gene modulation.},
}
@article {pmid41064997,
year = {2025},
author = {Chen, X and Ye, Q and Liang, Q and Li, J and Huang, Y and Xia, Q and Xiao, J and Liao, C and Lau, CH and Zhu, H},
title = {CRISPR-based platforms for detecting tumor-associated genetic materials in clinical samples.},
journal = {Bioanalysis},
volume = {},
number = {},
pages = {1-15},
doi = {10.1080/17576180.2025.2571023},
pmid = {41064997},
issn = {1757-6199},
abstract = {Tumor-associated genetic markers are useful for early cancer screening, diagnosis, and treatment monitoring. However, traditional detection methods are complex in operation procedures, time-consuming, and the equipment costs are expensive. CRISPR/Cas systems are becoming emerging detection tools for tumor detection due to their programmability, rapid reaction, high targeting specificity, and the ability to amplify the signals. CRISPR/Cas has made breakthroughs in the detection of tumor-associated genetic materials including gene mutations, DNA methylation, miRNA, lncRNA, and circRNA detection. Herein, we critically discuss these advancements and describe the key concepts of each CRISPR/Cas system for detecting tumor-associated genetic materials. The significance of these tumor-associated genetic materials in cancer diagnosis and prognosis is highlighted.},
}
@article {pmid41064763,
year = {2025},
author = {Ragulakollu, S and Loganathan, A and Swaminatham, M and Chellappan, G and Veeraswamy, R and Jegadeesan, R},
title = {Molecular breeding approaches for sustainable rice blast management: recent advances and challenges.},
journal = {Frontiers in plant science},
volume = {16},
number = {},
pages = {1551018},
pmid = {41064763},
issn = {1664-462X},
abstract = {Rice (Oryza sativa. L) is a staple crop globally, but blast disease caused by fungal pathogens Magnaporthe oryzae is one of the most devastating and results in severe economic losses in rice production worldwide. Recent technological advancements have opened new possibilities for developing blast resistance. The dynamic and highly adaptable nature of M. oryzae allows it to overcome plant defense mechanisms rapidly, posing a major threat to global food security and agricultural sustainability. While foundational to early resistance development, traditional breeding approaches have been limited by their time-consuming nature and reliance on phenotypic selection. These methods often require several generations to establish stable resistance traits. However, with the emergence of molecular breeding technologies, resistance breeding has experienced significant acceleration and precision. Tools such as marker-assisted selection (MAS), marker-assisted backcross breeding (MABB), and quantitative trait locus (QTL) mapping allow for the identification and introgression of resistance genes (R genes) more efficiently and accurately. Recent advances in genome engineering techniques, particularly CRISPR-Cas 9, have transformed the capability to manipulate resistance genes directly, enabling targeted editing and stacking of multiple genes (gene pyramiding) for durable resistance. Moreover, omics technologies-including genomics, transcriptomics, proteomics, and metabolomics-offer a comprehensive understanding of the molecular interactions between host and pathogen, facilitating the discovery of novel resistance mechanisms and regulatory pathways. The integration of allele mining with advanced biotechnological tools has further promoted the development of cisgenic and intragenic plants, where resistance genes from related cultivars or wild species are introduced without foreign DNA, thus addressing public concerns over transgenic crops. These strategies enhance resistance and help retain the desirable agronomic traits of elite rice varieties. Despite these advancements, the high mutation rate and genetic plasticity of M. oryzae enable it to evolve and overcome resistance provided by single R genes. Therefore, understanding host-pathogen interactions at the molecular and cellular levels remains essential. Emerging technologies such as nanotechnology show promise in developing targeted fungicide delivery systems and innovative diagnostic tools. Synthetic biology opens avenues for constructing synthetic resistance pathways or deploying plant biosensors. Additionally, machine learning and artificial intelligence (AI) algorithms are increasingly used to predict disease outbreaks, model gene interactions, and optimize breeding strategies based on large datasets. Thus, managing rice blast disease necessitates a holistic approach combining conventional breeding wisdom with modern molecular tools and emerging technologies. The synergy among these approaches holds promise to enhance resistance durability and protect global rice production against evolving fungal threats. This review emphasizes recent advancements in managing rice blast disease, offering valuable insights to sustain resilient breeding programs against this pathogen.},
}
@article {pmid41064460,
year = {2025},
author = {Rentz, L and Hellwig, L and Schneider, S and Schmitz, RA},
title = {Functional insights into Solo-Cas4 in Methanosarcina mazei Gö1.},
journal = {microLife},
volume = {6},
number = {},
pages = {uqaf024},
pmid = {41064460},
issn = {2633-6693},
abstract = {Solo-Cas4 homologs are Cas4-family proteins found outside of canonical CRISPR-Cas operons. Here, we present the biochemical characterization of Solo-Cas4 from Methanosarcina mazei Gö1. We found significantly upregulated solo-cas4 transcript levels during stationary phase, while remaining constant under oxygen exposure, temperature shifts, high salt conditions or virus challenge. Heterologously expressed as a SUMO-fusion, the purified tag-free protein displays an absorption peak at 420 nm, indicative of a [4Fe-4S]-cluster . Size-exclusion-chromatography revealed that Solo-Cas4 forms a higher oligomeric complex, with an apparent molecular mass of 318 kDa. In vitro nuclease activity assays demonstrated that Solo-Cas4 cleaves metal-dependent linear dsDNA, with highest cleavage activity in the presence of Mn[2+], followed by Mg[2+], while Ca[2+] and Cu[2+] result in negligible cleavage. Isoleucine169 was identified to be crucial for catalysis, mutating it to alanine completely abolished nuclease activity . Mutating any of the four conserved cysteines-proposed to coordinate the [4Fe-4S]-cluster did not affect nuclease activity; however, it abolishes metal cluster binding. Supercoiled circular dsDNA was preferentially nicked by Solo-Cas4 in the presence of Mg[2+], whereas Mn[2+] also led to linearization followed by complete degradation. Besides, ssDNA was cleaved by Solo-Cas4 but with lower activity. In agreement, Microscale thermophoresis analysis revealed strong dsDNA binding with highest affinity to supercoiled circular DNA, and weak ssDNA binding. Overall, these findings indicate that M. mazei Solo-Cas4 is a high oligomeric Cas4-family nuclease that preferentially targets supercoiled dsDNA and is upregulated during stationary growth.},
}
@article {pmid40988554,
year = {2025},
author = {Li, H and Gui, P and Li, X and Lin, Y and Ma, Z and Yu, H and Ma, F},
title = {CRISPR/Cas9-Mediated Construction of a YPS Gene-Deficient Komagataella phaffii Strain for Enhanced Expression of BIAP Ⅱ.},
journal = {Yeast (Chichester, England)},
volume = {42},
number = {8-10},
pages = {195-205},
doi = {10.1002/yea.70002},
pmid = {40988554},
issn = {1097-0061},
support = {//This work was supported by Youth Innovation Promotion Association Fellowship Program, CAS (2022327), Shandong Provincial Laboratory Project (SYS202209), Key Technologies R&amp;D Program of Guangdong Province (2022B1111050001), Natural Science Foundation of Shandong Province, China (ZR2021QB155)./ ; },
mesh = {Animals ; *CRISPR-Cas Systems ; *Alkaline Phosphatase/genetics/metabolism ; *Saccharomycetales/genetics/metabolism/enzymology ; Cattle ; *Aspartic Acid Endopeptidases/genetics/metabolism ; Recombinant Proteins/genetics/metabolism ; Gene Editing ; Proteolysis ; Gene Expression ; },
abstract = {Multiple isoforms of bovine intestinal alkaline phosphatase (BIAP) have been identified, among which type Ⅱ (BIAP Ⅱ) exhibits the highest specific activity. While Komagataella phaffii has been successfully employed for the secretory expression of recombinant BIAP Ⅱ, substantial proteolytic degradation during the secretion and expression processes has been observed, leading to reduced protein yield and challenging purification procedures. Our investigation demonstrates that the proteolytic cleavage of BIAP Ⅱ is predominantly mediated by secretory pathway proteases, particularly the aspartic protease yapsin (Yps), with Yps1 playing a crucial role. Genetic disruption of the YPS1 gene resulted in a remarkable 2.5-fold increase in BIAP Ⅱ production yield compared to the parental strain, accompanied by significantly reduced proteolytic degradation. Through detailed analysis, we have identified the Yps1 cleavage site within the BIAP Ⅱ peptide chain, located between Lys137 and Lys138. To further minimize BIAP Ⅱ proteolysis, we developed a YPS multigene-deficient engineered strain using CRISPR/Cas9-mediated triple gene editing technology. Additionally, we have established a novel dual-color quantitative PCR (DC-qPCR) method that enables rapid and precise determination of target gene dosage, thereby enhancing screening efficiency while reducing experimental errors associated with repeated sample processing. The strategies and methodologies developed in this study may serve as a valuable reference for optimizing the expression of various secretory heterologous proteins in Komagataella phaffii.},
}
@article {pmid40962170,
year = {2025},
author = {He, H and Huang, Z and Wen, F and Ge, N and Lin, X and Pan, J},
title = {PSPC1 knockout promotes radiosensitivity, inhibits EMT, and metastasis of nasopharyngeal carcinoma cells.},
journal = {Experimental cell research},
volume = {452},
number = {2},
pages = {114755},
doi = {10.1016/j.yexcr.2025.114755},
pmid = {40962170},
issn = {1090-2422},
mesh = {Humans ; *Radiation Tolerance/genetics ; *Nasopharyngeal Carcinoma/genetics/pathology/radiotherapy/metabolism ; Animals ; *Nasopharyngeal Neoplasms/pathology/genetics/radiotherapy ; Cell Line, Tumor ; *Epithelial-Mesenchymal Transition/genetics/radiation effects ; Cell Movement/genetics/radiation effects ; Apoptosis/genetics/radiation effects ; Mice ; Mice, Nude ; *RNA-Binding Proteins/genetics/metabolism ; Cell Proliferation/genetics ; *Nuclear Proteins/genetics/metabolism ; Gene Expression Regulation, Neoplastic/genetics ; Neoplasm Metastasis ; Mice, Inbred BALB C ; Gene Knockout Techniques ; CRISPR-Cas Systems ; },
abstract = {PURPOSE: Paraspeckle component 1 (PSPC1) is upregulated in numerous cancers and is associated with reduced patient survival rates. Our previous research indicated that elevated PSPC1 levels in nasopharyngeal carcinoma (NPC) are positively linked to radiation resistance and tumor metastasis, two primary clinical challenges in NPC treatment. However, the precise role of PSPC1 in radiation resistance and metastasis of NPC cells remains unclear. This study aimed to explore the molecular mechanisms by which PSPC1 influences radiation resistance and metastasis in NPC.<br><br>METHODS: Using the radiation-resistant R743 and radiosensitive CNE2 cell lines of NPC, we examined the impact of PSPC1 expression on post-radiation survival, cell cycle progression, apoptosis, migration, invasion, and tumor growth. CRISPR/Cas9 genome editing was employed to generate PSPC1 knockout (KO) lines in R743&#xa0;cells, while PSPC1 overexpression (pcD-PSPC1) was achieved in CNE2 cells via pcDNA3.1(+)-PSPC1 plasmid transfection.<br><br>RESULTS: PSPC1 knockout converted R743&#xa0;cells from radioresistant to radiosensitive, whereas PSPC1 overexpression decreased radiosensitivity in CNE2 cells. Cell cycle analysis revealed that PSPC1 KO arrested R743&#xa0;cells in the G2/M phase post-irradiation, while PSPC1 overexpression prevented G2/M phase arrest in CNE2 cells. PSPC1 KO increased irradiation-induced apoptosis in R743&#xa0;cells, whereas PSPC1 overexpression decreased it in CNE2 cells. Post-radiation, PSPC1 KO cells showed significantly reduced migration and invasion abilities. Bioinformatics analysis identified SFPQ as a PSPC1-interacting protein, with PSPC1 KO resulting in SFPQ downregulation. Additionally, PSPC1 KO enhanced the radiosensitivity of xenografted tumors in nude mice.<br><br>CONCLUSION: Our findings suggest that PSPC1 is a pivotal factor in enhancing the survival and spread of NPC cells post-radiation. Targeting PSPC1 or its downstream pathways could offer novel strategies to overcome radiation resistance and metastasis in NPC cells.},
}
@article {pmid40952014,
year = {2025},
author = {Llanga, T and Bush, K and Sun, Y and Yan, A and Zhou, J and Gorodkin, J and Sullenger, BA},
title = {Binding and Ligand Activation Driven Enrichment-Directed Evolution of SaCas9 gRNAs Improves Gene Editing Efficiency.},
journal = {Nucleic acid therapeutics},
volume = {35},
number = {5},
pages = {209-219},
doi = {10.1177/21593337251370553},
pmid = {40952014},
issn = {2159-3345},
mesh = {*Gene Editing/methods ; *RNA, Guide, CRISPR-Cas Systems/genetics/metabolism ; Staphylococcus aureus/genetics/enzymology ; *Directed Molecular Evolution/methods ; *CRISPR-Associated Protein 9/genetics/metabolism ; *CRISPR-Cas Systems/genetics ; Ligands ; Humans ; },
abstract = {Clustered regularly interspaced short palindromic repeats-based editing is inefficient at over two-thirds of genetic targets. A primary cause is ribonucleic acid (RNA) misfolding that can occur between the spacer and scaffold regions of the gRNA, which hinders the formation of functional Cas9 ribonucleoprotein (RNP) complexes. Here, we uncover hundreds of highly efficient gRNA variant scaffolds for Staphylococcus aureus (Sa)Cas9 utilizing an innovative binding and ligand activation driven enrichment (BLADE) methodology, which leverages asymmetrical product dissociation over rounds of evolution. SaBLADE-derived gRNA scaffolds contain 7%-42% of nucleotide variation relative to wild type. gRNA variants are able to improve gene editing efficiency at all targets tested, and they achieve their highest levels of editing improvement (>400%) at the most challenging DNA target sites for the wild-type SaCas9 gRNA. This arsenal of SaBLADE-derived gRNA variants showcases the power and flexibility of combinatorial chemistry and directed evolution to enable efficient gene editing at challenging, or previously intractable, genomic sites.},
}
@article {pmid40935921,
year = {2025},
author = {Roh, H and Shen, SP and Hu, Y and Kwok, HS and Siegenfeld, AP and Lee, C and Zepeda, MU and Guo, CJ and Roseman, SA and Comenho, C and Sankaran, VG and Buenrostro, JD and Liau, BB},
title = {Coupling CRISPR scanning with targeted chromatin accessibility profiling using a double-stranded DNA deaminase.},
journal = {Nature methods},
volume = {22},
number = {10},
pages = {2083-2093},
pmid = {40935921},
issn = {1548-7105},
support = {R35GM153476//U.S. Department of Health &amp; Human Services | National Institutes of Health (NIH)/ ; F31HL174076//U.S. Department of Health &amp; Human Services | National Institutes of Health (NIH)/ ; R01DK103794//U.S. Department of Health &amp; Human Services | National Institutes of Health (NIH)/ ; DGE1745303//National Science Foundation (NSF)/ ; },
mesh = {Humans ; *Chromatin/genetics/metabolism ; Hematopoietic Stem Cells/metabolism/cytology ; *Gene Editing/methods ; *CRISPR-Cas Systems ; *Cytidine Deaminase/genetics/metabolism ; *Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {Genome editing enables sequence-function profiling of endogenous cis-regulatory elements, driving understanding of their mechanisms. However, these approaches lack direct, scalable readouts of chromatin accessibility across long single-molecule chromatin fibers. Here we leverage double-stranded DNA cytidine deaminases to profile chromatin accessibility at endogenous loci of interest through targeted PCR and long-read sequencing, a method we term targeted deaminase-accessible chromatin sequencing (TDAC-seq). With high sequence coverage at targeted loci, TDAC-seq can be integrated with CRISPR perturbations to link genetic edits and their effects on chromatin accessibility on the same single chromatin fiber at single-nucleotide resolution. We employed TDAC-seq to parse CRISPR edits that activate fetal hemoglobin in human CD34[+] hematopoietic stem and progenitor cells (HSPCs) during erythroid differentiation as well as in pooled CRISPR and base-editing screens tiling an enhancer controlling the globin locus. We further scaled the method to interrogate 947 variants in a GFI1B-linked enhancer associated with myeloproliferative neoplasm risk in a single pooled CRISPR experiment in CD34[+] HSPCs. Together, TDAC-seq enables high-resolution sequence-function mapping of single-molecule chromatin fibers by genome editing.},
}
@article {pmid40921108,
year = {2026},
author = {Zhang, Z and Wang, J and Li, C and Sun, H and Bu, S and Jia, Q and Wan, Y and Zhao, Y and Zhou, H and Hao, Z and Li, N and Yu, S and Wang, L and Wan, J},
title = {An ultrasensitive biosensor for H1N1 virus coupled with 3D spherical DNA nanostructure and CRISPR-Cas12a.},
journal = {Spectrochimica acta. Part A, Molecular and biomolecular spectroscopy},
volume = {346},
number = {},
pages = {126905},
doi = {10.1016/j.saa.2025.126905},
pmid = {40921108},
issn = {1873-3557},
mesh = {*Influenza A Virus, H1N1 Subtype/isolation &amp; purification/genetics ; *Biosensing Techniques/methods ; *Nanostructures/chemistry ; *CRISPR-Cas Systems ; Limit of Detection ; *DNA/chemistry ; Animals ; *CRISPR-Associated Proteins/metabolism/chemistry ; Humans ; Aptamers, Nucleotide/chemistry ; Cattle ; Milk/virology ; Chickens ; Bacterial Proteins ; Endodeoxyribonucleases ; },
abstract = {To achieve ultrasensitive and real-time detection of the H1N1 influenza virus, this study designed a nucleic acid-free fluorescent biosensor based on 3D spherical DNA nanostructure and CRISPR/Cas12a (3D-SDNC). The biosensor constructs a rigid 3D nano-framework via self-assembly of six oligonucleotide chains, with H1N1-specific nucleic acid aptamers and Cas12a activator strands strategically positioned at multi-spined vertices for precise spatial coupling between viral recognition and signal transduction. Upon aptamer-virus binding, the induced conformational change liberates the activator strand, thereby activating the trans-cleavage activity of the Cas12a/crRNA complex to efficiently cleave the HEX/BHQ1 double-labeled fluorescent probe and initiate cascade signal amplification. Experimental results demonstrate a detection limit of 0.17 copies/μL (S/N = 3), achieving qPCR-comparable sensitivity, with spike recovery rates of 91.89 % to 104.03 % (RSD < 5.12 %) in chicken serum, bovine serum, and milk matrices. The innovative nucleic acid-free extraction design reduces the total detection time to 40 min; its efficiency is three times higher than qPCR. Notably, we not only discovered the ultra-high sensitivity of the sensor to H1N1 but also unexpectedly found that the rigid structure of the 3D spherical DNA nanostructure conferred enhanced stability under storage conditions. This work establishes a groundbreaking molecular engineering paradigm for rapid pathogen diagnosis, combining ultrahigh sensitivity, fast response, and clinical utility.},
}
@article {pmid40886669,
year = {2026},
author = {Liu, W and Xu, L and Lyu, Y and Yang, C},
title = {MXene-integrated CRISPR/Cas12a biosensor with Split activators for direct and rapid fluorescent detection of MicroRNA.},
journal = {Spectrochimica acta. Part A, Molecular and biomolecular spectroscopy},
volume = {346},
number = {},
pages = {126850},
doi = {10.1016/j.saa.2025.126850},
pmid = {40886669},
issn = {1873-3557},
mesh = {*MicroRNAs/analysis/genetics ; *Biosensing Techniques/methods ; *CRISPR-Cas Systems ; Humans ; Limit of Detection ; Spectrometry, Fluorescence/methods ; *Endodeoxyribonucleases/metabolism ; DNA, Single-Stranded/chemistry ; Bacterial Proteins ; Nitrites ; Transition Elements ; CRISPR-Associated Proteins ; },
abstract = {Early and accurate cancer diagnosis is essential for reducing cancer-related mortality, and miRNA-21 has emerged as a critical biomarker for the early detection of various malignancies In this study, we developed a novel fluorescence biosensor, termed the MXene-SNA-Cas12a, that enables direct and amplification-free detection of miRNA-21 by integrating the CRISPR/Cas12a system with a chimeric split nucleic acid (SNA) activator and MXene-assisted fluorescence modulation. Specifically, a split activator comprising S12 ssDNA hybridized with miRNA-21 was employed to activate the trans-cleavage activity of Cas12a, effectively overcoming the system's inherent limitation in RNA recognition. Simultaneously, MXene nanosheets served as efficient quenchers by adsorbing FAM-labeled ssDNA reporters through non-covalent interactions and facilitating target-induced strand release, enabling a robust fluorescence "on/off" mechanism. This biosensor demonstrated excellent linearity over a miRNA-21 concentration range of 50 pM-25 nM, with a detection limit as low as 16 pM. It exhibited high specificity and strong resistance to interference, making it well-suited for complex biological environments. Moreover, the programmable nature of the split activator allows for easy adaptation to detect other RNA targets through rational sequence redesign, offering a versatile platform for CRISPR/Cas12a-based RNA diagnostics.},
}
@article {pmid40851311,
year = {2025},
author = {Kittock, CM and Karia, K and Kc, P and Evans, C and Wollman, J and Meyerink, BL and Pilaz, LJ},
title = {Modeling MPPH syndrome in vivo using Breasi-CRISPR.},
journal = {HGG advances},
volume = {6},
number = {4},
pages = {100497},
pmid = {40851311},
issn = {2666-2477},
mesh = {Animals ; Mice ; *CRISPR-Cas Systems ; Disease Models, Animal ; *Gene Editing/methods ; Humans ; *Hydrocephalus/genetics/pathology ; *Megalencephaly/genetics/pathology ; *Polydactyly/genetics/pathology ; Neural Stem Cells/metabolism ; },
abstract = {The increasing availability and affordability of genetic testing has resulted in the identification of numerous novel variants associated with neurodevelopmental disorders. There remains a need for methods to analyze the functional impact of these variants. Some methods, like expressing these variants in cell culture, may be rapid, but they lack physiologic context. Other methods, like making a whole-mouse model, may provide physiologic accuracy, but these are costly and time-consuming. We recently developed a technique, Breasi-CRISPR (Brain Easi-CRISPR), which results in efficient genome editing of neural precursor cells via electroporation of CRISPR-Cas9 reagents into developing mouse brains. Since Breasi-CRISPR is extremely rapid and enables the analysis of targeted genes in vivo, we wondered whether this technique would accelerate the study of monogenic neurodevelopmental disorders. Here, we use Breasi-CRISPR to model megalencephaly postaxial polydactyly polymicrogyria hydrocephalus (MPPH) syndrome. We found that 2 days after Breasi-CRISPR, we were able to see neurodevelopmental phenotypes known to be associated with MPPH syndrome, including increased cyclin D2 protein abundance and an increase in neural progenitor proliferation. Thus, Breasi-CRISPR can efficiently model MPPH syndrome and may be a powerful method to add to the toolbox of those investigating the functional impact of patient variants in neurodevelopmental disorders.},
}
@article {pmid40706707,
year = {2025},
author = {Davydova, S and Meccariello, A},
title = {Engineering new clustered regularly interspaced short palindromic repeats-mediated population control for tephritid pests.},
journal = {Current opinion in insect science},
volume = {72},
number = {},
pages = {101415},
doi = {10.1016/j.cois.2025.101415},
pmid = {40706707},
issn = {2214-5753},
mesh = {Animals ; *Tephritidae/genetics ; *Pest Control, Biological/methods ; *CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Female ; Male ; Population Control ; },
abstract = {Tephritid fruit flies threaten the agricultural industry with a rising intensity on a worldwide scale. The application of clustered regularly interspaced short palindromic repeats (CRISPR) in insects has resulted in a current boost of CRISPR studies in tephritid pests. One of the primary pathways toward more efficient population management lies in genetic improvements to the Sterile Insect Technique. Herein, we review the pivotal advances in CRISPR application in non-model tephritid fruit flies in recent years. This consists of proof-of-principle studies to optimise CRISPR tools, applications for female elimination and male sterility, and the existing CRISPR-based systems for population control.},
}
@article {pmid40658346,
year = {2025},
author = {Uchigashima, M and Mikuni, T},
title = {Single-cell endogenous protein labeling via CRISPR-Cas9-mediated genome editing in the mouse brain.},
journal = {Anatomical science international},
volume = {100},
number = {4},
pages = {579-590},
pmid = {40658346},
issn = {1447-073X},
support = {20H05914//Japan Society for the Promotion of Science/ ; 20H05918//Japan Society for the Promotion of Science/ ; 23K18160//Japan Society for the Promotion of Science/ ; 24K02130//Japan Society for the Promotion of Science/ ; 22K21353//Japan Society for the Promotion of Science/ ; 23H04672//Japan Society for the Promotion of Science/ ; 23H02574//Japan Society for the Promotion of Science/ ; 23K27265//Japan Society for the Promotion of Science/ ; 24H01229//Japan Society for the Promotion of Science/ ; 24K22000//Japan Society for the Promotion of Science/ ; 25H02490//Japan Society for the Promotion of Science/ ; JP19dm0207080//Japan Agency for Medical Research and Development/ ; JP24wm0625117//Japan Agency for Medical Research and Development/ ; JPMJFR231M//Fusion Oriented REsearch for disruptive Science and Technology/ ; JPMJPR16F9//Precursory Research for Embryonic Science and Technology/ ; CDA00043/2019-C//Human Frontier Science Program/ ; },
mesh = {Animals ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; Mice ; *Brain/metabolism/cytology ; *Single-Cell Analysis/methods ; Electroporation ; Somatosensory Cortex/cytology/metabolism ; Pyramidal Cells/metabolism ; },
abstract = {High-precision mapping of endogenous proteins is essential for understanding the molecular mechanism underlying neuronal functions in the brain. The SLENDR (single-cell labeling of endogenous proteins by clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9-mediated homology-directed repair) technique provides single-cell endogenous protein labeling with genetically encoded tags within the mammalian brain through precise genome editing via homology-directed repair (HDR). This technique is based on the introduction of HDR-mediated genome editing into neuronal progenitors in embryonic brains by in utero electroporation. Subsequent histological analyses enable high-resolution interrogation of the subcellular distribution of endogenous proteins within a single neuron using conventional fluorescent microscopy. Here, we describe a step-by-step protocol for the SLENDR technique to label endogenous proteins with genetically encoded tags in single pyramidal cells of the mouse primary somatosensory cortex. This protocol would be helpful to visualize the molecular organization underlying biological processes at single-neuron levels in the brain, such as signal processing from synaptic inputs to neuronal outputs across different scales.},
}
@article {pmid41063348,
year = {2025},
author = {Newman, A and Saha, A and Starrs, L and Arantes, PR and Palermo, G and Burgio, G},
title = {CRISPR-Cas12a REC2-Nuc interactions drive target-strand cleavage and constrain trans cleavage.},
journal = {Nucleic acids research},
volume = {53},
number = {18},
pages = {},
doi = {10.1093/nar/gkaf988},
pmid = {41063348},
issn = {1362-4962},
support = {//National Health and Medical Research Council/ ; //The Gordon and Gretel Bootes/ ; //National Computing Infrastructure/ ; //NCRIS/ ; //Australian Government Research Training Program/ ; R01GM141329/NH/NIH HHS/United States ; CHE-2144823//National Science Foundation/ ; 2138259//National Science Foundation/ ; 2138286//National Science Foundation/ ; 2138307//National Science Foundation/ ; 2137603//National Science Foundation/ ; 2138296//National Science Foundation/ ; FG-2023-20431//Alfred P. Sloan Foundation/ ; TC-24-063//Camille and Henry Dreyfus Foundation/ ; },
mesh = {*CRISPR-Cas Systems ; *DNA Cleavage ; Humans ; *CRISPR-Associated Proteins/metabolism/genetics/chemistry ; Escherichia coli/genetics ; *Endodeoxyribonucleases/metabolism/genetics/chemistry ; Gene Editing ; Molecular Dynamics Simulation ; DNA/metabolism/chemistry/genetics ; *Bacterial Proteins/genetics/metabolism/chemistry ; Kinetics ; DNA, Single-Stranded/metabolism/genetics ; Mutation ; },
abstract = {CRISPR-Cas12a mediates RNA-guided cleavage of double-stranded DNA in cis, after which it remains catalytically active and non-specifically cleaves single-stranded DNA in trans. Native host defence by Cas12a employs cis cleavage, which can be repurposed for the genome editing of other organisms, and trans cleavage can be used for in vitro DNA detection. Cas12a orthologues have high structural similarity and a conserved mechanism of DNA cleavage, yet highly different efficacies when applied for genome editing or DNA detection. By comparing three well-characterized Cas12a orthologues (FnCas12a, LbCas12a, and AsCas12a), we sought to determine what drives their different cis and trans cleavage and how this relates to their applied function. We integrated in vitro DNA cleavage kinetics with molecular dynamics simulations, plasmid interference in Escherichia coli, and genome editing in human cell lines. We report large differences in cis cleavage kinetics between orthologues, which may be driven by dynamic REC2-Nuc interactions. We generated and tested REC2 and Nuc mutants, including a hitherto unstudied 'Nuc-loop', integrity of which is critical for the function of Cas12. In total, our in vitro, in vivo, and in silico survey of Cas12a orthologues highlights key properties that drive their function in biotechnology applications.},
}
@article {pmid41062879,
year = {2025},
author = {Paul, K and Raman K, V and Baaniya, M and Jadhav, I and Bhattacharjee, S and Tilgam, J and Saakre, M and Kumari, P and Das, S and Vijayan, J and Sreevathsa, R and Pattanayak, D},
title = {A novel recombinant CRISPR/Cas9 vector system for genome editing in plants.},
journal = {Transgenic research},
volume = {34},
number = {1},
pages = {45},
pmid = {41062879},
issn = {1573-9368},
mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; *Genetic Vectors/genetics ; Solanum tuberosum/genetics ; *Plants, Genetically Modified/genetics/growth &amp; development ; Genome, Plant ; Arabidopsis/genetics ; Nicotiana/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; Promoter Regions, Genetic ; },
abstract = {Genome editing employing CRISPR/Cas9 systems has found widespread applications for knocking out targeted genes. In spite of exponential applications in plants for trait improvement, low editing efficiency in plants is a major concern. We report construction of a pCAMBIA2300 based binary vector cassette (pCR) harbouring novel recombinant CRISPR/Cas9 system for efficient genome editing in plants. The Cas9 cDNA with sequence encoding nuclear localization signals at the N-terminal and C-terminal ends had been codon optimized for better expression in plants. Undesirable internal restriction sites were removed. Small stretch of 5' UTR sequence of Rubisco small subunit (rcbS) of potato, harbouring in between potato granule bound starch synthase (GBSS) intron, was added at the 5' end of the Cas9 cDNA to function as 5' UTR. The recombinant Cas9 gene (rdCas9) was placed under the transcriptional control of CaMV 35S promoter and NOS terminator. The single guide RNA cassette (sgRNA) was comprised of Arabidopsis U6 promoter, 20-21 nucleotide (nt) spacer sequence, sgRNA scaffold sequence and potato U6 RNA Pol-III termination sequence. The 20-21 nt sgRNA spacer sequence could be added to the sgRNA construct by AarI or PaqCI digestion. The sgRNA construct had been designed in such a way so that single or multiplexed sgRNA could be cloned into the pCR vector cassette in a single step. Moreover, modular nature of this vector system can help to derive different combination of promoter, terminator with Cas9 and sgRNA constructs. The efficacy of the pCR vector system had been validated in Nicotiana tabacum and Solanum tuberosum by knocking out phytoene desaturase gene (PDS), through Agrobacterium-mediated transformation. The pCR binary vector system can be utilized as a versatile tool box for efficient genome editing of plant to improve agriculturally important traits.},
}
@article {pmid41062549,
year = {2025},
author = {Sofras, D and Carolus, H and Subotić, A and Lobo Romero, C and Ennis, CL and Cuypers, L and Lagrou, K and Hernday, AD and Nobile, CJ and Rybak, JM and Van Dijck, P},
title = {A systematic comparison of CRISPR-Cas9 allele editing in Candida auris demonstrates unreliable cassette integration and effective episomal plasmid-based editing.},
journal = {Scientific reports},
volume = {15},
number = {1},
pages = {35105},
pmid = {41062549},
issn = {2045-2322},
support = {11J8122N//Fonds Wetenschappelijk Onderzoek/ ; 11D7620N//Fonds Wetenschappelijk Onderzoek/ ; G0L1622N//Joint Programming Initiative on Antimicrobial Resistance/ ; G0L1622N//Joint Programming Initiative on Antimicrobial Resistance/ ; G0L1622N//Joint Programming Initiative on Antimicrobial Resistance/ ; G0L1622N//Joint Programming Initiative on Antimicrobial Resistance/ ; C3/22/007//Onderzoeksraad, KU Leuven/ ; PDMT2/23/032//Onderzoeksraad, KU Leuven/ ; C3/22/007//Onderzoeksraad, KU Leuven/ ; C3/22/007//Onderzoeksraad, KU Leuven/ ; C3/22/007//Onderzoeksraad, KU Leuven/ ; C3/22/007//Onderzoeksraad, KU Leuven/ ; R35GM124594/GM/NIGMS NIH HHS/United States ; R35GM124594/GM/NIGMS NIH HHS/United States ; F31DE028488/DE/NIDCR NIH HHS/United States ; },
mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems ; *Plasmids/genetics ; *Candida auris/genetics ; Alleles ; Genome, Fungal ; RNA, Guide, CRISPR-Cas Systems/genetics ; },
abstract = {Candidozyma (Candida) auris is an emergent fungal pathogen of significant interest for molecular research. A handful of CRISPR-Cas9 based allele editing tools have been optimized for C. auris. Nonetheless, allele editing in this species remains a significant challenge, and different systems have different advantages and disadvantages. In this work, we compare four systems to introduce the genetic elements necessary for the production of Cas9 and the guide RNA molecule in the genome of C. auris, replacing the ENO1, LEU2 and HIS1 loci respectively, while the fourth system makes use of an episomal plasmid. We observed that the editing efficiency of all four systems was significantly different and strain-dependent. However, we did not detect correct integration of linear CRISPR cassette constructs in integration-based systems, in over 4,900 screened transformants. Still, all transformants, whether correctly edited or not, grew on selective nourseothricin media, suggesting ectopic integration of the CRISPR cassette, which was confirmed by long-read whole genome sequencing. The plasmid-based system showed the highest editing efficiency with an average of 41.9% correct transformants, despite yielding fewer transformants compared to the other systems. Transformation of protoplasts or silencing the non-homologous end joining (NHEJ) DNA repair pathway, by deleting two main NHEJ factors, KU70 and LIG4, did not improve the editing efficiency. While our research highlights important challenges in precise genome editing of C. auris by quantitatively evaluating the editing and targeting efficiencies of different methods, it also clearly shows the safety and usefulness of plasmid-based systems like EPIC, which we recommend for molecular work in this enigmatic fungal pathogen.},
}
@article {pmid41062528,
year = {2025},
author = {Smidler, AL and Marrogi, EA and Scott, S and Mameli, E and Abernathy, D and Akbari, OS and Church, GM and Catteruccia, F and Esvelt, K},
title = {Engineering gene drive docking sites in a haplolethal locus in Anopheles gambiae.},
journal = {Scientific reports},
volume = {15},
number = {1},
pages = {35074},
pmid = {41062528},
issn = {2045-2322},
support = {AI120480-02//Foundation for the National Institutes of Health/ ; R01 AI104956/AI/NIAID NIH HHS/United States ; R01 AI104956/AI/NIAID NIH HHS/United States ; R01AI151004//Foundation for the National Institutes of Health/ ; R01 AI104956/AI/NIAID NIH HHS/United States ; R00-DK102669-04//Foundation for the National Institutes of Health/ ; IRSA1016432//Burroughs Wellcome Fund/ ; IRSA1016432//Burroughs Wellcome Fund/ ; IRSA1016432//Burroughs Wellcome Fund/ ; OPP1158190/HHMI/Howard Hughes Medical Institute/United States ; },
mesh = {*Anopheles/genetics ; Animals ; *Gene Drive Technology/methods ; Proteasome Endopeptidase Complex/genetics ; *Genetic Engineering/methods ; Mosquito Vectors/genetics ; *Genetic Loci ; CRISPR-Cas Systems ; },
abstract = {Gene drives are selfish genetic elements which promise to be powerful tools in the fight against vector-borne diseases such as malaria. We previously proposed population replacement gene drives designed to better withstand the evolution of resistance by homing through haplolethal loci. Because most mutations in the wild-type allele that would otherwise confer resistance are lethal, only successful drive homing and functional r1 alleles permits the cell to survive. Here we outline the development and characterization of two ΦC31-Recombination mediated cassette exchange gene drive docking lines with these features in Anopheles gambiae, a first step towards construction of robust gene drives in this important malaria vector. We outline adaption of the technique HACK (Homology Assisted CRISPR knockin) to knock-in two docking site sequences into a paired putative haplolethal-haplosufficient (Ribosome-Proteasome) locus, and confirm that these docking lines permit insertion of drive-relevant transgenes. We report the first anopheline proteasome knockouts, and identify ribosome mutants in the process reveal a major lethality and infertility hurdle that such designs must overcome to develop robust drives in the future. Although we do not achieve drive, this work provides a new tool for constructing future evolution-robust drive systems and reveals critical challenges that must be overcome for development of future gene drives designed to target haplolethal loci in anophelines and, potentially, other metazoans.},
}
@article {pmid41062469,
year = {2025},
author = {Han, J and Min, Y and Hu, L and Chen, JJ and Zhang, SX and Li, XF and Cheng, ZH and Liu, DF and Yu, HQ},
title = {Tailoring Cas12a functionality with a user-friendly and versatile crRNA variant toolbox.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {8939},
pmid = {41062469},
issn = {2041-1723},
support = {52322002//National Natural Science Foundation of China (National Science Foundation of China)/ ; },
mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; *CRISPR-Associated Proteins/genetics/metabolism ; Mutation ; *Endodeoxyribonucleases/genetics/metabolism ; *Bacterial Proteins/genetics/metabolism ; Escherichia coli/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; },
abstract = {Cas12a, with its unique targeting and cleavage activity towards DNA, has been widely applied in gene editing and molecular diagnostics. However, there currently lacks an activity regulation strategy that combines flexibility and simplicity to adapt Cas12a to different demands across various application scenarios. In this study, we present a simple yet effective strategy, wherein we systematically mutate the crRNA direct repeat (DR) sequence to uncover a range of distinct crRNA mutants, which are then compiled into a crRNA toolbox to enable flexible regulation of Cas12a activity. By harnessing the complementarity and synergy between these mutants, we successfully enhance Cas12a performance across various application scenarios. Our crRNA toolbox enables fine-tuned control over expression levels, improves base editing accuracy, enhances transformation and editing efficiency in prokaryote homologous recombination-mediated gene editing, and facilitates rapid, accurate, one-pot, semi-quantitative nucleic acid diagnostics. In summary, the DR sequence mutation strategy provides simple, flexible, and diverse options for Cas12a activity regulation and functional optimization.},
}
@article {pmid41061057,
year = {2025},
author = {Oonuma, K and Kuroda, R and Uchida, T and Zhang, SM},
title = {CRISPR/Cas9-germline editing of Biomphalaria glabrata: A breakthrough in genetic modification of snails that transmit schistosomiasis.},
journal = {Science advances},
volume = {11},
number = {41},
pages = {eadx5889},
pmid = {41061057},
issn = {2375-2548},
mesh = {Animals ; *Biomphalaria/genetics/parasitology ; *CRISPR-Cas Systems ; *Gene Editing/methods ; Schistosoma mansoni ; *Schistosomiasis/transmission/parasitology ; Humans ; },
abstract = {Human schistosomiasis remains one of the most devastating parasitic diseases worldwide, and the development of genetically modified vector snails has long been a goal in the field. Here, we report the successful creation of genetically modified Biomphalaria glabrata, an important intermediate host, using CRISPR/Cas9 gene editing. We targeted the fibrinogen-related protein 3.1 (FREP3.1) gene, confirmed stable germline transmission of the mutated gene, and established two different homozygous FREP3.1-edited lines. Disruption of the FREP 3.1 gene did not alter snail susceptibility to Schistosoma mansoni infection, possibly due to a limited role of FREP3.1 in resistance or to functional redundancy and/or compensatory expression within the highly diverse FREP gene family. Our study demonstrates successful germline editing, effective ex ovo culture of decapsulated embryos, and the generation of viable, genetically modified B. glabrata snails, thereby establishing a foundation for future genetic strategies to control schistosomiasis.},
}
@article {pmid41060691,
year = {2025},
author = {Gao, F and Colles, FM and Ko, S and Luo, J and Sheppard, SK and Chen, M},
title = {Genomic epidemiology and the evolution of erm(B)-mediated macrolide resistance in Campylobacter.},
journal = {Microbial genomics},
volume = {11},
number = {10},
pages = {},
doi = {10.1099/mgen.0.001528},
pmid = {41060691},
issn = {2057-5858},
mesh = {*Campylobacter/genetics/drug effects/isolation &amp; purification/classification ; *Macrolides/pharmacology ; Humans ; *Anti-Bacterial Agents/pharmacology ; Phylogeny ; Animals ; *Campylobacter Infections/epidemiology/microbiology ; *Drug Resistance, Bacterial/genetics ; China/epidemiology ; *Methyltransferases/genetics ; Genome, Bacterial ; Microbial Sensitivity Tests ; *Bacterial Proteins/genetics ; Gene Transfer, Horizontal ; Poultry/microbiology ; Evolution, Molecular ; },
abstract = {Campylobacter is a major foodborne bacterial pathogen that has become increasingly resistant to clinically important antimicrobials. Of particular concern is the emergence of erm(B)-mediated macrolide resistance, which has been increasingly documented across Campylobacter isolates from diverse ecological reservoirs. In this study, we investigated the genomic characteristics and epidemiology of erm(B)-carrying clinical Campylobacter isolates from Shanghai, alongside a globally representative dataset of all publicly available strains. Among clinical isolates obtained from a diarrhoeal outpatient surveillance programme between 2020 and 2023 in Shanghai, China, 16% (80/500) were erythromycin-resistant, with 23.8% (19/80) testing positive for erm(B). The genomes of these isolates were sequenced to identify erm(B) gene alleles. Phylogenetic analyses, pairwise comparisons of core and accessory genomes and examination of shared alleles revealed horizontal gene transfer as the predominant mechanism driving the transmission of erm(B) between isolates from various sources. Poultry was identified as a key reservoir for human infections caused by erm(B)-positive Campylobacter isolates. Comparative pangenome analyses of erm(B)-positive and negative isolates identified multiple accessory elements associated with erm(B) acquisition, among which the IS607 family transposon-associated tnpB gene exhibited sequence and structural homology to functional progenitors of CRISPR-Cas nucleases. These findings expand our understanding of the epidemiology of erm(B)-mediated macrolide resistance in Campylobacter and underscore the urgent need for enhanced antimicrobial stewardship in poultry production and targeted surveillance programmes to curb the spread of resistance.},
}
@article {pmid41042795,
year = {2025},
author = {Shen, Y and Fan, K and Gökbağ, B and Sun, N and Yang, C and Cheng, L and Li, L},
title = {A multi-layer encoder prediction model for individual sample specific gene combination effect (MLEC-iGeneCombo).},
journal = {PLoS computational biology},
volume = {21},
number = {10},
pages = {e1013547},
doi = {10.1371/journal.pcbi.1013547},
pmid = {41042795},
issn = {1553-7358},
mesh = {Humans ; Gene Knockout Techniques ; CRISPR-Cas Systems/genetics ; Computational Biology/methods ; Deep Learning ; *Models, Genetic ; Systems Biology/methods ; RNA, Guide, CRISPR-Cas Systems/genetics ; },
abstract = {Using data from gene combination double knockout (CDKO) experiments, top ranked synthetic lethal (SL) gene pairs were highly inconsistent among different SL scores. This leads to a significant concern that SL prediction models highly depend on SL scores. In this paper, we introduce a new gene combination effect (GCE) measurement, log-fold change of dual-gRNA expression before and after CRISPR-cas9 lentivirus transfection. We show it is a direct and highly consistent measurement of GCE in all CDKO experiments. We therefore develop a multi-layer encoder model for individual sample specific GCE prediction, MLEC-iGeneCombo. Under a deep learning framework, MLEC-iGeneCombo is a systems biology model that contains sample specific multi-omics encoder, network encoder and cell-line encoder. For the first time, MLEC-iGeneCombo predicts GCE for a new cell. Using data from 18 CDKO experiments, MLEC-iGeneCombo achieves an average GCE prediction performance, 71.9%. All three encoders significantly improve the model's prediction performance (p[Formula: see text]), and their combined use yields the best GCE prediction performance. Our source code is available at https://github.com/karenyun/MLEC-iGeneCombo.},
}
@article {pmid40930101,
year = {2025},
author = {Vučićević, D and Hsu, CW and Lopez Zepeda, LS and Burkert, M and Hirsekorn, A and Bilić, I and Kastelić, N and Landthaler, M and Lacadie, SA and Ohler, U},
title = {Sensitive dissection of a genomic regulatory landscape using bulk and targeted single-cell activation.},
journal = {Cell genomics},
volume = {5},
number = {10},
pages = {100984},
doi = {10.1016/j.xgen.2025.100984},
pmid = {40930101},
issn = {2666-979X},
mesh = {Humans ; *Single-Cell Analysis/methods ; Transcription Factors/genetics/metabolism ; Neuroblastoma/genetics ; Homeodomain Proteins/genetics ; Gene Regulatory Networks/genetics ; Enhancer Elements, Genetic/genetics ; CRISPR-Cas Systems/genetics ; *Genomics/methods ; Cell Line, Tumor ; },
abstract = {Enhancers are known to spatiotemporally regulate gene transcription, yet the identification of enhancers and their target genes is often indirect, low resolution, and/or assumptive. To identify and functionally perturb enhancers at their endogenous sites, we performed a pooled tiling CRISPR activation (CRISPRa) screen surrounding PHOX2B, a master regulator of neuronal cell fate and a key player in neuroblastoma, and found many CRISPRa-responsive elements (CaREs) that alter cellular growth. To determine CaRE target genes, we developed TESLA-seq (targeted single-cell activation), which combines CRISPRa screening with targeted single-cell RNA sequencing and enables the parallel readout of the effect of hundreds of enhancers on all genes in the locus. While most TESLA-revealed CaRE-gene relationships involved neuroblastoma-related regulatory elements, we found many CaREs and target connections normally active only in other tissues. This highlights the power of TESLA-seq to reveal gene regulatory networks, including edges active outside of a given experimental system.},
}
@article {pmid40888415,
year = {2025},
author = {Zhao, T and Yu, L and Yin, M and Huang, S and Tian, R and Zhong, C and Nan, F and Zhang, H and Tian, X and Hu, Z},
title = {Enhanced One-Pot Cas12a-Based Nucleic Acid Detection via Epitope Insertion and Recruitment of Rad51.},
journal = {Small (Weinheim an der Bergstrasse, Germany)},
volume = {21},
number = {40},
pages = {e02417},
doi = {10.1002/smll.202502417},
pmid = {40888415},
issn = {1613-6829},
support = {32171465//National Natural Science Foundation of China/ ; 32371541//National Natural Science Foundation of China/ ; 82102392//National Natural Science Foundation of China/ ; 82172584//National Natural Science Foundation of China/ ; 2023M734091//China Postdoctoral Science Foundation/ ; 2023M734090//China Postdoctoral Science Foundation/ ; 2023M744121//China Postdoctoral Science Foundation/ ; 2024BCB057//Key Technology R&amp;D Program of Hubei/ ; 0820250//Guangdong Special Support Plan Young Top-notch Talent/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; *Epitopes/metabolism ; *Rad51 Recombinase/metabolism ; Humans ; *Nucleic Acids/analysis ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {The CRISPR-Cas12a system has emerged as a promising tool for nucleic acid-based diagnostics. However, its multi-step workflow and limited sensitivity hinder its integration into point-of-care testing (POCT). Here, the ECOT system (Engineered Cas12a for One-pot Test), a novel approach that combines protein engineering with one-pot detection, offering high sensitivity, specificity, and rapid response is introduced. By introducing GCN4 epitope insertions into LtCas12a and LbCas12a variants, their cis-cleavage activity, promoting efficient accumulation of amplification products is reduced. Additionally, the inclusion of scFv-Rad51 (single-chain variable fragment-Rad51) enhances Cas12a's trans-cleavage activity, amplifying signal intensity. The ECOT-Lb system demonstrated superior sensitivity in detecting low-copy HPV DNA samples, outperforming traditional qPCR in clinical tests. Achieving detection limits as low as 3 copies in under 30 min, the ECOT-Lb system is well-suited for home-based self-testing and widespread clinical diagnostics. This work provides a versatile and scalable protein engineering strategy that enhances the performance of CRISPR-based diagnostic tools, offering a promising platform for rapid molecular detection in diverse applications.},
}
@article {pmid40811280,
year = {2025},
author = {Chen, H and Jiang, Z},
title = {CrisprDA: A Data Augmentation Method Enhancing the Efficiency of sgRNA on-Target Activity Prediction.},
journal = {IEEE transactions on computational biology and bioinformatics},
volume = {22},
number = {5},
pages = {2313-2319},
doi = {10.1109/TCBBIO.2025.3591871},
pmid = {40811280},
issn = {2998-4165},
mesh = {*RNA, Guide, CRISPR-Cas Systems/genetics/metabolism ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; Humans ; *Computational Biology/methods ; *Deep Learning ; Neural Networks, Computer ; },
abstract = {The CRISPR/Cas9 system has emerged as a revolutionary technology in genome editing, yet the efficiency of this system is often limited by the activity level of single-guide RNAs (sgRNAs). In recent years, deep learning models have been increasingly utilized to predict sgRNA targeting activity. Notably, data scarcity rather than model architecture has become the predominant bottleneck in accurately predicting sgRNA activity. To overcome this challenge and enhance the performance of deep learning models, we propose Automix, a straightforward yet effective data augmentation method grounded in autoencoder technology. This method is complemented by CNLC (Confidence-based Nearest Label Correction), a pseudo-label correction technique designed to improve both the quality and quantity of training data. Additionally, we develop CrisprDA, a novel parallel architecture that integrates convolutional neural networks (CNNs) with attention mechanisms, for the precise prediction of sgRNA activity. Comprehensive experiments conducted on nine high-throughput datasets and eight functional datasets demonstrate that CrisprDA outperforms five compared methods, showing its superior predictive ability. Moreover, the application of Automix and CNLC to other comparative methods in our experiments further validates the effectiveness and generalizability of the proposed data augmentation strategy.},
}
@article {pmid41058711,
year = {2025},
author = {Barraclough, A and Bär, I and van Duijl, T and Fijnvandraat, K and Eikenboom, JCJ and Leebeek, FWG and Bierings, R and Voorberg, J and Trasanidou, D},
title = {Rewriting the script: gene therapy and genome editing for von Willebrand Disease.},
journal = {Frontiers in genome editing},
volume = {7},
number = {},
pages = {1620438},
pmid = {41058711},
issn = {2673-3439},
abstract = {In recent years gene therapy has emerged as a powerful technology for treatment of a large variety of inherited disorders. With the FDA approval of in vivo gene therapy of hemophilia A and B using AAV-mediated transgene delivery to hepatocytes, the path towards a new treatment era seemed paved. Also, CRISPR-Cas based approaches have reached the clinic, as in the ex vivo treatment of hematopoietic stem cells for sickle cell disease and thalassemia patients. The question arises whether these innovative strategies will also be suitable for patients with von Willebrand Disease (VWD). Whilst in and ex vivo delivery to endothelial cells (ECs) has been demonstrated, and CRISPR-Cas9 gene editing has been successful in ECs, there are currently no gene therapy options available for VWD. The wide variety of pathogenic VWF mutations makes development of broadly applicable, cost-effective gene therapies challenging. While delivery of von Willebrand factor (VWF) as a therapeutic transgene would be optimal, the size of VWF challenges efficient delivery. Therefore, treatment of VWD requires targeted, personalized gene therapy; for instance by using the newest CRISPR-Cas technologies which can be tailored to facilitate alteration and restoration of various pathogenic VWD variants. This review describes the inherited bleeding disorder VWD and potential gene therapy approaches for management of the disease. Thereby we are exploring different CRISPR-Cas technologies and recent developments in the field. Moreover, we will discuss the ongoing advances of in vivo delivery systems, all with the scope on ECs.},
}
@article {pmid41057395,
year = {2025},
author = {Osgood, JA and Brown, AC and Burnham, KL and Mielczarek, O and Migliorini, G and Tay, C and Zhang, P and Palmer, MH and Davies, B and Cowley, SA and Knight, JC},
title = {Evidence for enhancer activity in intron 1 of TNFRSF1A using CRISPR/Cas9 in human induced pluripotent stem cell-derived macrophages.},
journal = {Scientific reports},
volume = {15},
number = {1},
pages = {34885},
pmid = {41057395},
issn = {2045-2322},
support = {20773/VAC_/Versus Arthritis/United Kingdom ; 204969/Z/16/Z/WT_/Wellcome Trust/United Kingdom ; 2018-I2M-2-002//Chinese Academy of Medical Sciences Innovation 537 Fund for Medical Science/ ; LC0910-004//Oxford Martin School, University of Oxford/ ; },
mesh = {Humans ; *CRISPR-Cas Systems ; *Induced Pluripotent Stem Cells/cytology/metabolism ; *Macrophages/metabolism/cytology ; *Introns/genetics ; *Enhancer Elements, Genetic ; *Receptors, Tumor Necrosis Factor, Type I/genetics ; Gene Editing ; Cell Differentiation ; Genome-Wide Association Study ; Gene Expression Regulation ; },
abstract = {TNFα is a common drug target in the treatment of autoimmune diseases, with pro-inflammatory functions that are primarily mediated through its receptor, TNFRSF1A. TNFRSF1A has been genetically associated with many immune-mediated diseases including ankylosing spondylitis, multiple sclerosis, and inflammatory bowel disease. Many of the genetic variants within or near TNFRSF1A that have been associated with disease through genome-wide association studies (GWAS) lie in non-coding regions of the genome. Understanding the functional consequences of these genetic variants is limited by incomplete understanding of TNFRSF1A gene regulation, including for specific cellular contexts relevant to inflammation and immunity such as macrophages. This work used CRISPR/Cas9 in human induced pluripotent stem cells followed by differentiation into macrophages to investigate putative regulatory elements in the TNFRSF1A gene locus. Through gene editing, with functional genomic readouts including the assay for transposase-accessible chromatin using sequencing (ATAC-Seq), chromatin immunoprecipitation with sequencing (ChIP-Seq), and RNA-Seq to assess the consequences of these edits, we present evidence for an enhancer of TNFRSF1A contained within an intron of the gene. Understanding gene regulation and the genomic context in which GWAS variants lie could bring us closer to deconvoluting the genetic basis of common disease aetiology and uncover effective drug targets.},
}
@article {pmid41057263,
year = {2025},
author = {Yang, B and Wang, K},
title = {CRISPR-Cas-Directed Genome Editing in Maize.},
journal = {Cold Spring Harbor protocols},
volume = {},
number = {},
pages = {},
doi = {10.1101/pdb.top108448},
pmid = {41057263},
issn = {1559-6095},
abstract = {Genetic engineering techniques are essential for both plant science and agricultural biotechnology, enabling functional genomics studies, dissection of complex traits, and targeted crop improvement. Among the various genetic tools currently in use, clustered regularly interspaced short palindromic repeats-CRISPR-associated protein (CRISPR-Cas)-based genome editing has emerged as a transformative technology due to its precision, versatility, and ease of use. In particular, CRISPR-Cas9 has become the most widely adopted platform for genome manipulation in plant systems, including maize, owing to its high editing efficiency, multiplexing capabilities, and scalability for diverse applications. This review highlights the biological significance and technical considerations necessary to implement CRISPR-Cas9 in maize. We discuss critical components for successful editing, including the selection of strong and tissue-appropriate promoters for Cas gene and guide RNA expression, codon optimization of Cas nuclease genes, effective guide RNA design, and multiplexing strategies using RNA polymerase III (Pol III)- or Pol II-dependent promoter-driven polycistronic expression systems. Additionally, we provide insights into vector construction methodologies and reliable genotyping techniques to detect and validate genome edits. Together, these elements constitute a practical framework for deploying genome editing in maize research and breeding. By optimizing these parameters, researchers can enhance the efficiency and accuracy of CRISPR-mediated genome modifications, accelerating functional genomic discovery and the development of improved maize varieties tailored to meet future agricultural demands.},
}
@article {pmid41056201,
year = {2025},
author = {Weber, VJ and Reschigna, A and Gerhardt, MJ and Heigl, T and Hinrichsmeyer, KS and van den Engel, S and Otify, DY and Gavrilov, Z and Blaser, F and Meneau, I and Betz, C and Bolz, HJ and Biel, M and Michalakis, S and Becirovic, E},
title = {CRISPR/Cas-mediated activation of genes associated with inherited retinal dystrophies in human cells for diagnostic purposes.},
journal = {JCI insight},
volume = {},
number = {},
pages = {},
doi = {10.1172/jci.insight.189615},
pmid = {41056201},
issn = {2379-3708},
abstract = {Many patients suffering from inherited diseases do not receive a genetic diagnosis and are therefore excluded as candidates for treatments, such as gene therapies. Analyzing disease-related gene transcripts from patient cells would improve detection of mutations that have been missed or misinterpreted in terms of pathogenicity during routine genome sequencing. However, the analysis of transcripts is complicated by the fact that a biopsy of the affected tissue is often not appropriate, and many disease-associated genes are not expressed in tissues or cells that can be easily obtained from patients. Here, using CRISPR/Cas-mediated transcriptional activation (CRISPRa) we developed a robust and efficient approach to activate genes in skin-derived fibroblasts and in freshly isolated peripheral blood mononuclear cells (PBMCs) from healthy individuals. This approach was successfully applied to blood samples from patients with inherited retinal dystrophies (IRD). We were able to efficiently activate several IRD-linked genes and detect the corresponding transcripts using different diagnostically relevant methods such as RT-qPCR, RT-PCR and long- and short-read RNA sequencing. The detection and analysis of known and unknown mRNA isoforms demonstrates the potential of CRISPRa-mediated transcriptional activation in PBMCs. These results will contribute to ceasing the critical gap in the genetic diagnosis of IRD patients and other inherited diseases.},
}
@article {pmid41055650,
year = {2025},
author = {Che, X and Wei, Y and Wang, X and Wang, X and Wu, Z and Deng, J and Ge, S and Liu, X and Cai, Z and Zhang, H and He, L and Xu, J},
title = {Lipoxygenase ZmLOX3 Enhances Salt Tolerance of Maize Under the Regulation of ZmNAC032.},
journal = {Journal of agricultural and food chemistry},
volume = {},
number = {},
pages = {},
doi = {10.1021/acs.jafc.5c02349},
pmid = {41055650},
issn = {1520-5118},
abstract = {Lipoxygenase (LOX) plays a critical role in plant biotic and abiotic stress responses by mediating lipid peroxidation and the production of jasmonic acid (JA). In this study, maize ZmLOX3 was identified as a positive regulator in salt stress tolerance. Overexpression of ZmLOX3 enhanced the salt tolerance of Arabidopsis. When maize seedlings were subjected to salt stress, the ZmLOX3[OE] lines exhibited a better growth phenotype than the control (B104) and the zmlox3[CR] (CRISPR/Cas) knockout mutants. Overexpression of ZmLOX3 improved ROS scavenging, Na[+]/K[+] homeostasis, and cell membrane stability. Transcriptome analyses revealed that ZmLOX3[OE] triggered the expression of genes involved in both the JA synthesis and signaling pathways. A transcription factor ZmNAC032 was identified via Y1H screening and was able to bind to the C[A/G]CG[T/G] sequence in the ZmLOX3 promoter and activate its expression. These findings are helpful for deciphering the function and regulatory status of ZmLOX in improving salt tolerance.},
}
@article {pmid41054568,
year = {2025},
author = {Xia, B and Wang, Z and Fei, T and Ma, Y and Guo, Y and Fei, D and Shu, X and Zhao, G and Ma, M and Yuan, H},
title = {Development and application of a CRISPR/Cas12a-based reverse transcription-recombinase polymerase amplification assay with lateral flow dipstick and fluorescence detection for Getah virus.},
journal = {PeerJ},
volume = {13},
number = {},
pages = {e20119},
pmid = {41054568},
issn = {2167-8359},
mesh = {*CRISPR-Cas Systems ; *Alphavirus/isolation &amp; purification/genetics ; Animals ; *Nucleic Acid Amplification Techniques/methods ; Sensitivity and Specificity ; Fluorescence ; *Alphavirus Infections/diagnosis/virology/veterinary ; Swine ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {Getah virus (GETV), a mosquito-borne alphavirus classified as a zoonotic disease, primarily infects livestock, particularly pigs and horses. In recent years, it has re-emerged in multiple Asian countries, posing a potential threat to animal husbandry and public health. In this study, we developed a rapid and sensitive GETV detection method based on reverse transcription-recombinase polymerase amplification (RT-RPA) and the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas12a system combined with a lateral flow dipstick (LFD) for visual readout. By leveraging sequence conservation in the GETV E2 envelope protein-coding regions, we engineered matched crRNA guides and amplification primers to develop a rapid CRISPR-Cas12a diagnostic workflow. The optimized platform combines RT-RPA (42 °C/20 min) with Cas12a's trans-nuclease activity, permitting multiplex detection via real-time fluorescence quantification or immunochromatographic strip visualization. Analytical evaluation demonstrated a detection capability of 10 copies/µL and exclusive specificity against four pathogen controls, including Japanese encephalitis virus and pseudorabies virus. Validation performed using simulated clinical samples revealed 100% concordance between the results of RT-RPA-CRISPR/Cas12a-LFD and quantitative polymerase chain reaction (PCR), while reducing the total detection time to 50 minutes. This approach eliminated the need for advanced instrumentation owing to its simplified operational design, enabling field-deployable rapid detection capabilities that establish essential technical infrastructure for initiating timely GETV containment measures. This approach has broad application potential in the fields of food safety, clinical diagnostics, and environmental science.},
}
@article {pmid41026092,
year = {2025},
author = {Toyonishi, G and Nakazawa, T and Koshi, D and Horii, M and An, GH and Kawauchi, M and Honda, Y},
title = {CRISPR/Cas9-directed disruption of wc-2 leads to the absence of fruiting body development in Pleurotus ostreatus.},
journal = {FEMS microbiology letters},
volume = {372},
number = {},
pages = {},
doi = {10.1093/femsle/fnaf104},
pmid = {41026092},
issn = {1574-6968},
support = {18KK0178//Japan Society for the Promotion of Science/ ; 22H00380//Japan Society for the Promotion of Science/ ; 22KK0090//Japan Society for the Promotion of Science/ ; PJ0175072025//Rural Development Administration/ ; },
mesh = {*Pleurotus/genetics/growth &amp; development/radiation effects ; *Fruiting Bodies, Fungal/growth &amp; development/genetics/radiation effects ; *CRISPR-Cas Systems ; *Fungal Proteins/genetics/metabolism ; Light ; },
abstract = {Light, particularly blue light, is a key environmental factor that induces fruiting in certain agaricomycetes. In this study, we characterized mutant strains of Pleurotus ostreatus with disrupted wc-2, which encodes one of the white-collar proteins, Wc-2, to investigate the role of light in fruiting in P. ostreatus. We introduced two different plasmids containing expression cassettes for Cas9 and two different gRNAs targeting wc-2 separately into the dikaryotic P. ostreatus strain PC9×#64. Among the 11 dikaryotic hygromycin-resistant transformants, six strains did not form fruiting bodies. Genomic PCR followed by sequencing analysis suggested that all six fruitless strains were dikaryotic wc-2 disruptants. Small aggregate structures were not observed in the dikaryotic wc-2 disruptants grown under light conditions, as in PC9×#64 grown in a red box. These results suggest that Wc-2 is essential for the initiation of blue light-induced fruiting in P. ostreatus.},
}
@article {pmid41002250,
year = {2025},
author = {Gillmore, JD and Gane, E and Täubel, J and Pilebro, B and Echaniz-Laguna, A and Kao, J and Litchy, W and Shahda, S and Haagensen, A and Walsh, L and Smith, D and Kachadourian, J and Ward, JH and Lebwohl, D and Zhu, P and Xu, Y and Leung, A and Sonderfan, A and Gutstein, DE and Manvelian, G and Adams, D},
title = {Nexiguran Ziclumeran Gene Editing in Hereditary ATTR with Polyneuropathy.},
journal = {The New England journal of medicine},
volume = {393},
number = {14},
pages = {1375-1386},
doi = {10.1056/NEJMoa2510209},
pmid = {41002250},
issn = {1533-4406},
mesh = {Humans ; *Amyloid Neuropathies, Familial/therapy/genetics ; Male ; Female ; Middle Aged ; *Prealbumin/genetics/metabolism/antagonists &amp; inhibitors ; Adult ; Aged ; *Gene Editing ; *Genetic Therapy/adverse effects/methods ; CRISPR-Cas Systems ; Neurofilament Proteins/blood ; Body Mass Index ; },
abstract = {BACKGROUND: Hereditary transthyretin amyloidosis with polyneuropathy (ATTRv-PN) is a rare, multisystem, progressive, debilitating, and fatal disease characterized by tissue deposition of misfolded transthyretin (TTR) in peripheral nerves. Nexiguran ziclumeran (nex-z) is an investigational in vivo therapy based on CRISPR-Cas9 (clustered regularly interspaced short palindromic repeats and associated Cas9 endonuclease) that is designed to reduce serum TTR levels through selective inactivation of TTR in the liver.<br><br>METHODS: In this phase 1, open-label study, we administered one infusion of nex-z to patients with ATTRv-PN. Primary objectives included assessment of the safety and pharmacodynamics of nex-z. Secondary end points included changes in the familial amyloid polyneuropathy stage, polyneuropathy disability score, serum neurofilament light chain (NfL) level, modified body-mass index (modified BMI, defined as the conventional BMI [weight in kilograms divided by square of height in meters] multiplied by the albumin level in grams per liter), and modified Neuropathy Impairment Score+7 (mNIS+7; range, 0 to 304, with higher scores indicating more impairment).<br><br>RESULTS: A total of 36 patients received nex-z; the mean follow-up was 27 months. The mean percent change from baseline in the serum TTR level was -90% at day 28, which was sustained through month 24 (-92%). Treatment-related adverse events included transient infusion-related reactions (in 21 patients), decreased thyroxine level without hypothyroidism or elevated thyrotropin level (in 8), and headache (in 4). One participant died from cardiac amyloidosis, and one withdrew owing to progressive decline in motor function. Serious adverse events were reported in 11 patients. At month 24, the familial amyloid polyneuropathy stage and polyneuropathy disability score remained stable in 29 and 27 patients, respectively; improved in 2 and 5, respectively; and worsened in 2 and 2, respectively. The mean change in the serum NfL level was -9.0 pg per milliliter, and the change in the modified BMI was 24.7. The mean change from baseline in the mNIS+7 was -8.5.<br><br>CONCLUSIONS: A single administration of nex-z in patients with ATTRv-PN was associated with rapid, deep, and durable reductions in serum TTR levels. The results support further investigation of nex-z to treat ATTRv-PN. (Funded by Intellia Therapeutics and Regeneron Pharmaceuticals; ClinicalTrials.gov number, NCT04601051.).},
}
@article {pmid40990297,
year = {2025},
author = {Sheng, J and Dong, Y and Sun, S and Zhang, Y and Li, C and Xu, X and Wang, H},
title = {Construction of a Sensing Platform Integrated with a CRISPR/Cas12a-Triggered Colorimetric Strategy for the Quantitative Detection of Meat Freshness.},
journal = {Journal of agricultural and food chemistry},
volume = {73},
number = {40},
pages = {25604-25614},
doi = {10.1021/acs.jafc.5c04851},
pmid = {40990297},
issn = {1520-5118},
mesh = {*Colorimetry/methods/instrumentation ; *Meat/analysis/microbiology ; CRISPR-Cas Systems ; Animals ; *Pseudomonas/genetics/isolation &amp; purification/metabolism ; *Bacterial Proteins/genetics/metabolism ; *Biosensing Techniques/instrumentation/methods ; *Endodeoxyribonucleases/genetics/metabolism ; CRISPR-Associated Proteins ; },
abstract = {Monitoring microbial determinants, such as Pseudomonas spp., is thus essential for assessing meat freshness. Here, a novel colorimetric sensing platform based on magnetic enzyme-labeled nanoparticles combined with clustered regularly interspaced short palindromic repeats (CRISPR)/Cas12a without nucleic acid molecule preamplification was developed for detecting meat freshness. Under optimal conditions, a high-specificity crRNA was systematically verified, and the colorimetric sensor could accurately quantify Pseudomonas spp. loads with levels ranging from 1 × 10[3.7] to 1 × 10[8.7] CFU/mL, with a color change from colorless to yellow. A smart colorimetric platform, including a self-designed image acquisition device and self-programmed image analysis software, was developed and applied to the integrated determination of meat freshness by using the B-value in the RGB channel. The platform has been applied to both consumers and producers and has been validated by 48 actual samples of chilled meat. These findings provide new insights into the exploration of reliable tools for monitoring meat freshness.},
}
@article {pmid40985907,
year = {2025},
author = {Fei, S and Zhang, C and Zhang, X and Xie, Y and Fu, S and Wu, J},
title = {An Ultrasensitive Immunocapture (IC)-RPA-CRISPR/Cas12a Assay with Three Readout Modes for Detecting Xanthomonas oryzae pv. oryzicola of Rice Bacterial Leaf Streak.},
journal = {Journal of agricultural and food chemistry},
volume = {73},
number = {40},
pages = {25664-25675},
doi = {10.1021/acs.jafc.5c04360},
pmid = {40985907},
issn = {1520-5118},
mesh = {*Oryza/microbiology ; *Xanthomonas/genetics/isolation &amp; purification ; *Plant Diseases/microbiology ; CRISPR-Cas Systems ; *Nucleic Acid Amplification Techniques/methods/instrumentation ; Plant Leaves/microbiology ; Bacterial Proteins/genetics/metabolism ; },
abstract = {Xanthomonas oryzae pv oryzicola (Xoc) is the causal agent of rice bacterial leaf streak (BLS) and causes enormous losses of rice yields in many countries every year. Development of sensitive diagnostic techniques is crucial for its prevention and control. Here, we developed an ultrasensitive IC-RPA-CRISPR/Cas12a assay with three readout modes [qPCR machine, UV lamp, and lateral flow strip (LFS)] for Xoc detection in rice, which combined advantages of immunocapture, recombinase polymerase amplification (RPA), and CRISPR/Cas12a-based cleavage. Especially, the immunocapture step allows to capture and enrich Xoc from samples, which minimizes the interference from rice debris to benefit nucleic acid release and amplification and enhances the specificity and sensitivity of this assay. The detection limits of its three readout modes for Xoc bacterial suspension is 2, 6, and 60 CFU/mL, respectively. Collectively, this study provides a specific, ultrasensitive, practical approach for quarantine and detection of Xoc that will benefit the prevention and control of BLS.},
}
@article {pmid40970715,
year = {2025},
author = {Zhao, C and Li, G and Shen, C and Xie, Y and Chen, Y and Ying, X and Chen, Y and Zhang, C},
title = {An extraction-free and one-pot two-temperature CRISPR/Cas12b system for visual detection of Group B Streptococcus by integrating with RPA.},
journal = {Journal of clinical microbiology},
volume = {63},
number = {10},
pages = {e0081925},
doi = {10.1128/jcm.00819-25},
pmid = {40970715},
issn = {1098-660X},
support = {2024KY1444//Medical and Health Research Project of Zhejiang Province/ ; LTGC23H200004//Zhejiang Provincial Natural Science Foundation of China/ ; },
mesh = {*Streptococcus agalactiae/isolation &amp; purification/genetics ; Humans ; *Streptococcal Infections/diagnosis/microbiology ; *Nucleic Acid Amplification Techniques/methods ; Sensitivity and Specificity ; *CRISPR-Cas Systems ; *Molecular Diagnostic Techniques/methods ; Female ; Recombinases/metabolism ; Temperature ; Vagina/microbiology ; },
abstract = {UNLABELLED: Group B Streptococcus (GBS) is a major cause of neonatal infections, and rapid detection is essential for timely clinical intervention. In this study, we developed an extraction-free, one-pot CRISPR/Cas12b assay for visual detection of GBS by combining with isothermal amplification, including loop-mediated isothermal amplification (LAMP) and recombinase polymerase amplification (RPA). The results showed that LAMP-CRISPR/Cas12b outperformed RPA-CRISPR/Cas12b system across all template concentrations, especially in low-copy template (30 and 10 copies/test) detection. To enhance the detection performance of RPA-CRISPR/Cas12b, we introduced a two-temperature protocol, with RPA reaction at 39°C followed by Cas12b activation at 62°C. Through the two-temperature approach, the detection rate of RPA-CRISPR/Cas12b system was significantly improved even in low-copy samples, achieving a sensitivity of 10 copies/test (1 copy/μL). Clinical validation using 60 vaginal-rectal swab samples showed 96.7% and 98.3% of concordance when compared to culture and qPCR methods, respectively. This assay offers a rapid (<1 h), highly sensitive, and user-friendly solution without requiring nucleic acid extraction or sophisticated instruments. Its compatibility with visual signal detection makes it ideal for point-of-care testing, especially in low-resource or time-sensitive settings. The platform can be adapted for broader pathogen detection in future field diagnostics.<br><br>IMPORTANCE: This study presents a rapid, convenient, and highly accurate method for Group B Streptococcus (GBS) detection by integrating the CRISPR/Cas12b system with recombinase polymerase amplification, an isothermal nucleic acid amplification technique. To streamline the workflow, we established a one-pot, extraction-free assay that significantly reduces the detection time. Through the systematic optimization of the dual-temperature conditions, we enhanced the amplification efficiency of target DNA, thereby improving the sensitivity of the CRISPR/Cas12b system. Additionally, the incorporation of a UV-visible detection system enables visual readout, facilitating instrument-free testing suitable for point-of-care (POC) applications.},
}
@article {pmid40899880,
year = {2025},
author = {Liu, P and Zhang, J and Gong, Y and Liu, W and Xiao, G and Liang, J and Wang, X and Bi, J and Zhang, G},
title = {Application of engineered CRISPR/Cas12a variants with altered protospacer adjacent motif specificities for the detection of isoniazid resistance mutations in Mycobacterium tuberculosis.},
journal = {Microbiology spectrum},
volume = {13},
number = {10},
pages = {e0016525},
pmid = {40899880},
issn = {2165-0497},
mesh = {*Isoniazid/pharmacology ; *Mycobacterium tuberculosis/genetics/drug effects/isolation &amp; purification ; *CRISPR-Cas Systems/genetics ; *Antitubercular Agents/pharmacology ; Bacterial Proteins/genetics ; Humans ; *Drug Resistance, Bacterial/genetics ; Mutation ; Tuberculosis, Multidrug-Resistant/microbiology/diagnosis ; *Endodeoxyribonucleases/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats ; Catalase/genetics ; Sensitivity and Specificity ; CRISPR-Associated Proteins/genetics ; Microbial Sensitivity Tests ; },
abstract = {UNLABELLED: Drug-resistant tuberculosis (TB) is a major global public health concern. Although isoniazid is currently considered one of the most effective first-line drugs for TB treatment, its efficacy is limited by the emergence of resistance. Therefore, it is imperative to develop new methods for detecting drug-resistant TB. In this study, we developed a nucleic acid detection system based on the clustered regularly interspaced short palindromic repeat (CRISPR) Cas12a_RR protein. The system combines recombinase polymerase amplification with an engineered CRISPR/Cas12a_RR protein to enable rapid and specific detection of the katG G944C mutation in isoniazid-resistant Mycobacterium tuberculosis (Mtb). It could detect the target DNA at concentrations as low as 1% in a mixed sample. Compared with TaqMan quantitative polymerase chain reaction and DNA sequencing, the CRISPR/Cas12a_RR system demonstrated superior detection performance in terms of sensitivity, specificity, and cost-effectiveness. Furthermore, it effectively differentiated between drug-resistant Mtb strains from wild-type Mtb strains in clinically isolated samples, with the entire detection process completed in 60 min. In conclusion, the CRISPR/Cas12a_RR detection system offers a novel, rapid, simple, sensitive, and specific approach for identifying isoniazid-resistant Mtb, with significant potential for clinical application, particularly in resource-limited settings.<br><br>IMPORTANCE: This study presents a novel method for detecting isoniazid-resistant Mycobacterium tuberculosis (Mtb) using clustered regularly interspaced short palindromic repeat (CRISPR)/Cas12a mutants, offering rapid detection, cost-effectiveness, and high specificity, and thereby providing a promising new avenue for detecting isoniazid-resistant Mtb.},
}
@article {pmid40866699,
year = {2025},
author = {Pacesa, M and Nickel, L and Schellhaas, C and Schmidt, J and Pyatova, E and Kissling, L and Barendse, P and Choudhury, J and Kapoor, S and Alcaraz-Serna, A and Cho, Y and Ghamary, KH and Vinué, L and Yachnin, BJ and Wollacott, AM and Buckley, S and Westphal, AH and Lindhoud, S and Georgeon, S and Goverde, CA and Hatzopoulos, GN and Gönczy, P and Muller, YD and Schwank, G and Swarts, DC and Vecchio, AJ and Schneider, BL and Ovchinnikov, S and Correia, BE},
title = {One-shot design of functional protein binders with BindCraft.},
journal = {Nature},
volume = {646},
number = {8084},
pages = {483-492},
pmid = {40866699},
issn = {1476-4687},
support = {R35 GM138368/GM/NIGMS NIH HHS/United States ; },
mesh = {Humans ; Gene Editing ; Protein Binding ; CRISPR-Cas Systems/genetics ; Allergens/immunology/metabolism/chemistry ; Immunoglobulin E/immunology/metabolism ; *Protein Engineering/methods ; *Proteins/metabolism/chemistry ; Receptors, Cell Surface/metabolism/chemistry ; Binding Sites ; Models, Molecular ; CRISPR-Associated Protein 9/metabolism ; Animals ; },
abstract = {Protein-protein interactions are at the core of all key biological processes. However, the complexity of the structural features that determine protein-protein interactions makes their design challenging. Here we present BindCraft, an open-source and automated pipeline for de novo protein binder design with experimental success rates of 10-100%. BindCraft leverages the weights of AlphaFold2 (ref. [1]) to generate binders with nanomolar affinity without the need for high-throughput screening or experimental optimization, even in the absence of known binding sites. We successfully designed binders against a diverse set of challenging targets, including cell-surface receptors, common allergens, de novo designed proteins and multi-domain nucleases, such as CRISPR-Cas9. We showcase the functional and therapeutic potential of designed binders by reducing IgE binding to birch allergen in patient-derived samples, modulating Cas9 gene editing activity and reducing the cytotoxicity of a foodborne bacterial enterotoxin. Last, we use cell-surface-receptor-specific binders to redirect adeno-associated virus capsids for targeted gene delivery. This work represents a significant advancement towards a 'one design-one binder' approach in computational design, with immense potential in therapeutics, diagnostics and biotechnology.},
}
@article {pmid40862592,
year = {2025},
author = {Shen, Y and Yi, C and Wang, H and Tang, Y and Li, J},
title = {Development of a rapid and sensitive RPA-CRISPR/Cas12a-based assay for the detection of Brucella melitensis.},
journal = {Microbiology spectrum},
volume = {13},
number = {10},
pages = {e0099825},
pmid = {40862592},
issn = {2165-0497},
mesh = {*Brucellosis/diagnosis/microbiology ; *Brucella melitensis/genetics/isolation &amp; purification ; Humans ; *CRISPR-Cas Systems ; *Nucleic Acid Amplification Techniques/methods ; Sensitivity and Specificity ; Recombinases/genetics/metabolism ; *Molecular Diagnostic Techniques/methods ; Limit of Detection ; Reproducibility of Results ; Bacterial Proteins/genetics ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {Brucellosis, a zoonotic disease caused by Brucella species, presents significant public health challenges due to its complex diagnosis and the limited availability of rapid detection methods. To address these challenges, we developed a novel detection method that integrates recombinase polymerase amplification (RPA) with the CRISPR/Cas12a system, enabling dual readout through fluorescence (FL) and lateral flow strip (LFS) detection. The RPA-CRISPR/Cas12a-FL assay demonstrated an impressive detection limit of 1 copy/μL, which is 10 times more sensitive than quantitative polymerase chain reaction, while the RPA-CRISPR/Cas12a-LFS method achieved a detection limit of 10 copies/μL, comparable to nested PCR. Specificity testing confirmed the robustness of the assay, as it produced strong signals exclusively for Brucella without cross-reactivity with other bacterial species. Clinical validation using serum samples from 24 confirmed brucellosis patients and six healthy controls demonstrated a 100% concordance with serological results, underscoring the reliability of this method for clinical applications. This assay provides a rapid, sensitive, and specific tool for Brucella detection, suitable for both laboratory and field settings, and holds significant potential for enhancing the diagnosis and control of brucellosis.IMPORTANCEBrucellosis is a significant zoonotic disease, and rapid and accurate diagnosis is crucial for its treatment and control. To address this need, we developed a novel detection method that combines recombinant enzyme polymerase amplification with a CRISPR/Cas12a system, achieving dual readout through fluorescence and lateral flow strips. The test demonstrates excellent sensitivity and specificity, with clinical validation confirming complete concordance with serological results. This approach offers a fast, reliable, and field-deployable solution for brucellosis diagnosis, significantly enhancing disease management and public health outcomes.},
}
@article {pmid40856920,
year = {2025},
author = {Thiel, J and Sürün, D and Brändle, DC and Teichert, M and Künzel, SR and Friedrich, U and Dahl, A and Schubert, K and Rzagalinski, I and Shevchenko, A and Traikov, S and Mirtschink, P and Wagenführ, L and Buchholz, F and Hölig, K and Tonn, T and Kronstein-Wiedemann, R},
title = {Knock Out of miRNA-30a-5p and Reconstitution of the Actin Network Dynamics Partly Restores the Impaired Terminal Erythroid Differentiation during Blood Pharming.},
journal = {Stem cell reviews and reports},
volume = {21},
number = {8},
pages = {2637-2653},
pmid = {40856920},
issn = {2629-3277},
support = {530364326//Deutsche Forschungsgemeinschaft/ ; },
mesh = {*MicroRNAs/genetics/metabolism ; Humans ; *Cell Differentiation/genetics ; *Actins/metabolism ; *Erythroid Cells/metabolism/cytology ; *Erythropoiesis/genetics ; Proto-Oncogene Mas ; Hematopoietic Stem Cells/metabolism/cytology ; CRISPR-Cas Systems ; Erythrocytes/metabolism/cytology ; bcl-X Protein/metabolism/genetics ; Gene Knockout Techniques ; Cell Line ; },
abstract = {In vitro red blood cell (RBC) production offers a promising complement to conventional blood donation, particularly for patients with rare blood types. Previously, we developed imBMEP-A, the first erythroid cell line derived from reticulocyte progenitors, which maintains robust hemoglobin expression and erythroid differentiation in the presence of erythropoietin (EPO) despite its immortalized state. However, clinical translation remains hindered by the inability to scale up production due to impaired in vitro enucleation of RBC progenitor cell lines. Enhancing enucleation efficiency in imBMEP-A cells involved CRISPR/Cas9-mediated knockout (K.O.) of miR-30a-5p, a key enucleation inhibitor, moderately increasing rates to 3.3 ± 0.4%- 8.9 ± 1.7%. Further investigation of enucleation inefficiencies led to transcriptome and proteome comparisons between imBMEP-miR30a-K.O. cells and hematopoietic stem cells (HSCs). These analyses revealed altered gene expression and protein abundances linked to metabolic transitions, apoptosis promotion, and cytoskeletal regulation. Notably, forced expression of the proto-oncogene c-Myc, required for cell immortalization, emerged as a key driver of these physiological changes. Counteracting these effects required optimization of imBMEP-A cells by activating BCL-XL transcription and knocking out SCIN, which encodes the actin-severing protein scinderin. While BCL-XL is upregulated in normal erythropoiesis, it is downregulated in imBMEP-A. Conversely, SCIN, typically absent in erythroid cells, is highly expressed in imBMEP-A, disrupting actin organization. These interventions improved viability, restored actin network formation, and increased terminal erythropoiesis, yielding 22.1 ± 1.7% more orthochromatic erythroblasts. These findings establish a foundation for optimizing imBMEP-A cells for therapeutic use and advancing the understanding the pathophysiology of erythroleukemia.},
}
@article {pmid40833103,
year = {2025},
author = {Omachi, R and Imai, K and Sato, A and Tanaka, M and Mizushina, H and Takeuchi, K and Maeda, T},
title = {Development and clinical evaluation of a novel SHERLOCK test for Mycoplasma genitalium.},
journal = {Microbiology spectrum},
volume = {13},
number = {10},
pages = {e0044525},
pmid = {40833103},
issn = {2165-0497},
support = {24K10540//KAKEN/ ; 2024 Research grant//Charitable Trust Laboratory Medicine Research Foundation of Japan/ ; Grant-in-Aid for Young Researchers//Saitama Medical University Hospital/ ; },
mesh = {*Mycoplasma genitalium/genetics/isolation &amp; purification ; Humans ; *Mycoplasma Infections/diagnosis/microbiology/urine ; Male ; *Nucleic Acid Amplification Techniques/methods ; Sensitivity and Specificity ; *Urethritis/microbiology/diagnosis ; *Molecular Diagnostic Techniques/methods ; Japan ; Point-of-Care Testing ; DNA, Bacterial/genetics ; CRISPR-Cas Systems ; },
abstract = {Mycoplasma genitalium (MG) is a sexually transmitted pathogen associated with urethritis. Nucleic acid amplification tests are the gold standard for its diagnosis but often require specialized equipment, which limits their use in point-of-care testing. This study aimed to develop a rapid, sensitive detection method for MG using a specific high-sensitivity enzymatic reporter unlocking (SHERLOCK) test, which combines isothermal recombinase polymerase amplification and a clustered regularly interspaced short palindromic repeats (CRISPR)-Cas13a reaction. We developed a novel SHERLOCK test targeting the Mg219 gene in MG. The SHERLOCK method was evaluated using 128 first-void urine samples collected from male patients who were suspected of MG urethritis in Japan. The results of SHERLOCK were compared to those of the cobas TV/MG test and in-house quantitative PCR. SHERLOCK was optimized for use with crude DNA extracted from clinical urine samples. The results were detected via a lateral flow assay, allowing for visual interpretation within 60 min. The method demonstrated a limit of detection of 10 copies/reaction and showed no cross-reactivity with other pathogens. In clinical evaluations of 128 urine samples, SHERLOCK showed an overall agreement rate of 91.4% with the cobas TV/MG PCR test; the positive and negative agreement rates were 79.6 and 100%, respectively. SHERLOCK showed superior performance to quantitative PCR. This study demonstrates that the novel SHERLOCK assay for MG has potential as a point-of-care test in the clinical setting. Further evaluation in prospective studies is needed to confirm its clinical value.IMPORTANCEMycoplasma genitalium (MG) is a causative agent of sexually transmitted infections and is associated with urethritis and prostatitis in men. To prevent the transmission of MG, it is essential to identify infected individuals through diagnostic testing and provide appropriate treatment. Nucleic acid amplification tests are commonly used for MG diagnosis in the clinical setting, but the point-of-care testing (POCT) for MG remains limited. In this study, we developed a novel nucleic acid amplification test-specific high-sensitivity enzymatic reporter unlocking (SHERLOCK)-for MG, combining crude DNA extraction with a lateral flow assay. Our SHERLOCK assay successfully detected MG in approximately 1 h, with a detection limit of 10 copies/reaction. Clinical evaluations using urine samples showed a high agreement rate with the cobas TV/MG test. SHERLOCK is expected to be a useful tool for POCT for MG.},
}
@article {pmid40819286,
year = {2025},
author = {Beck, CW and Reily-Bell, M and Bicknell, LS},
title = {Unilateral loss of recql4 function in Xenopus laevis tadpoles leads to ipsilateral ablation of the forelimb, hypoplastic Meckel's cartilage, and vascular defects.},
journal = {G3 (Bethesda, Md.)},
volume = {15},
number = {10},
pages = {},
doi = {10.1093/g3journal/jkaf179},
pmid = {40819286},
issn = {2160-1836},
support = {PRG1732//Neurological Foundation of New Zealand/ ; },
mesh = {Animals ; *Xenopus laevis/genetics ; Larva/genetics ; *RecQ Helicases/genetics/metabolism ; *Forelimb/abnormalities ; *Cartilage/abnormalities/metabolism ; *Xenopus Proteins/genetics/metabolism ; Gene Knockdown Techniques ; Phenotype ; CRISPR-Cas Systems ; },
abstract = {RECQL4 encodes a RecQ helicase, one of a family of DNA unwinding enzymes with roles in DNA replication, double-strand break repair, and genomic stability. Pathogenic variants in RECQL4 are clinically associated with 3 rare autosomal recessive conditions: Rothmund-Thomson syndrome type II, Baller-Gerold syndrome, and RAPADILINO syndrome. These 3 syndromes show overlapping growth retardation, low bone density, and skeletal defects affecting the arms and hands. Here, we take advantage of the ability to generate one-sided CRISPR knockdowns of recql4 in Xenopus laevis tadpoles. Tadpoles develop normally until feeding starts, after which growth slows on the edited side, leading to a curved posture, smaller eyes (microphthalmia), and reduced head size (microcephaly). Forelimb buds fail to develop, leading to complete absence of the forelimb on the edited side. Additionally, Meckel's cartilage (lower jaw) ossification is absent or reduced and the hyoid cartilage is smaller, but this is not due to deficiencies in cranial neural crest migration on the edited side. Knockdown of recql4 also results in hypoplastic vasculature, with reduced branching from the aorta on the edited side. Taken together, our results clearly show the utility of unilateral CRISPR editing in Xenopus for understanding the specific phenotypic developmental effects of mutations affecting cell proliferation.},
}
@article {pmid40794772,
year = {2025},
author = {Després, PC and Gervais, NC and Fogal, M and Rogers, RKJ and Cuomo, CA and Shapiro, RS},
title = {Targeted loss of heterozygosity in Candida albicans using CRISPR-Cas9 reveals the functional impact of allelic variation.},
journal = {Genetics},
volume = {231},
number = {2},
pages = {},
doi = {10.1093/genetics/iyaf154},
pmid = {40794772},
issn = {1943-2631},
support = {//FRQS/ ; //NSERC/ ; //N.C.G./ ; //M.F./ ; PJT 162195//CIHR/ ; //R.S.S./ ; //CIFAR/ ; //Fungal Kingdom/ ; //C.A.C./ ; U19 AI110818/AI/NIAID NIH HHS/United States ; //Canada Research Chair/ ; },
mesh = {*Candida albicans/genetics/drug effects ; *Loss of Heterozygosity ; *CRISPR-Cas Systems ; Alleles ; Genome, Fungal ; RNA, Guide, CRISPR-Cas Systems/genetics ; Drug Resistance, Fungal/genetics ; Gene Editing ; Genetic Variation ; Antifungal Agents/pharmacology ; },
abstract = {The diploid genome of the fungal pathogen Candida albicans is highly heterozygous, with most allele pairs diverging at either the coding or regulatory level. When faced with selection pressure like antifungal exposure, this hidden genetic diversity can provide a reservoir of adaptive mutations through loss of heterozygosity (LOH) events. Validating the potential phenotypic impact of LOH events observed in clinical or experimentally evolved strains can be difficult due to the challenge of precisely targeting one allele over the other. Here, we show that a CRISPR-Cas9 system can be used to overcome this challenge. By designing allele-specific guide RNA sequences, we can induce targeted, directed LOH events, which we validate by whole-genome long-read sequencing. Using this approach, we efficiently recapitulate a recently described LOH event that increases resistance to the antifungal fluconazole. Additionally, we find that the recombination tracts of these induced LOH events have similar lengths to those observed naturally. To facilitate future use of this method, we provide a database of allele-specific sgRNA sequences for Cas9 that provide near genome-wide coverage of heterozygous sites through either direct or indirect targeting. This approach will be useful in probing the adaptive role of LOH events in this important human pathogen.},
}
@article {pmid40758833,
year = {2025},
author = {Stuecker, TN and Hood, SE and Molina Pineda, J and Lenaduwe, S and Winter, J and Sadhu, MJ and Lewis, JA},
title = {Improved vectors for retron-mediated CRISPR-Cas9 genome editing in Saccharomyces cerevisiae.},
journal = {G3 (Bethesda, Md.)},
volume = {15},
number = {10},
pages = {},
doi = {10.1093/g3journal/jkaf175},
pmid = {40758833},
issn = {2160-1836},
support = {P20 GM103429/GM/NIGMS NIH HHS/United States ; MCB-1941824//National Science Foundation/ ; /HG/NHGRI NIH HHS/United States ; 1ZIAHG200401/GF/NIH HHS/United States ; //Arkansas IDeA Network of Biomedical Research Excellence/ ; //Summer Research Fellowship/ ; P20 GM103429/GM/NIGMS NIH HHS/United States ; },
mesh = {*Saccharomyces cerevisiae/genetics ; *CRISPR-Cas Systems ; *Gene Editing/methods ; *Genetic Vectors/genetics ; Plasmids/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; *Genome, Fungal ; Escherichia coli/genetics ; },
abstract = {In vivo site-directed mutagenesis is a powerful genetic tool for testing the effects of specific alleles in their normal genomic context. While the budding yeast Saccharomyces cerevisiae possesses classical tools for site-directed mutagenesis, more efficient recent CRISPR-based approaches use Cas "cutting" combined with homologous recombination of a "repair" template that introduces the desired edit. However, current approaches are limited for fully prototrophic yeast strains and rely on relatively low-efficiency cloning of short gRNAs. We were thus motivated to simplify the process by combining the gRNA and its cognate repair template in cis on a single oligonucleotide. Moreover, we wished to take advantage of a new approach that uses an Escherichia coli retron (EcRT) to amplify repair templates as multi-copy single-stranded (ms)DNA in vivo, which are more efficient templates for homologous recombination. To this end, we have created a set of plasmids that express Cas9-EcRT, allowing for co-transformation with the gRNA-repair template plasmid in a single step. Our suite of plasmids contains different antibiotic (Nat, Hyg, Kan) or auxotrophic (HIS3, URA3) selectable markers, allowing for editing of fully prototrophic wild yeast strains. In addition to classic galactose induction, we generated a β-estradiol-inducible version of each plasmid to facilitate editing in yeast strains that grow poorly on galactose. The plasmid-based system results in >95% editing efficiencies for point mutations and >50% efficiencies for markerless deletions, in a minimum number of steps and time. We provide a detailed step-by-step guide on how to use this system.},
}
@article {pmid40495695,
year = {2025},
author = {Wang, H and Zhan, H and Pan, B and Zeng, L and Chen, Z and Liu, S and Zhang, Q and Hong, X and Lu, J and Lin, X and Zhao, X and Lai, J and Jie, K and Li, Y and Zhong, J and Peng, S and Chen, S and Chen, C and Zhong, W and Wu, S and Pan, Y and Lin, T and Chen, X},
title = {Engineering CRISPR System-Based Bacterial Outer Membrane Vesicle Potentiates T Cell Immunity for Enhanced Cancer Immunotherapy.},
journal = {Advanced materials (Deerfield Beach, Fla.)},
volume = {37},
number = {39},
pages = {e2501565},
doi = {10.1002/adma.202501565},
pmid = {40495695},
issn = {1521-4095},
support = {2018YFA0902800//National Key Research and Development Program of China/ ; 82322056//National Natural Science Foundation of China/ ; 82341018//National Natural Science Foundation of China/ ; 82072827//National Natural Science Foundation of China/ ; 82273421//National Natural Science Foundation of China/ ; 82303405//National Natural Science Foundation of China/ ; 82472137//National Natural Science Foundation of China/ ; 2023A03J0718//Science and Technology Program of Guangzhou/ ; 2024B03J1234//Science and Technology Program of Guangzhou/ ; 2024A04J6558//Science and Technology Program of Guangzhou/ ; //Fundamental Research Funds for the Central Universities/ ; 23ykbj002//Sun Yat-sen University/ ; 2020B1111170006//Guangdong Provincial Clinical Research Centre for Urological Diseases/ ; 2020B1212060018//Guangdong Science and Technology Department/ ; 2018B030317001//Guangdong Science and Technology Department/ ; 2017B030314026//Guangdong Science and Technology Department/ ; },
mesh = {Animals ; Mice ; *Immunotherapy/methods ; *T-Lymphocytes/immunology ; Humans ; Cell Line, Tumor ; *Bacterial Outer Membrane/metabolism ; *CRISPR-Cas Systems ; *Neoplasms/therapy/immunology ; Escherichia coli/genetics ; Interleukin-12/genetics ; Chemokine CXCL9/genetics ; Genetic Engineering ; },
abstract = {Immune checkpoint blockade (ICB) therapy has revolutionized cancer treatment but only benefits a subset of patients because of insufficient infiltration and inactivation of effector T cells. Bacterial outer membrane vesicles (OMVs) can activate immunity and deliver therapeutic agents for immunotherapy. However, efficiently targeting and packaging therapeutic molecules into OMVs remains challenging. Here, the engineered E. coli BL21-derived OMVs enable the packaging of multiple genes, resulting in a 7-fold increase in DNA enrichment efficiency and gene silencing in vitro. Moreover, the engineered OMVs carrying genes encoding CXCL9 and IL12 (OMV-C9I12) reprogram tumor cells to secrete these factors, significantly enhancing T-cell chemotaxis and activation. More importantly, this system markedly inhibits tumors, extends survival, and synergizes with anti-PD-1/PD-L1 therapy in murine MB49 and B16F10 tumor models. Single-cell RNA sequencing (scRNA-seq) further reveals significant upregulation of T-cell chemotaxis and activation-related pathways following OMV-C9I12 treatment. Finally, OMV-C9I12 potentiates T cell-mediated immunotherapy and suppresses the growth of bladder and breast cancer tumors in humanized mouse models. These findings highlight the potential of this engineered OMV platform for cancer gene therapy and provide novel strategies to overcome resistance to immunotherapy.},
}
@article {pmid40436751,
year = {2025},
author = {Bold-Erdene, A and Miura, K and Yamasaki, N and Miura, S and Ogata, S and Sasatani, M and Yamamoto, T and Kaminuma, O},
title = {Effect of gamma-ray exposure on the genome-editing efficiency of improved genome-editing via oviductal nucleic acids delivery (i-GONAD).},
journal = {Experimental animals},
volume = {74},
number = {4},
pages = {457-462},
doi = {10.1538/expanim.25-0036},
pmid = {40436751},
issn = {1881-7122},
mesh = {Animals ; Female ; *Gamma Rays/adverse effects ; *Gene Editing/methods ; Pregnancy ; Mice ; *Oviducts ; CRISPR-Cas Systems ; DNA Breaks, Double-Stranded/radiation effects ; *Nucleic Acids/administration &amp; dosage ; RNA, Guide, CRISPR-Cas Systems ; },
abstract = {DNA double-strand breaks (DSBs) are among the most hazardous cellular damages, potentially leading to cell death or oncogenesis if unrepaired. Genome editing methods, such as the CRISPR/Cas9 system, induce DSBs and utilize these repair pathways for gene knockout and knock-in. Although ionizing radiation also induces DSBs, it is not clear whether the efficiency of genome editing is affected by ionizing radiation. This study investigated the impact of gamma-ray exposure on the genome editing efficiency of the improved genome editing via oviductal nucleic acid delivery (i-GONAD) method. Gamma-rays were exposed to pregnant mice receiving i-GONAD targeting the Hr gene, whose mutation causes hair loss in mice. The exposure on the fertilization day (Day 0) decreased natural delivery rates and litter sizes, with notable effects at 0.3 Gy or higher. Although the proportions of hairless offspring obtained by i-GONAD differed greatly between single-guide RNAs (sgRNAs) used, total mutation rates, including hairless, mosaic, and indel, were equivalent. Gamma-ray exposure on Day 0 and the day after fertilization (Day 1) similarly and almost dose-dependently enhanced the genome editing efficiency evaluated by the total mutation rate. This study suggests the improvement of genome editing efficiency by gamma-ray exposure, at least in i-GONAD method, potentially facilitating the creation of diverse experimental animal models.},
}
@article {pmid40029006,
year = {2025},
author = {Mattoscio, D and Baeza, LA and Bai, H and Colangelo, T and Castagnozzi, S and Marzotto, M and Cufaro, MC and Lotti, V and Yuan, YC and Mucci, M and Si, L and Zuccarini, M and Tredicine, M and D'Orazio, S and Pieragostino, D and Del Boccio, P and Sorio, C and Trerotola, M and Romano, M and Plebani, R},
title = {Inflammation and epithelial-mesenchymal transition in a CFTR-depleted human bronchial epithelial cell line revealed by proteomics and human organ-on-a-chip.},
journal = {The FEBS journal},
volume = {292},
number = {19},
pages = {5086-5104},
doi = {10.1111/febs.70050},
pmid = {40029006},
issn = {1742-4658},
support = {AT2021 to Roberto Plebani//Italian Ministry of Health (fund ex60%)/ ; AT2022 to Roberto Plebani//Italian Ministry of Health (fund ex60%)/ ; AIRC; #19548 to Tommaso Colangelo//Associazione Italiana per la Ricerca sul Cancro/ ; MFAG 2022 - ID. 27060 to Domenico Mattoscio//Associazione Italiana per la Ricerca sul Cancro/ ; //Fondazione Umberto Veronesi/ ; GMSG#01/2023 to Roberto Plebani//Fondazione per la Ricerca sulla Fibrosi Cistica/ ; FFC#11/2022 to Domenico Mattoscio//Fondazione per la Ricerca sulla Fibrosi Cistica/ ; },
mesh = {Humans ; *Cystic Fibrosis Transmembrane Conductance Regulator/genetics/metabolism ; Proteomics/methods ; *Epithelial-Mesenchymal Transition/genetics ; *Epithelial Cells/metabolism/pathology ; *Inflammation/pathology/genetics/metabolism ; *Cystic Fibrosis/pathology/genetics/metabolism ; *Bronchi/pathology/metabolism/cytology ; Cell Line ; Cell Movement/genetics ; Lab-On-A-Chip Devices ; CRISPR-Cas Systems ; Neutrophils/metabolism/pathology ; Signal Transduction ; Microphysiological Systems ; },
abstract = {Cystic fibrosis (CF) is a genetic disease caused by mutations in the CF transmembrane conductance regulator (CFTR) gene, leading to chronic, unresolved inflammation of the airways due to uncontrolled recruitment of polymorphonuclear leukocytes (PMNs). Evidence indicates that CFTR loss-of-function, in addition to promoting a pro-inflammatory phenotype, is associated with an increased risk of developing cancer, suggesting that CFTR can exert tumor-suppressor functions. Three-dimensional (3D) in vitro culture models, such as the CF lung airway-on-a-chip, can be suitable for studying PMN recruitment, as well as events of cancerogenesis, that is epithelial cell invasion and migration, in CF. To gather insight into the pathobiology of CFTR loss-of-function, we generated CFTR-knockout (KO) clones of the 16HBE14o- human bronchial cell line by CRISPR/Cas9 gene editing, and performed a comparative proteomic analysis of these clones with their wild-type (WT) counterparts. Systematic signaling pathway analysis of CFTR-KO clones revealed modulation of inflammation, PMN recruitment, epithelial cell migration, and epithelial-mesenchymal transition. Using a latest-generation organ-on-a-chip microfluidic platform, we confirmed that CFTR-KO enhanced PMN recruitment and epithelial cell invasion of the endothelial layer. Thus, a dysfunctional CFTR affects multiple pathways in the airway epithelium that ultimately contribute to sustained inflammation and cancerogenesis in CF.},
}
@article {pmid41053552,
year = {2025},
author = {Qin, Z and Surnido, W and Mizuta, H and Uji, T},
title = {Stable transgene expression and CRISPR-mediated knock-in system of a bacteria-derived antibiotic selection gene in the green alga Ulva prolifera.},
journal = {BMC plant biology},
volume = {25},
number = {1},
pages = {1323},
pmid = {41053552},
issn = {1471-2229},
support = {JPMJOP1851//Japan Science and Technology Agency/ ; },
mesh = {*Ulva/genetics/drug effects ; *Gene Knock-In Techniques/methods ; *Transgenes/genetics ; Kanamycin Kinase/genetics ; CRISPR-Cas Systems ; Hygromycin B/pharmacology ; Anti-Bacterial Agents/pharmacology ; Edible Seaweeds ; },
abstract = {Ulva prolifera is a fast-growing green seaweed that has garnered considerable interest in both fundamental and applied research. Here, we established a molecular tool by employing a selectable marker gene that allowed the isolation of U. prolifera cells integrating exogenous DNA. We developed a modular plasmid for expressing exogenous genes in U. prolifera based on the bacterial antibiotic-resistance marker, aminoglycoside phosphotransferase gene (aph7"). Integration of aph7" in macroalgae can generate transformants resistant to hygromycin B. In addition, we characterized the promoter region of the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase gene (pUpRbcS) to drive the expression of aph7". The transcripts were consistently confirmed from antibiotic-selected transformants, stably retaining the exogenous gene in the succeeding generations. Subsequently, a CRISPR-based knock-in system was established, facilitating the integration of aph7" cassette in the endogenous selection gene encoding for adenine phosphoribosyltransferase (UpAPT). APT gene can serve as an endogenous marker in algae that exhibits a lethal phenotype under cultivation with 2-fluoroadenine. The resulting knock-in mutants could resist the co-selection of the antibiotic hygromycin B and 2-fluoroadenine. Our results advance U. prolifera as a genetic platform, enabling functional research to elucidate Ulva biology, and to bring forth biotechnological utilization of algal resources.},
}
@article {pmid41053297,
year = {2025},
author = {Vanderperre, B and Muraleedharan, A and Dorion, MF and Larroquette, F and Del Cid Pellitero, E and Rajakulendran, N and Chen, CX and Larivière, R and Michaud-Tardif, C and Goiran, T and Chidiac, R and Lipuma, D and MacLeod, G and Thomas, R and Wang, Z and Reintsch, WE and Luo, W and Shlaifer, I and Zhang, F and Xia, K and Steinhart, Z and Linhardt, RJ and Trempe, JF and Liu, J and Durcan, TM and Angers, S and Fon, EA},
title = {Novel regulators of heparan sulfate proteoglycans modulate cellular uptake of α-synuclein fibrils.},
journal = {Communications biology},
volume = {8},
number = {1},
pages = {1426},
pmid = {41053297},
issn = {2399-3642},
support = {021129//Michael J. Fox Foundation for Parkinson's Research (Michael J. Fox Foundation)/ ; MFE-152571//Gouvernement du Canada | Canadian Institutes of Health Research (Instituts de Recherche en Sant&#xe9; du Canada)/ ; Canada Research Chair (Tier 1) in Parkinson's disease//Gouvernement du Canada | Canadian Institutes of Health Research (Instituts de Recherche en Sant&#xe9; du Canada)/ ; },
mesh = {*alpha-Synuclein/metabolism/genetics ; Humans ; *Heparan Sulfate Proteoglycans/metabolism ; Golgi Apparatus/metabolism ; Animals ; CRISPR-Cas Systems ; Cation Transport Proteins/genetics/metabolism ; Mice ; },
abstract = {Synucleinopathies are characterized by the accumulation and propagation of α-synuclein (α-syn) aggregates throughout the brain, leading to neuronal dysfunction and death. In this study, we used an unbiased FACS-based genome-wide CRISPR/Cas9 knockout screening to identify genes that regulate the entry and accumulation of α-syn preformed fibrils (PFFs) in cells. We identified key genes and pathways specifically implicated in α-syn PFFs intracellular accumulation, including heparan sulfate proteoglycans (HSPG) biosynthesis and Golgi trafficking. All confirmed hits affected heparan sulfate (HS), a post-translational modification known to act as a receptor for proteinaceous aggregates including α-syn and tau. Intriguingly, deletion of SLC39A9 and C3orf58 genes, encoding respectively a Golgi-localized exporter of Zn[2+], and the Golgi-localized putative kinase DIPK2A, specifically impaired the uptake of α-syn PFFs, by preventing the binding of PFFs to the cell surface. Mass spectrometry-based analysis of HS chains in SLC39A9[-/-] and C3orf58[-/-] cells indicated major defects in HS homeostasis. Additionally, Golgi accumulation of NDST1, a prime HSPG biosynthetic enzyme, was detected in C3orf58[-/-] cells. Interestingly, C3orf58[-/-] human iPSC-derived microglia and dopaminergic neurons exhibited a strong reduction in their ability to internalize α-syn PFFs. Altogether, our data identifies new modulators of HSPGs that regulate α-syn PFFs cell surface binding and uptake.},
}
@article {pmid41051826,
year = {2025},
author = {McCallum, GE and Ho, SFS and Cummins, EA and Wildsmith, AJ and McInnes, RS and Weigel, C and Tong, LYS and Quick, J and van Schaik, W and Moran, RA},
title = {The Kocurious case of Noodlococcus: genomic insights into Kocuria rhizophila from characterisation of a laboratory contaminant.},
journal = {Microbial genomics},
volume = {11},
number = {10},
pages = {},
doi = {10.1099/mgen.0.001526},
pmid = {41051826},
issn = {2057-5858},
mesh = {*Genome, Bacterial ; Phylogeny ; Genomics ; *Micrococcaceae/genetics/classification/isolation &amp; purification ; Whole Genome Sequencing ; Sequence Analysis, DNA ; },
abstract = {The laboratory contaminant strain Noodlococcus was named for its coccoid cells and unusual colony morphology, which resembled a pile of noodles. Along with laboratory characterisation and electron microscopy, we generated a complete Noodlococcus genome sequence using Illumina and Oxford Nanopore data. The genome consisted of a single, circular, 2,732,108 bp chromosome that shared 97.5% average nucleotide identity (ANI) with the Kocuria rhizophila type strain TA68. We identified genomic features involved in replication (oriC), carotenoid synthesis (crt) and genome defence (CRISPR-Cas) and discovered four novel mobile elements (ISKrh4-7). Despite its environmental ubiquity and relevance to food production, bioremediation and human medicine, there have been few genomic studies of the Kocuria genus. We conducted a comparative, phylogenetic and pangenomic examination of all 257 publicly available Kocuria genomes, with a particular focus on the 56 that were identified as K. rhizophila. We found that there are two phylogenetically distinct clades of K. rhizophila, with within-clade ANI values of 96.7-100.0% and between-clade values of 89.5-90.4%. The second clade, which we refer to as Kocuria pseudorhizophila, exhibited ANI values of <95% relative to TA68 and should constitute a separate species. Delineation of the two clades would be consistent with the rest of the genus, where all other species satisfy the 95% ANI threshold criteria. Differences in the K. rhizophila and K. pseudorhizophila pangenomes likely reflect phenotypic as well as evolutionary divergence. This distinction is relevant to clinical and industrial settings, as strains and genomes from both clades are currently used interchangeably, which may lead to reproducibility issues and phenotype-genotype discordance. Investigating an innocuous laboratory contaminant has therefore provided useful insights into the understudied species K. rhizophila, prompting an unexpected reassessment of its taxonomy.},
}
@article {pmid41051673,
year = {2026},
author = {Kumar, P and Verma, V and Irfan, M},
title = {Target-Specific Single Guide RNA (sgRNA) Design and In Vitro Validation of Target-Specific sgRNAs for CRISPR/Cas9-Mediated Editing of a Plant Stress Memory-Associated Gene.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2988},
number = {},
pages = {145-156},
pmid = {41051673},
issn = {1940-6029},
mesh = {*CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; *RNA, Guide, CRISPR-Cas Systems/genetics ; *Arabidopsis/genetics ; *Stress, Physiological/genetics ; Arabidopsis Proteins/genetics ; Transcription Factors/genetics ; },
abstract = {CRISPR/Cas9 genome editing has emerged as a transformative tool in plant biology, enabling precise manipulation of genes involved in stress responses. In the context of plant stress memory, where prior exposure to environmental stress enhances subsequent stress tolerance. CRISPR-based approaches offer a powerful means to dissect and engineer underlying regulatory genes. A critical factor determining the success of CRISPR/Cas9 editing is the careful design and validation of single guide RNAs (sgRNAs), which guide the Cas9 nuclease to specific genomic targets. This chapter provides a detailed, step-by-step protocol for the design, in vitro transcription, and in vitro cleavage assay to check efficiency of target-specific sgRNAs for plant genome editing applications. As a case study, we describe the design and validation of sgRNAs targeting the Arabidopsis thaliana DREB2A gene, a key transcription factor associated with drought stress memory. Emphasis is placed on strategies to maximize on-target efficiency, minimize off-target effects, and assess sgRNA functionality in vitro prior to in planta applications. This chapter serves as a practical guide for researchers aiming to functionally characterize stress memory-associated genes using CRISPR/Cas9 technology.},
}
@article {pmid41051363,
year = {2025},
author = {Zhytnik, L and Ventura, L and Sclocco, A and Verhage, M and Bakker, AD and Shim, JH and Beaino, W and Pereira, PM and Hoogeland, ME and Heine, VM and Maas, H and Jaspers, RT and Niehoff, A and Zaucke, F and de Waard, V and Eekhoff, EMW and Micha, D},
title = {New Lens On Congenital Mild Bone Fragility: a Novel Col1a1 Knockout Mouse Model for Osteogenesis Imperfecta Type 1.},
journal = {Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research},
volume = {},
number = {},
pages = {},
doi = {10.1093/jbmr/zjaf138},
pmid = {41051363},
issn = {1523-4681},
abstract = {Osteogenesis imperfecta (OI) is a genetic disorder characterized by bone fragility. It is one of the most prevalent rare skeletal dysplasias. The mildest form, OI type 1, predominantly results from collagen type I haploinsufficiency due to pathogenic variants in the COL1A1 gene, leading to reduced collagen type I. Despite OI type 1 representing approximately half of the OI population, the lack of an effective mouse model has hindered research and therapy development(1). To address this gap, we developed a genetically engineered mouse model harbouring a heterozygous deletion of the Col1a1 allele using the CRISPR/Cas system. The bone phenotype was characterised in 8- and 24-week-old mice, assessing transcriptomics and serum markers for bone formation (procollagen type I N-terminal propeptide) and resorption (tartrate-resistant acid phosphatase 5b). Bone volume, microarchitecture, and strength were evaluated by micro-computed tomography, histomorphometry and three-point bending test. We showed that the decreased Col1a1 to Col1a2 mRNA ratio determines reduced collagen type I production in OI mice bones as the underlying mechanism of haploinsufficient OI. This was supported by COL1A1 to COL1A2 mRNA ratio findings in human OI cell models, including fibroblasts and induced mesenchymal stem cells, as well as in induced pluripotent and mesenchymal stem cell models that were edited to carry a heterozygous COL1A1 allele. Our findings indicate for the first time that reduced bone volume and altered bone microarchitecture in haploinsufficient OI depends on the Col1a1 to Col1a2 mRNA ratio regulation. This novel mouse model faithfully recapitulates OI type 1 and provides a vital tool for investigating the disease mechanism and developing targeted therapeutic strategies for this large neglected OI patient population.},
}
@article {pmid41048399,
year = {2025},
author = {Huang, S and Qin, H and Dai, B and Liu, M and Shen, J},
title = {Establishment and evaluation of a circAdpgk-0001 knockdown method using CRISPR-Cas13d RNA-targeting technology.},
journal = {PeerJ},
volume = {13},
number = {},
pages = {e20123},
pmid = {41048399},
issn = {2167-8359},
mesh = {*RNA, Circular/genetics ; *CRISPR-Cas Systems/genetics ; *Gene Knockdown Techniques/methods ; RNA, Small Interfering/genetics ; Humans ; Cell Line ; Actins/genetics/metabolism ; },
abstract = {BACKGROUND: The small interfering RNA (siRNA) method has been used to knock down circular RNAs (circRNAs). However, issues such as low efficiency and off-target effects have become increasingly recognized. Recent studies have demonstrated that CRISPR-Cas13 can specifically target and cleave RNA. In this study, we established a CRISPR-Cas13d-based RNA-targeting method to specifically knock down circRNAs, such as circAdpgk-0001, and compared its performance with the siRNA method.<br><br>METHODS: Four clustered regularly interspaced short palindromic repeats (CRISPR) RNAs (crRNAs) of different nucleotide lengths spanning the back-splicing junction (BSJ) of circAdpgk-0001 were designed. A CRISPR-RfxCas13d plasmid capable of specifically cleaving circAdpgk-0001 was constructed and transfected into the JS-1 cell line. Knockdown efficiency was assessed using quantitative real-time PCR (qRT-PCR) and compared with that of the siRNA method. The expression of activation-related factors alpha-smooth muscle actin (&#x3b1;-SMA) and collagen I in JS-1 cells was further evaluated using qRT-PCR and Western blot.<br><br>RESULTS: CRISPR-Cas13d with a 24-nucleotide crRNA showed the highest knockdown efficiency (&#x223c;50%). After further optimization, the knockdown efficiency of CRISPR-Cas13d reached 70%, significantly higher than that of the siRNA method (40%). Knockdown of circAdpgk-0001 using Cas13d reduced the expression of collagen I and &#x3b1;-SMA by approximately 40%, which was greater than the reduction achieved by siRNA-mediated knockdown.<br><br>CONCLUSION: CRISPR-Cas13d demonstrated higher efficiency than the siRNA method in knocking down circRNAs, providing a promising tool for investigating circRNA functions.},
}
@article {pmid41020566,
year = {2025},
author = {Xue, J and Mao, K and Tang, Z and Hu, J and Zhang, H},
title = {Machine-Learning-Assisted CRISPR/Cas12a Biosensors for Monitoring Organophosphorus Pesticide Degradation.},
journal = {Analytical chemistry},
volume = {97},
number = {39},
pages = {21491-21501},
doi = {10.1021/acs.analchem.5c03596},
pmid = {41020566},
issn = {1520-6882},
mesh = {*Biosensing Techniques/methods ; *Pesticides/analysis/metabolism ; *CRISPR-Cas Systems ; *Organophosphorus Compounds/analysis/metabolism ; *Machine Learning ; *Water Pollutants, Chemical/analysis/metabolism ; Smartphone ; Acetylcholinesterase/metabolism ; Manganese Compounds/chemistry ; Oxides/chemistry ; Bacterial Proteins/metabolism/genetics ; Limit of Detection ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {Owing to the severe environmental and health issues posed by organophosphorus pesticides (OPs), a dual-enzyme cascade biosensing platform based on manganese dioxide (MnO2) and CRISPR/Cas12a was developed in this study. Smartphones were innovatively integrated with a stacked ensemble learning (SEL) model for ultrasensitive detection and dynamic monitoring of OPs in environmental water samples. A dual-enzyme cascade signal amplification strategy was used to construct this sensing platform. Acetylcholinesterase (AChE) catalyzes the generation of thiocholine (TCh), which, in turn, regulates the degradation of MnO2 nanosheets, releasing Cas12a activators and generating a fluorescence signal. Owing to the irreversible inhibition of AChE activity by OPs, dichlorvos (DDVP) was successfully detected, with a detection limit as low as 4.62 pg/mL. Additionally, the SEL model, integrated into the smartphone biosensing platform and incorporating random forest (RF), XGBoost, and ridge regression algorithms, exhibited strong performance in detecting OPs after optimization (R[2] = 0.9985). In real water samples, the SEL model achieved a recovery rate of 93.1-103.1%, and the degradation kinetics of DDVP were successfully monitored over 24 h, revealing significant differences in DDVP degradation rates across various water matrices. This study is the first to report the integration of CRISPR/Cas12a biosensing technology with an SEL model-driven smartphone detection platform, providing a novel approach for sensitive, portable, and intelligent monitoring of OPs and offering new insights for water quality monitoring and early detection of environmental risks.},
}
@article {pmid40923283,
year = {2025},
author = {Man, Y and Posey, RR and Bai, H and Jiang, A and Dosta, P and Ocampo-Alvarado, D and Plebani, R and Ji, J and Belgur, C and Artzi, N and Ingber, DE},
title = {Preclinical assessment of pan-influenza A virus CRISPR RNA therapeutics in a human lung alveolus chip.},
journal = {Lab on a chip},
volume = {25},
number = {20},
pages = {5240-5254},
doi = {10.1039/d5lc00156k},
pmid = {40923283},
issn = {1473-0189},
mesh = {Humans ; *RNA, Viral/genetics ; *Pulmonary Alveoli/virology/cytology ; *CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; *Influenza A virus/genetics ; *Influenza A Virus, H3N2 Subtype/genetics ; *Lab-On-A-Chip Devices ; Influenza, Human/therapy ; },
abstract = {CRISPR technology offers an entirely new approach to therapeutic development because it can target specific nucleotide sequences with high specificity, however, preclinical animal models are not useful for evaluation of their efficacy and potential off-target effects because of high gene sequence variations between animals and humans. Here, we explored the potential of using the CRISPR effector Cas13 to develop a new therapeutic approach for influenza A virus (IAV) infections based on its ability to specifically and robustly cleave single-strand viral RNA using a complementary CRISPR RNA (crRNA). We engineered crRNAs to target highly conserved regions in the IAV genome to create a potential pan-viral treatment strategy. A human lung alveolus chip (Lung Chip) lined by human primary alveolar epithelial cells interfaced with human primary pulmonary microvascular endothelial cells and infected with a pandemic IAV H3N2 strain was used to evaluate the on-target and off-target effects of these antiviral crRNA therapeutics. Our data show that the crRNAs targeting highly conserved regions in the IAV genome potently reduced viral replication in the alveolar airspace in the Lung Chip, and this was accompanied by suppression of the human host inflammatory response as indicated by a significant reduction in cytokine production and recruitment of immune cells. Importantly, only minimal off-target effects were observed based on transcriptomic analyses. As these crRNAs inhibit replication of influenza H1N1 and H3N2 in A549 cells as well as H3N2 in Lung Chips, these findings support use of CRISPR-Cas13 as a potentially viable approach to develop pan-IAV therapeutics for combating future influenza pandemics. The results also demonstrate that human Organ Chips be useful as more clinically relevant preclinical models for testing the efficacy and safety of crRNA therapeutics.},
}
@article {pmid40910953,
year = {2025},
author = {Feng, D and Guo, J and Yan, J and Chen, J and Ding, L and Zhu, X and Chen, Z and Hu, Y and Zhang, M and Liu, J and Zhu, C and Liu, M and Zhao, C and Zhang, X and Xu, J},
title = {COG6 is an essential host factor for influenza A virus infection.},
journal = {Microbiology spectrum},
volume = {13},
number = {10},
pages = {e0136225},
doi = {10.1128/spectrum.01362-25},
pmid = {40910953},
issn = {2165-0497},
support = {2022YFC2604100//National Key Research and Development Program of China/ ; 2023YFC2605602//National Key Research and Development Program of China/ ; 82072273//National Natural Science Foundation of China/ ; },
mesh = {*Influenza A virus/physiology/genetics ; Humans ; Virus Replication ; Golgi Apparatus/metabolism ; Lysosomes/metabolism ; Animals ; *Influenza, Human/virology/metabolism ; *Host-Pathogen Interactions ; *Adaptor Proteins, Vesicular Transport/metabolism/genetics ; Viral Proteins/metabolism ; CRISPR-Cas Systems ; Madin Darby Canine Kidney Cells ; Dogs ; HEK293 Cells ; A549 Cells ; Virus Internalization ; },
abstract = {Influenza A virus (IAV) relies on the host cellular machinery to support its replication. Understanding these host dependencies can inform the development of novel antiviral strategies. In this study, we identified conserved oligomeric Golgi complex subunit 6 (COG6) as a novel host factor critical for IAV replication through a genome-wide clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) knockout screen. Disruption of COG6 significantly impaired viral replication. Mechanistically, COG6 supports IAV replication via two distinct means. First, consistent with the role of the COG complex in Golgi homeostasis, COG6 is required for the proper presentation of surface sialic acids, the primary receptor for IAV entry. Second, COG6 deficiency unexpectedly led to lysosome-dependent degradation of viral proteins. Notably, lysosomal activity was also upregulated in IAV-infected wild-type cells, albeit to a lesser extent than in COG6-deficient cells. Treatment with lysosomal inhibitors rescued viral protein stability in COG6 knockout cells. Protein interaction analysis further demonstrated that COG6-mediated stabilization of viral proteins did not rely on viral protein-COG6 interaction, refuting the hypothesis that COG6 acts as a shield factor to protect viral protein from lysosomal degradation. Moreover, knockout of other COG subunits produced similar antiviral effects, suggesting that an intact COG complex is required for IAV replication. Together, these findings uncover a critical role of the COG complex in regulating IAV replication and highlight a previously unappreciated functional link between the Golgi and lysosomes that could be exploited for treating IAV infections.IMPORTANCEDespite advances in virology, numerous host determinants facilitating influenza A virus (IAV) pathogenesis remain uncharacterized. Our study establishes conserved oligomeric Golgi complex subunit 6 (COG6) as a critical host factor promoting IAV infection through complementary mechanisms: receptor modulation and viral protein stabilization. This represents the first demonstration that the COG complex regulates viral pathogenesis through proteostasis mechanisms, fundamentally expanding our understanding of host-virus interactions at the organelle interface. These findings not only provide new perspectives on viral exploitation of Golgi trafficking networks but also identify potential therapeutic targets against evolving influenza strains.},
}
@article {pmid40865226,
year = {2025},
author = {Zhang, X and Zhao, J and Dong, Y and Zhang, C and Hu, X and Wang, S and Chen, Y},
title = {Magnetic relaxation switching biosensor based on CRISPR-mediated cascade reaction for the amplification-free detection of Salmonella.},
journal = {Journal of hazardous materials},
volume = {497},
number = {},
pages = {139664},
doi = {10.1016/j.jhazmat.2025.139664},
pmid = {40865226},
issn = {1873-3336},
mesh = {*Biosensing Techniques/methods ; *Salmonella typhimurium/isolation &amp; purification/genetics ; Alkaline Phosphatase/chemistry ; *CRISPR-Cas Systems ; Food Microbiology ; Limit of Detection ; Magnetite Nanoparticles/chemistry ; DNA, Single-Stranded ; DNA, Bacterial/analysis ; Food Contamination/analysis ; },
abstract = {Sensitive and accurate detection of foodborne pathogens is crucial for preventing foodborne outbreaks and ensuring public health safety, but challenged by extremely low infectious doses of many foodborne pathogens and matrix interference effects. In this study, we developed a magnetic relaxation switching biosensor boosted by CRISPR/Cas12a-mediated enzymatic cascade reaction (CMCR-MRS) for amplification-free detection of Salmonella typhimurium (S. typhimurium). CRISPR/Cas12a was designed to precisely target the pathogen-specific DNA and efficiently cleaved single-stranded DNA (ssDNA) immobilized on the magnetic nanoparticle-alkaline phosphatase (MNP-ALP) probes through trans-cleavage activity. Subsequently, the released ALP facilitated the conversion of paramagnetic Mn(VII) to Mn(II), which resulted in the change of transverse relaxation time (T2), achieving a high signal-to-background ratio with superior biocompatibility and minimal background interference, making them highly advantageous for sensitive detection in complex biological samples. Our assay showed a broad dynamic range from 40 to 10[7] CFU/mL and a limit of detection (LOD) of 10 CFU/mL for S. typhimurium without amplification. Furthermore, it has been successfully validated in real food samples, demonstrating strong consistency (R[2] = 0.989) with the quantitative real-time polymerase chain reaction (qPCR) test. CMCR-MRS can serve as a highly effective and reliable strategy for achieving sensitive and accurate pathogen detection.},
}
@article {pmid40843899,
year = {2025},
author = {Stuible, M and Alpuche-Lazcano, SP and Gervais, C and Ouimet, M and Lippens, J and Pagé, M and Morasse, A and Moraitis, AN and Durocher, Y},
title = {Endogenous Retrovirus-Like Particle-Deficient CHO Cells Can be Generated by CRISPR or shRNA and Enriched Based on Cell-Surface Expression of Retroviral Envelope Protein.},
journal = {Biotechnology and bioengineering},
volume = {122},
number = {11},
pages = {3192-3204},
doi = {10.1002/bit.70043},
pmid = {40843899},
issn = {1097-0290},
support = {//The authors received no specific funding for this work./ ; },
mesh = {CHO Cells ; Cricetulus ; Animals ; *Viral Envelope Proteins/genetics/metabolism ; *RNA, Small Interfering/genetics/metabolism ; *Endogenous Retroviruses/genetics ; *CRISPR-Cas Systems ; Cricetinae ; *Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {Despite evidence that they are not functional or infective, retrovirus-like particles (RVLPs), originating from endogenous proviral sequences in Chinese hamster ovary (CHO) cells, present a safety risk for biotherapeutics manufactured using this cell line due to their resemblance to other mammalian leukemia viruses. Here, we demonstrate that CRISPR- and shRNA-based cell engineering strategies can be used to disrupt RVLP production by targeting the RVLP nucleotide sequences. Additionally, specific antibodies were generated to monitor RVLP protein expression, including RVLP envelope (Env) protein localized on the surface of CHO cells, greatly facilitating selection of RVLP-deficient clones. These modified CHO cells showed reduced RVLP production while maintaining or enhancing the ability to produce recombinant virus-like particles (VLPs), highlighting their potential application in biomanufacturing, especially for complex biologics that are incompatible with standard RVLP mitigation procedures, namely viral inactivation and nanofiltration.},
}
@article {pmid40829398,
year = {2025},
author = {Song, D and Xu, C and Sang, P and Liu, Y and Huang, X},
title = {Rapid and contamination-free detection of cucumber green mottle mosaic virus as a viral indicator in wastewater via UDG-RT-LAMP combined with CRISPR/Cas12a.},
journal = {Journal of hazardous materials},
volume = {497},
number = {},
pages = {139571},
doi = {10.1016/j.jhazmat.2025.139571},
pmid = {40829398},
issn = {1873-3336},
mesh = {*Wastewater/virology ; CRISPR-Cas Systems ; *Nucleic Acid Amplification Techniques/methods ; *Tobamovirus/isolation &amp; purification/genetics ; Uracil-DNA Glycosidase/genetics ; Biosensing Techniques ; CRISPR-Associated Proteins/genetics ; Bacterial Proteins ; Endodeoxyribonucleases ; Molecular Diagnostic Techniques ; },
abstract = {The removal of viruses by wastewater treatment plants plays a pivotal role in ensuring water environment safety, where precise evaluation of elimination efficiency is essential for controlling viral dissemination. Current bacterial indicators exhibit limited correlations with virological safety parameters, whereas conventional viral detection methods face practical constraints such as high instrumentation requirements and long detection cycles. To overcome these limitations, this study presents an on-site detection method for cucumber green mottle mosaic virus (CGMMV) in wastewater as a viral indicator, integrating uracil-DNA glycosylase (UDG)-reverse transcription loop-mediated isothermal amplification (RT-LAMP) with CRISPR/Cas12a (clustered regularly interspaced short palindromic repeats/CRISPR-associated nuclease 12a)-mediated biosensing system. The developed method achieved effective prevention of aerosol contamination from residual amplicons via the dUTP-UDG system, rapid amplification via RT-LAMP, and improved sensitivity and visualization by CRISPR/Cas12a-mediated biosensing system. The potential of CGMMV as a viral indicator in wastewater treatment process was demonstrated, and the rapid detection was realized using the proposed method. This integrated approach achieves sensitive CGMMV detection (limit of detection of 1.13 copies/μL) within 35 min, demonstrating field applicability through equipment independence, contamination resistance, and rapid operation. The proposed assay offers a promising tool for rapid viral monitoring in wastewater treatment system for resource-limited settings.},
}
@article {pmid40810567,
year = {2025},
author = {Ji, C and Ru, L and Han, T and Mai, G and Zheng, L and Jiang, Y},
title = {VGRCOT: a one-tube visual detection method for group B Streptococcus combining RPA and CRISPR/Cas12a for point-of-care testing in reproductive health.},
journal = {Microbiology spectrum},
volume = {13},
number = {10},
pages = {e0139525},
doi = {10.1128/spectrum.01395-25},
pmid = {40810567},
issn = {2165-0497},
support = {2025ZNSFSC1561//Natural Science Foundation of Sichuan Province/ ; 2024SZY001//Key Research and Developmet Guidance Projects of Deyang City/ ; },
mesh = {Humans ; *Streptococcal Infections/diagnosis/microbiology ; *Streptococcus agalactiae/genetics/isolation &amp; purification ; *Point-of-Care Testing ; *CRISPR-Cas Systems ; Female ; Pregnancy ; Sensitivity and Specificity ; Pregnancy Complications, Infectious/diagnosis/microbiology ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {Group B Streptococcus (GBS) is a significant pathogen that causes perinatal infections, seriously threatening the health of pregnant women and newborns. Prophylactic antibiotic treatment for pregnant women who screen positive for GBS can notably reduce the incidence and fatality of neonatal infections. Herein, we developed a visual nucleic acid method for GBS that integrates RPA and CRISPR/Cas12a in a one-tube setup, termed VGRCOT. The VGRCOT method achieved one-tube detection by adding the appropriate reagents to the bottom and lid of the EP tube, respectively. By rigorous optimization of ssDNA-FQ reporter concentration, crRNA concentration, RPA reaction time, and CRISPR/Cas12a cleavage time, VGRCOT can exhibit fluorescence under ultraviolet light, enabling visual detection. Under optimal conditions, VGRCOT has a satisfactory selectivity, and the detection limit was determined as 10[1] copies/reaction. Finally, VGRCOT also showed good performance comparable to qPCR in the actual detection of clinical specimens. Due to its ease of operation and convenient signal acquisition, VGRCOT shows promise for point-of-care testing in reproductive health.IMPORTANCEThis study presents a convenient, sensitive, and accurate visual detection method (VGRCOT) for GBS, combining RPA and CRISPR/Cas12a in a single reaction vessel. Through optimization of experimental conditions, VGRCOT enables detection within 60 min, with a minimum detection limit of 10[1] copies per reaction. VGRCOT offers several advantages by adding the appropriate reagents to the bottom and lid of the EP tube. The one-tube visualization method effectively prevents aerosol contamination, simplifies procedures, and enables visual detection without complex instruments, making it ideal for resource-limited environments. Additionally, its editable crRNA and the use of commonly available laboratory reagents allow for easy reprogramming to detect various pathogens, supporting scalable and low-cost batch production.},
}
@article {pmid40645879,
year = {2025},
author = {Liang, Y and Tong, S and Zhang, J and Tan, GY and Zhang, L and Lee, SY and Tong, Y},
title = {Expanding horizons of CRISPR applications beyond genome editing.},
journal = {Trends in genetics : TIG},
volume = {41},
number = {10},
pages = {934-953},
doi = {10.1016/j.tig.2025.06.003},
pmid = {40645879},
issn = {0168-9525},
mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; Humans ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Biosensing Techniques ; Single-Cell Analysis ; },
abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR) technologies have rapidly evolved beyond genome editing, transforming fields such as molecular diagnostics, biosensing, transcriptional regulation, molecular imaging, protein interaction mapping, and single-cell analysis. Emerging CRISPR-based diagnostics harness the collateral cleavage activity of CRISPR-associated (Cas) enzymes for rapid nucleic acid detection. Advanced biosensors extend CRISPR's capabilities to detect ions, metabolites, and proteins by integrating synthetic biology components. Catalytically inactive Cas proteins enable precise gene regulation and live-cell imaging of nucleic acids, whereas CRISPR-guided proximity labeling has revolutionized the mapping of biomolecular interactions. Recent single-cell CRISPR screens provide unprecedented resolution of cellular heterogeneity. Future research will focus on overcoming current limitations. The integration of CRISPR technologies with artificial intelligence (AI), spatial omics, and microfluidics is expected to further amplify their impact.},
}
@article {pmid40032999,
year = {2025},
author = {Sahu, SU and Castro, M and Muldoon, JJ and Asija, K and Wyman, SK and Krishnappa, N and de Oñate, L and Eyquem, J and Nguyen, DN and Wilson, RC},
title = {Peptide-enabled ribonucleoprotein delivery for CRISPR engineering (PERC) in primary human immune cells and hematopoietic stem cells.},
journal = {Nature protocols},
volume = {20},
number = {10},
pages = {2735-2770},
pmid = {40032999},
issn = {1750-2799},
support = {K08 AI153767/AI/NIAID NIH HHS/United States ; L40 AI140341/AI/NIAID NIH HHS/United States ; K08AI153767//U.S. Department of Health &amp; Human Services | NIH | Office of Extramural Research, National Institutes of Health (OER)/ ; UG3AI150552//U.S. Department of Health &amp; Human Services | NIH | Office of Extramural Research, National Institutes of Health (OER)/ ; L40AI140341//U.S. Department of Health &amp; Human Services | NIH | Office of Extramural Research, National Institutes of Health (OER)/ ; },
mesh = {Humans ; *Hematopoietic Stem Cells/metabolism ; *Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; *Ribonucleoproteins/genetics/metabolism ; *Peptides/chemistry ; T-Lymphocytes/metabolism ; Cells, Cultured ; },
abstract = {Peptide-enabled ribonucleoprotein delivery for CRISPR engineering (PERC) is a new approach for ex vivo genome editing of primary human cells. PERC uses a single amphiphilic peptide reagent to mediate intracellular delivery of the same pre-formed CRISPR ribonucleoprotein enzymes that are broadly used in research and therapeutics, resulting in high-efficiency editing of stimulated immune cells and cultured hematopoietic stem and progenitor cells (HSPCs). PERC facilitates nuclease-mediated gene knockout, precise transgene knock-in and base editing. The protocol involves mixing the CRISPR ribonucleoprotein enzyme with peptide and then incubating with cultured cells. For efficient transgene knock-in, adeno-associated virus (AAV) homology-directed repair template (HDRT) DNA may be included. In contrast to electroporation, PERC is appealing because it needs no dedicated hardware and has less impact on cell phenotype and viability. Because of the gentle nature of PERC, delivery can be performed multiple times without substantial impact to cell health or phenotype. Editing efficiencies can surpass 90% when using either Cas9 or Cas12a in primary T cells or HSPCs. After 3 h dedicated to reagent preparation, the PERC delivery step can be completed in 1 h, with the associated cell culture steps taking 3-7 d total. Because the protocol calls for only three readily available reagents (protein, RNA and peptide) and does not require dedicated hardware for any step, PERC demands no special expertise and is exceptionally straightforward to adopt. The inherent compatibility of PERC with established cell engineering pipelines makes the protocol appealing for rapid deployment in research and clinical settings.},
}
@article {pmid41046988,
year = {2025},
author = {Chen, Y and Qi, ZD and Ji, R and Shi, N and Chen, H and Wei, DX},
title = {Synthetic biology for scalable production of medical polyhydroxyalkanoates: Advances and applications.},
journal = {Biotechnology advances},
volume = {},
number = {},
pages = {108722},
doi = {10.1016/j.biotechadv.2025.108722},
pmid = {41046988},
issn = {1873-1899},
abstract = {Polyhydroxyalkanoates (PHAs), characterized by their biodegradability and biocompatibility, present a promising, sustainable alternative to conventional synthetic polymers for biomedical applications. This study highlights the diversity of PHA monomers and structures, controllable biodegradability, and excellent biocompatibility, emphasizing their suitability for tissue engineering (bone, skin, cardiovascular, oral), anti-hair loss treatments, and drug delivery systems. Significant advancements in synthetic biology, encompassing CRISPR/Cas genome editing, promoter engineering, ribosome binding site optimization, metabolic pathway fine-tuning, and morphology engineering, have led to substantial improvements in PHA production efficiency and a reduction in associated costs. The adoption of next-generation industrial biotechnology (NGIB) using halophiles further enhances economic viability and simplifies the production process. The current commercial landscape and the future prospects of medical-grade PHAs, poised to become mainstream biodegradable materials, are also critically discussed.},
}
@article {pmid41046905,
year = {2025},
author = {Jaballah, SA and Ali, LM and Jehad, MA and Akhlaq, S and Rizvi, TA and Mustafa, F},
title = {Retroviral Vector Technology for Gene Therapy: History, Current Landscape, and Future Prospects.},
journal = {Journal of molecular biology},
volume = {},
number = {},
pages = {169473},
doi = {10.1016/j.jmb.2025.169473},
pmid = {41046905},
issn = {1089-8638},
abstract = {The concept of gene therapy and its practice has been prevalent for over five decades. The first successful retroviral vector-based gene therapy trial took place ∼35 years ago, followed by several setbacks. However, recent years have seen a surge in successes, offering new hope to patients with genetic and other disorders once deemed untreatable. Over the past decade, rapid advancements in molecular biology have led to the development of safer and more effective gene therapy strategies with various gene delivery systems now in use. Among these, viral vectors such as retroviruses, adenoviruses, and adeno-associated viruses are the most widely employed in both research and clinical settings. This is due to their natural efficiency in delivering genetic material into target cells. Among these viral vectors, retroviruses stand out for their unique ability to reverse-transcribe and integrate their genetic material into the host genome, ensuring stable and long-term gene expression. This review highlights advances in retroviral vector development, examining both their therapeutic potential and associated challenges. It also explores strategies for vector production, including transient and stable systems tailored to meet clinical and regulatory demands. Significant progress is discussed in mitigating insertional mutagenesis and vector silencing. As a result, next-generation retroviral vectors with improved safety and efficacy have made it past regulatory-approval and are commercially available. Current innovations include replication-competent, non-integrating, integration-re-targeted, and hybrid CRISPR/Cas-expressing retroviral vectors undergoing pre-clinical and clinical investigations. This reflects a new era in gene therapy, with retroviral vectors reimagined for greater precision, control, and therapeutic impact.},
}
@article {pmid41045024,
year = {2025},
author = {Thomson, G and Mermaz, B and Sagawa, CHD and Lin, CI and Tachev, M and Joly, V and Irish, VF and Jacob, Y},
title = {Enzymatic depletion of transposable elements in sequencing libraries and its application for genotyping multiplexed CRISPR-edited plants.},
journal = {The Plant journal : for cell and molecular biology},
volume = {124},
number = {1},
pages = {e70501},
doi = {10.1111/tpj.70501},
pmid = {41045024},
issn = {1365-313X},
support = {2023-70029-41277//U.S. Department of Agriculture/ ; },
mesh = {*DNA Transposable Elements/genetics ; Zea mays/genetics ; *Gene Editing/methods ; Genome, Plant/genetics ; DNA Methylation ; *Citrus/genetics ; Gene Library ; CRISPR-Cas Systems/genetics ; *Genotyping Techniques/methods ; Genotype ; High-Throughput Nucleotide Sequencing/methods ; Sequence Analysis, DNA/methods ; },
abstract = {Whole-genome sequencing has become a common strategy to genotype individual plants of interest. Although a limited number of genomic regions usually need to be surveyed with this strategy, excess sequencing information is almost always generated at an appreciable financial cost. Repetitive sequences (e.g., transposons), which can account for more than 80% of the genome of some plants, are often not required in these genotyping projects. Therefore, strategies that enrich DNA coding for the protein-coding genes prior to sequencing can lower the cost to obtain sufficient sequence information. Here, we present the development and application of methylation-sensitive reduced representation sequencing (MsRR-Seq), which relies on the cytosine methylation-sensitive restriction enzyme MspJI to deplete constitutive heterochromatic DNA before library construction. By applying MsRR-Seq to citrus and maize, we show that protein-coding genes can be enriched in sequencing datasets. We then describe the application of MsRR-Seq to facilitate the identification of complex mutants from populations of citrus plants resulting from multiplex CRISPR/Cas9 editing of four genes. Overall, this work demonstrates an easy and low-cost method to enrich non-repetitive DNA in high-throughput sequencing libraries, an approach that is especially useful for large plant genomes with an excessively high proportion of methylated repetitive sequences.},
}
@article {pmid41044600,
year = {2025},
author = {Conery, M and Pippin, JA and Wagley, Y and Trang, K and Pahl, MC and Villani, DA and Favazzo, LJ and Ackert-Bicknell, CL and Zuscik, MJ and Katsevich, E and Wells, AD and Zemel, BS and Voight, BF and Hankenson, KD and Chesi, A and Grant, SFA},
title = {GWAS-informed data integration and non-coding CRISPRi screen illuminate genetic etiology of bone mineral density.},
journal = {Genome biology},
volume = {26},
number = {1},
pages = {331},
pmid = {41044600},
issn = {1474-760X},
support = {Gates Grubstake Award//University of Colorado/ ; DMS 2113072//National Science Foundation, United States/ ; R01DK122586//National Institute of Diabetes and Digestive and Kidney Diseases,United States/ ; UM1 DK126194/DK/NIDDK NIH HHS/United States ; R01AI154773//National Institute of Allergy and Infectious Diseases/ ; R01 HD100406/HD/NICHD NIH HHS/United States ; UL1 TR001878/TR/NCATS NIH HHS/United States ; Henry Ruppenthal Family Professorship for Bioengineering and Orthopaedic Surgery//University of Michigan/ ; R01 AG072705/AG/NIA NIH HHS/United States ; Daniel B. Burke Endowed Chair for Diabetes Research//Children's Hospital of Philadelphia/ ; },
mesh = {Humans ; *Bone Density/genetics ; *Genome-Wide Association Study ; Osteoblasts/metabolism ; *CRISPR-Cas Systems ; Single-Cell Analysis ; Quantitative Trait Loci ; },
abstract = {BACKGROUND: Over 1100 independent signals have been identified with genome-wide association studies (GWAS) for bone mineral density (BMD), a key risk factor for mortality-increasing fragility fractures; however, the effector gene(s) for most remain unknown.<br><br>RESULTS: We execute a CRISPRi screen in human fetal osteoblasts (hFOBs) with single-cell RNA-seq read-out for 89 non-coding elements predicted to regulate osteoblast gene expression at BMD GWAS loci. The BMD relevance of hFOBs is supported by heritability enrichment from stratified LD-score regression involving 98 cell types grouped into 15 tissues. Twenty-three genes show perturbation in the screen, with four (ARID5B, CC2D1B, EIF4G2, and NCOA3) exhibiting consistent effects upon siRNA knockdown on three measures of osteoblast maturation and mineralization. Lastly, additional heritability enrichments, genetic correlations, and multi-trait fine-mapping unexpectedly reveal that many BMD GWAS signals are pleiotropic and likely mediate their effects via non-bone tissues.<br><br>CONCLUSIONS: Our results provide a roadmap for how single-cell CRISPRi screens may be applied to the challenging task of resolving effector gene identities at all BMD GWAS loci. Extending our CRISPRi screening approach to other tissues could play a key role in fully elucidating the etiology of BMD.},
}
@article {pmid41041610,
year = {2025},
author = {de Mello Fiallos, N and Irfan, M and Solbiati, J and R Walker, A and Frias-Lopez, J and Gibson, FC},
title = {CRISPR cas7 influences the host-pathogen interaction of Porphyromonas gingivalis.},
journal = {Journal of oral microbiology},
volume = {17},
number = {1},
pages = {2561790},
pmid = {41041610},
issn = {2000-2297},
abstract = {INTRODUCTION: Porphyromonas gingivalis, a Gram-negative anaerobe, is a key contributor to periodontal disease. Emerging evidence suggests a role for the P. gingivalis CRISPR-Cas system in disease progression, although the specific roles of its components remain unclear.<br><br>OBJECTIVES: Here we investigate the role of cas7, a Class 1 type I-B CRISPR-Cas system component, in P. gingivalis physiology and host interaction.<br><br>METHODS: We compared P. gingivalis wild-type and &#x2206;cas7 strains for growth, biofilm formation, oxidative stress resistance, and hemagglutination. Host interactions were assessed using THP-1 macrophage-like cells to evaluate intracellular survival and cytokine response. Dual RNA-seq enabled host and microbe transcriptomic profiling during cellular infection, and Galleria mellonella was used to assess virulence.<br><br>RESULTS: The &#x2206;cas7 mutant showed similar planktonic growth and biofilm formation compared to wild-type but was more sensitive to oxidative stress and had reduced hemagglutination. Although intracellular survival was unaffected, &#x2206;cas7 altered the host cytokine production profile. Transcriptomic analysis revealed differential gene expression linked to oxidative stress and disease progression. In vivo, &#x2206;cas7 infection led to a trend of increased larval mortality.<br><br>CONCLUSION: These findings reveal a previously unrecognized role for cas7 in modulating P. gingivalis virulence, offering new insights into CRISPR-Cas system functions in bacterial pathogenesis.},
}
@article {pmid40974079,
year = {2025},
author = {Mariki, A and Kohlmeier, KA and Mousavi, SM and Shabani, M},
title = {CRISPR and Myelin regeneration: a systematic review of applications in demyelinating CNS Disorders, with a focus on MS.},
journal = {Regenerative medicine},
volume = {20},
number = {9},
pages = {431-443},
doi = {10.1080/17460751.2025.2561451},
pmid = {40974079},
issn = {1746-076X},
mesh = {Humans ; Animals ; *Myelin Sheath/metabolism ; *Multiple Sclerosis/therapy/genetics ; *CRISPR-Cas Systems ; Gene Editing ; *Demyelinating Diseases/therapy/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; *Regeneration/genetics ; },
abstract = {AIMS: Current treatments for demyelinating disorders focus on slowing progression but fail to repair damaged myelin. Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) -based technology has the potential to address key challenges in myelin repair by targeting genetic dysfunctions, modulating immune responses, and promoting oligodendrocyte differentiation. This systematic review aimed to evaluate CRISPR applications for myelin regeneration.<br><br>METHODS: A comprehensive search of PubMed, Scopus, and other databases identified 48 studies. The included studies employed CRISPR in diverse experimental models, targeting genes associated with immune regulation and astrocyte activity, as well as correcting RNA splicing dysfunctions linked to neurodegeneration.<br><br>RESULTS: CRISPR-edited stem cells showed significant potential in promoting myelin regeneration, with enhanced functional recovery in animal models of multiple sclerosis (MS). While most research focused on MS, promising applications were also observed in neuromyelitis optica spectrum disorder (NMOSD), such as reducing astrocytic damage via AQP4 targeting, and in progressive multifocal leukoencephalopathy (PML), where CRISPR disrupted JC polyomavirus replication.<br><br>CONCLUSIONS: Despite its promise, challenges remain. Future research should prioritize optimizing CRISPR delivery systems, expanding applications to underexplored disorders, and conducting long-term safety assessments. Early results are encouraging, but further studies are essential to translate preclinical success into clinical therapies.},
}
@article {pmid40972450,
year = {2026},
author = {Eom, KH and Yum, SY and Gim, GM and Kim, YC and Moon, B and Jang, G},
title = {SpCas9-mediated gene editing in bovine embryo via single adeno-associated virus infection using a novel micro-sized promoter.},
journal = {Theriogenology},
volume = {249},
number = {},
pages = {117676},
doi = {10.1016/j.theriogenology.2025.117676},
pmid = {40972450},
issn = {1879-3231},
mesh = {Animals ; Cattle/embryology/genetics ; *Gene Editing/veterinary/methods ; *Promoter Regions, Genetic ; *Dependovirus/genetics ; Genetic Vectors ; Embryo, Mammalian ; *CRISPR-Associated Protein 9/genetics/metabolism ; CRISPR-Cas Systems ; Fertilization in Vitro/veterinary ; },
abstract = {Genome editing in livestock offers practical solutions to address challenges related to land use, climate change, and food production. However, conventional delivery methods such as electroporation and microinjection impose physical stress on embryos, limiting scalability. This study aimed to develop a simplified, non-invasive, and scalable genome editing system for bovine embryos by designing an all-in-one adeno-associated virus (AAV) vector. A novel micro-sized promoter (50 bp), derived from the core regulatory region upstream of the bovine MSTN gene, was constructed to enable expression of Streptococcus pyogenes Cas9 (spCas9) within the AAV packaging limit (∼4.7 kb). This promoter was incorporated into an AAV cassette containing spCas9, a polyadenylation signal, a U6 promoter, and a single-guide RNA (sgRNA) targeting the bovine ALB gene. After confirming editing activity in bovine fibroblasts, the AAV6 vector was added directly to in vitro fertilization (IVF) cultures without physical manipulation. Genome editing was successfully induced, with insertion/deletion (indel) mutations detected in 33.8 ± 23.2 % of the blastocysts. Although blastocyst development was moderately reduced, gene editing was achieved without invasive techniques. These results demonstrate that a micro-promoter-based AAV system can support spCas9-mediated genome editing in bovine embryos through a single-vector infection strategy. The system presents a promising platform for producing gene-edited livestock and may contribute to more efficient and less labor-intensive applications in animal biotechnology.},
}
@article {pmid40650655,
year = {2025},
author = {Li, Y and Zhang, Y and Li, C and Chen, G and Muhammad, P and Yao, Y and Gao, L and Liu, Z and Wang, Y},
title = {Advanced Cancer Immunotherapy via SMARCAL1 Blockade Using a Glucose-Responsive CRISPR Nanovaccine.},
journal = {Advanced science (Weinheim, Baden-Wurttemberg, Germany)},
volume = {12},
number = {37},
pages = {e02929},
doi = {10.1002/advs.202502929},
pmid = {40650655},
issn = {2198-3844},
support = {81901684//National Natural Science Foundation of China/ ; },
mesh = {*Immunotherapy/methods ; Animals ; Mice ; *Glucose/metabolism ; Humans ; *Cancer Vaccines/immunology/genetics ; *Neoplasms/therapy/immunology ; CRISPR-Cas Systems/genetics ; Glucose Oxidase ; Gene Editing/methods ; Membrane Proteins/metabolism ; Nanovaccines ; },
abstract = {Cancer immunotherapy that activates the stimulator of interferon genes (STING) signaling pathway to resist tumors has recently attracted considerable attention. However, STING activation can induce opposing interferon functions that contribute to T-cell exhaustion via programmed death-ligand 1 (PD-L1). In particular, effectively using the immune system to combat tumors remains a substantial challenge due to tumor immunosuppressive factors such as SMARCAL1. Here, a glucose-responsive CRISPR nanovaccine is developed for enhancing STING signaling while inhibiting interferon-mediated immunosuppressive feedback. The formulation encapsulates a bimetallic zeolitic imidazolate framework with glucose oxidase (GOx) and CRISPR-mediated SMARCAL1 gene-editing plasmids. The dual enzyme-driven cascade reactions of peroxidase and GOx generate reactive oxygen species (ROS) and gluconic acid, which release and activate the genome-editing system. The silencing of SMARCAL1 enhances STING activity and inhibits PD-L1 expression, resulting in the termination of PD-L1-mediated opposing functions of interferon. Zinc ions and double-stranded DNA formed via ROS further activate the STING pathway, effectively inducing dendritic cell maturation and immune system activation. This is a critical report of in situ CRISPR nanovaccination driven by dual enzymes. The work highlights the potential of glucose-responsive CRISPR nanovaccination in bolstering antitumor immunity and extends the implementation of gene editing in cancer immunotherapy.},
}
@article {pmid40642954,
year = {2025},
author = {Fu, X and Wang, N and Li, L and Qiao, D and Qi, X and Liu, C and Gao, Z and Xie, C and Zhu, J},
title = {Development of cytosine and adenine base editors for maize precision breeding.},
journal = {Journal of integrative plant biology},
volume = {67},
number = {10},
pages = {2731-2743},
doi = {10.1111/jipb.13964},
pmid = {40642954},
issn = {1744-7909},
support = {No. 2023YFD1202901//National Key Research and Development Program of China Stem Cell and Translational Research/ ; 241111112300//Henan Province key research and development project/ ; //Xinjiang "Leading the Charge with Open Competition" project/ ; No. 2022ZD04006//Science and Technology Innovation 2030 Major projects/ ; },
mesh = {*Zea mays/genetics/drug effects ; *Gene Editing/methods ; *Adenine/metabolism ; *Cytosine/metabolism ; *Plant Breeding/methods ; Plants, Genetically Modified ; Mutation/genetics ; CRISPR-Cas Systems/genetics ; },
abstract = {Base editing technologies can improve crops, but their efficiency in maize remains suboptimal. This study attempts to overcome these limitations by examining optimized cytosine and adenine base editors (CBEs and ABEs), namely evoAPOBEC1, evoFERNY, evoCDA1, TadA8.20, and TadA8e, for precise genome editing in transient and stable expression maize cells. Employing a seed fluorescence reporter (SFR) system for rapid screening of BE transformants and transgene-free progenies, we enhanced editing efficiencies and heritability. Notably, TadA8.20 and evoCDA1 attained multiplexed editing efficiencies of up to 100.0% and 79.0% at the tested loci, respectively, with some homozygous and bi-allelic mutants exceeding 72.4% and 73.7%. Precise editing of ZmACC1/2 (acetyl-CoA carboxylase) improved herbicide resistance, with ZmACC2 mutants displaying improved performance. This study advances crop genetic engineering by facilitating robust, multi-locus modifications without altered agronomic performance, enhancing herbicide tolerance in maize. The successful utilization of these BE is a significant step forward in agricultural biotechnology and precision breeding.},
}
@article {pmid39994413,
year = {2025},
author = {Oliynyk, RT and Church, GM},
title = {Circular Vectors as an efficient, fully synthetic, cell-free approach for preparing small circular DNA as a plasmid substitute for guide RNA expression in CRISPR-Cas9 genome editing.},
journal = {Nature protocols},
volume = {20},
number = {10},
pages = {2942-2959},
pmid = {39994413},
issn = {1750-2799},
mesh = {*RNA, Guide, CRISPR-Cas Systems/genetics ; *Plasmids/genetics ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; *Genetic Vectors/genetics ; *DNA, Circular/genetics ; Cell-Free System ; Humans ; },
abstract = {Robust expression of guide RNA (gRNA) is essential for successful implementation of CRISPR-Cas9 genome-editing methods. The gRNA components, such as an RNA polymerase promoter followed by the gRNA coding sequence and an RNA polymerase terminator sequence, and the Cas9 protein are expressed either via an all-in-one plasmid or separate dedicated plasmids. The preparation of such plasmids involves a laborious multi-day process of DNA assembly, bacterial cloning, validation, purification and sequencing. Our Circular Vector (CV) protocol introduces an efficient, fully synthetic, cell-free approach for preparing gRNA expression templates suitable for transfection, marking a significant advancement over traditional plasmid-based approaches. This protocol consists of the circularization and purification of linear double-stranded DNA (dsDNA) containing gRNA expression elements into compact, bacterial-backbone-free circular DNA expression vectors in as little as 3 h. We provide a guide to the design of the dsDNA template coding for gRNA elements for CRISPR-Cas9 base and prime editing, along with step-by-step instructions for the efficient preparation of gRNA-expressing CVs. In addition to rapid preparation, CVs created via this protocol offer several key advantages: a compact size, absence of a bacterial backbone, absence of bacterial endotoxins and no contamination by bacterial RNA or DNA fragments. These features make gRNA-expressing CVs a superior choice over plasmid-based gRNA expression templates.},
}
@article {pmid41039221,
year = {2025},
author = {Ilmi, AFN and Kaewsapsak, P and Rotcheewaphan, S},
title = {Repression of mab_1999 impairs growth and alters cellular morphology of Mycobacterium abscessus.},
journal = {BMC microbiology},
volume = {25},
number = {1},
pages = {599},
pmid = {41039221},
issn = {1471-2180},
mesh = {*Mycobacterium abscessus/growth &amp; development/genetics/drug effects/cytology/metabolism ; *Bacterial Proteins/genetics/metabolism ; Anti-Bacterial Agents/pharmacology ; Microbial Sensitivity Tests ; Cell Division/genetics ; Gene Expression Regulation, Bacterial ; Mycobacterium smegmatis/genetics ; Gene Knockdown Techniques ; CRISPR-Cas Systems ; },
abstract = {BACKGROUND: Cell division is essential for bacterial survival and represents a promising target for the development of novel antibiotics, particularly in mycobacteria. The role of the division protein FtsL in Mycobacterium abscessus remains poorly understood. This study investigated the effects of MAB_1999, a predicted homolog of FtsL, on the growth and cell division of M. abscessus.<br><br>METHOD: To investigate the function of mab_1999, a knockdown mutant was generated via CRISPR interference (CRISPRi). The phenotypic impact of mab_1999 suppression was evaluated, with a focus on its effects on M. abscessus growth, cellular morphology, and antibiotic susceptibility.<br><br>RESULTS: The putative homolog of FtsL in M. abscessus (MAB_1999) shares 54% amino acid sequence identity with FtsL from M. smegmatis (MSMEG_4234). CRISPRi-mediated repression of mab_1999 expression resulted in cell elongation and growth defects, although complete growth arrest was not observed. Furthermore, reduced mab_1999 expression increased the susceptibility of M. abscessus to &#x3b2;-lactam antibiotics, including ceftriaxone and imipenem.<br><br>CONCLUSIONS: Our findings suggest that mab_1999 is involved in cell division and cell wall integrity in M. abscessus. However, further investigation is necessary to confirm its identity as FtsL and to fully elucidate its role in the cell division process and cell wall synthesis.},
}
@article {pmid41039127,
year = {2025},
author = {Safenkova, IV and Kamionskaya, MV and Ivanov, AV and Zherdev, AV and Dzantiev, BB},
title = {A novel tripod probe and lateral flow test to improve CRISPR/Cas12a assay: benefits of branched probe based on trebler phosphoramidite modification.},
journal = {Mikrochimica acta},
volume = {192},
number = {11},
pages = {711},
pmid = {41039127},
issn = {1436-5073},
support = {23-46-10011//Russian Science Foundation/ ; },
mesh = {*CRISPR-Cas Systems ; *DNA Probes/chemistry ; *Biosensing Techniques/methods ; *Organophosphorus Compounds/chemistry ; Fluorescent Dyes/chemistry ; Limit of Detection ; Gold/chemistry ; *CRISPR-Associated Proteins/metabolism ; Metal Nanoparticles/chemistry ; *Bacterial Proteins/metabolism ; *Endodeoxyribonucleases/metabolism ; },
abstract = {CRISPR/Cas12a-based assays, when integrated with lateral flow tests (LFTs), provide highly specific nucleic acid detection in a simple, rapid, and equipment-free format. Nevertheless, traditional DNA probes utilized for cleavage by Cas12a have limitations as the cleaved probe only has one label. To overcome this challenge, we engineered a novel type of DNA probe with multiple fluorescein (FAM) labels and a biotin-labeled single-stranded DNA fragment (polyFAM probe). The cleaved polyFAM parts of the probes were detected using a specially designed sandwich LFT, where FAM-specific antibodies were immobilized in the test zone and conjugated with gold nanoparticles. The LFT ensured accurate recognition of the cleaved polyFAM fragments within 10 min. A comparison of five distinct polyFAM probes revealed that the highest signal-to-noise ratio was achieved with a tripod-branched probe synthesized via trebler phosphoramidite modification. Each arm of the tripod probe consists of a hexaethylene glycol spacer ending in a FAM label. Upon Cas12a cleavage, the tripod structure carrying three FAMs is released and detected by LFT. A rapid magnetic separation strategy was subsequently implemented, facilitating the efficient removal of uncleaved probes via biotin-streptavidin capture within 5 min. The CRISPR/Cas12a-tripod-LFT strategy demonstrated excellent sensitivity without preamplification, with a detection Limit of 1.4 pM for DNA target of Salmonella Typhimurium. The CRISPR/Cas12a-tripod-LFT with preliminary loop-mediated isothermal amplification enabled the detection of as few as 0.3 cells per reaction. This innovative tripod probe with corresponding LFT creates a universal, sensitive, rapid, and equipment-free biosensing platform for CRISPR/Cas12a-based diagnostics in point-of-care applications.},
}
@article {pmid40847019,
year = {2025},
author = {Ramani, B and Rose, IVL and Teyssier, N and Pan, A and Danner-Bocks, S and Sanghal, T and Yadanar, L and Tian, R and Ma, K and Palop, JJ and Kampmann, M},
title = {CRISPR screening by AAV episome-sequencing (CrAAVe-seq): a scalable cell-type-specific in vivo platform uncovers neuronal essential genes.},
journal = {Nature neuroscience},
volume = {28},
number = {10},
pages = {2129-2140},
pmid = {40847019},
issn = {1546-1726},
support = {R01 AG082141/AG/NIA NIH HHS/United States ; K99 AG062776/AG/NIA NIH HHS/United States ; RF1 AG062234/AG/NIA NIH HHS/United States ; R25 NS070680/NS/NINDS NIH HHS/United States ; K08 NS133300/NS/NINDS NIH HHS/United States ; T32 NS115706/NS/NINDS NIH HHS/United States ; EDUC4-12812//California Institute for Regenerative Medicine (CIRM)/ ; UCSF Hillblom/BARI Graduate Fellowship Award//Larry L. Hillblom Foundation (Larry L. Hillblom Foundation, Inc.)/ ; R01 AG082141/AG/NIA NIH HHS/United States ; K99 AG062776/AG/NIA NIH HHS/United States ; RF1 AG062234/AG/NIA NIH HHS/United States ; R25 NS070680/NS/NINDS NIH HHS/United States ; K08 NS133300/NS/NINDS NIH HHS/United States ; T32 NS115706/NS/NINDS NIH HHS/United States ; },
mesh = {Animals ; *Dependovirus/genetics ; *Neurons/metabolism ; Mice ; *CRISPR-Cas Systems/genetics ; *Genes, Essential/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Mice, Inbred C57BL ; *Genetic Testing/methods ; Humans ; },
abstract = {There is a substantial need for scalable CRISPR-based genetic screening methods that can be applied in mammalian tissues in vivo while enabling cell-type-specific analysis. Here we developed an adeno-associated virus (AAV)-based CRISPR screening platform, CrAAVe-seq, that incorporates a Cre-sensitive sgRNA construct for pooled screening within targeted cell populations in mouse tissues. We used this approach to screen two large sgRNA libraries, which collectively target over 5,000 genes, in mouse brains and uncovered genes essential for neuronal survival, of which we validated Rabggta and Hspa5. We highlight the reproducibility and scalability of the platform and show that it is sufficiently sensitive for screening in a restricted subset of neurons. We systematically characterize the impact of sgRNA library size, mouse cohort size, the size of the targeted cell population, viral titer, and coinfection rate on screen performance to establish general guidelines for large-scale in vivo screens.},
}
@article {pmid41038958,
year = {2025},
author = {Chen, J and Huang, H and Chen, C and Xia, G and Huang, H and Xiong, Y and Luo, P and Chen, Y and Li, J and Wen, L and Li, L and Lin, J and Xu, G and Ji, C and Tian, W and Zhou, J and Wei, P and Shen, C and Wang, X},
title = {ABCC4 impairs the clearance of plasma LDL cholesterol through suppressing LDLR expression in the liver.},
journal = {Communications biology},
volume = {8},
number = {1},
pages = {1414},
pmid = {41038958},
issn = {2399-3642},
support = {8217051361//National Natural Science Foundation of China (National Science Foundation of China)/ ; },
mesh = {*Receptors, LDL/metabolism/genetics ; Animals ; *Liver/metabolism ; Mice ; *Cholesterol, LDL/blood/metabolism ; *Multidrug Resistance-Associated Proteins/metabolism/genetics ; Humans ; Male ; Proprotein Convertase 9/metabolism/genetics ; Hepatocytes/metabolism ; Mice, Inbred C57BL ; Hypercholesterolemia/metabolism/genetics ; CRISPR-Cas Systems ; Signal Transduction ; Hep G2 Cells ; },
abstract = {Low expression level of low-density lipoprotein receptor (LDLR) in hepatocytes leads to hypercholesterolemia and eventually contributes to atherosclerotic cardiovascular disease (ASCVD). Here, we report that inhibition of hepatocyte ABCC4, identified as a top hit from large-scale CRISPR/Cas9 screens, significantly increases hepatic LDLR abundance and enhances LDL cholesterol clearance. As a hepatic transporter for cAMP efflux, ABCC4 silencing alters its intracellular distribution and activates the downstream Epac2/Rap1a signaling pathway, which ultimately blocks PCSK9 protein expression, thereby preventing lysosomal degradation of LDLR. Furthermore, in both male mice and cell models, we demonstrate that liver-specific disruption and pharmacological inhibition of ABCC4 elevate hepatic plasma membrane LDLR levels and reduce plasma LDL cholesterol through ABCC4-cAMP-PCSK9 pathway. Collectively, our genome-wide CRISPR screening offers a valuable resource for identifying LDLR modifiers, providing potential insights for therapeutic strategies in hypercholesterolemia and atherosclerosis.},
}
@article {pmid41038616,
year = {2025},
author = {Aliciaslan, M and Erbasan, E and Erendor, F and Sanlioglu, S},
title = {Prime Editing: The Next Frontier in Precision Gene Therapy.},
journal = {The journal of gene medicine},
volume = {27},
number = {10},
pages = {e70040},
doi = {10.1002/jgm.70040},
pmid = {41038616},
issn = {1521-2254},
support = {//Akdeniz &#xdc;niversitesi/ ; },
mesh = {*Gene Editing/methods ; Humans ; *Genetic Therapy/methods ; *Precision Medicine/methods ; CRISPR-Cas Systems ; Animals ; RNA, Guide, CRISPR-Cas Systems/genetics ; },
abstract = {Prime editing (PE) represents a significant advancement in genome editing, offering high precision for diverse genetic modifications without inducing double-strand breaks or requiring exogenous donor DNA templates. This "search-and-replace" technology employs a Cas9 nickase-reverse transcriptase fusion protein, guided by a PE guide RNA (pegRNA), to directly install specified edits including all 12 base-to-base conversions and targeted insertions/deletions with high fidelity. Since its introduction, PE systems have undergone rapid evolution (e.g., PE2-PE6, PEmax), markedly improving editing efficiency, product purity, and targeting scope. Although PE efficacy is context dependent, influenced by pegRNA design, cellular milieu, and DNA repair pathway engagement, ongoing research focuses on comprehensive system optimization. These efforts include engineering the Cas9 nickase and reverse transcriptase components for enhanced performance and processivity, alongside developing improved pegRNA architectures and chemical modifications to increase their stability and editing efficiency. Furthermore, strategies to modulate the cellular environment, such as transiently altering DNA repair pathway activities, particularly mismatch repair, are being explored to boost the accuracy and yield of precise edits. PE holds substantial promise for basic research, including precise disease modeling, and has demonstrated successful correction of pathogenic mutations in preclinical models of various genetic disorders like sickle cell disease, cystic fibrosis, and inherited retinal diseases. A significant milestone was the US Food and Drug Administration's granting of Investigational New Drug (IND) clearance for the first clinical trial of PM359, a therapeutic based on PE. This agent employs an ex vivo strategy, correcting the NCF1 gene in patient-derived hematopoietic stem cells for the treatment of chronic granulomatous disease. Despite considerable progress, unlocking the complete therapeutic promise of PE requires overcoming significant hurdles, particularly in developing effective in vivo delivery systems for its sizable components, with ongoing research actively investigating diverse viral and nonviral approaches. The translation of this versatile platform into transformative precision gene therapies is critically dependent upon its continued responsible advancement under robust ethical and regulatory oversight.},
}
@article {pmid41037600,
year = {2025},
author = {de Alba, EL and Salguero, I and Giménez-Llorente, D and Montes-Torres, J and Fernández-Sanromán, &#xc1; and Casajús-Pelegay, E and Terrón-Bautista, J and Barroso-González, J and Bernal, JA and Macintyre, G and Fernández-Leiro, R and Losada, A and Cortés-Ledesma, F},
title = {A comprehensive genetic catalog of human double-strand break repair.},
journal = {Science (New York, N.Y.)},
volume = {390},
number = {6768},
pages = {eadr5048},
doi = {10.1126/science.adr5048},
pmid = {41037600},
issn = {1095-9203},
mesh = {Humans ; *DNA Breaks, Double-Stranded ; CRISPR-Cas Systems ; *DNA Repair/genetics ; Von Hippel-Lindau Tumor Suppressor Protein/genetics ; INDEL Mutation ; Gene Editing ; DNA End-Joining Repair/genetics ; Kidney Neoplasms/genetics ; DNA-Binding Proteins/genetics ; Transcription Factors/genetics ; CRISPR-Associated Protein 9 ; Genome, Human ; Gene Knockout Techniques ; Carcinoma, Renal Cell/genetics ; },
abstract = {The analysis of DNA sequence outcomes provides molecular insights into double-strand break (DSB) repair mechanisms. Using parallel in-pool profiling of Cas9-induced insertions and deletions (indels) within a genome-wide knockout library, we present a comprehensive catalog that assesses the influence of nearly every human gene on DSB repair outcomes. This REPAIRome resource uncovers uncharacterized mechanisms, pathways, and factors involved in DSB repair, including opposing roles for XLF and PAXX, a molecular explanation for Cas9-induced multinucleotide insertions, HLTF functions in Cas9-induced DSB repair, the involvement of the SAGA complex in microhomology-mediated end joining, and an indel mutational signature linked to VHL loss, renal carcinoma, and hypoxia. These results exemplify the potential of REPAIRome to drive future discoveries in DSB repair, CRISPR-Cas gene editing and the etiology of cancer mutational signatures.},
}
@article {pmid41035653,
year = {2025},
author = {Naumovas, D and Rojas-Araya, B and Polanco, CM and Andrade, V and Čekauskienė, R and Valatkaitė-Rakštienė, B and Laurinaitytė, I and Jakubauskas, A and Stoškus, M and Griškevičius, L and Nalvarte, I and Inzunza, J and Baltriukienė, D and Arias, J},
title = {Identification of HLA-A, HLA-B, and HLA-C triple homozygous and double homozygous donors: a path toward synthetic superdonor advanced therapeutic medicinal products.},
journal = {Frontiers in immunology},
volume = {16},
number = {},
pages = {1626787},
pmid = {41035653},
issn = {1664-3224},
mesh = {Humans ; *Homozygote ; *HLA-B Antigens/genetics ; *HLA-C Antigens/genetics ; *HLA-A Antigens/genetics ; Alleles ; Gene Frequency ; Induced Pluripotent Stem Cells/immunology ; *Tissue Donors ; Genotype ; CRISPR-Cas Systems ; Female ; Male ; },
abstract = {Human-induced pluripotent stem cells with broad immune compatibility are highly desirable for regenerative medicine applications. Human leukocyte antigen (HLA) class I homozygous cell sources are ideal for immune compatibility modeling. Here, we profile HLA-A, HLA-B, and HLA-C alleles in 3,496 Lithuanian donors genotyped at three-field resolution. The five most frequent alleles constitute 74.6% of HLA-A, 43.2% of HLA-B, and 59.2% of HLA-C, with HLA-A*02:01:01, HLA-B*07:02:01, and HLA-C*07:02:01 being the most common. Lithuanian allele frequencies closely resemble those of European-American and British populations. We identified 153 double homozygotes and 51 triple homozygotes for HLA-A, HLA-B, and HLA-C. Compatibility modeling showed that triple homozygous profiles match 60.5% of Lithuanians, 13.4% of the British population, and 7.4% of European-Americans. CRISPR-Cas9 guide RNA design yielded 54 candidates predicted to disrupt HLA-A or HLA-B while preserving HLA-C, producing edited profiles matching over 97.9% of Lithuanians, 95.7% of European-Americans, and 95.5% of the British population. Finally, we established 15 fibroblast lines from triple homozygotes as a bioresource for the derivation of human-induced pluripotent stem cells and immune compatibility studies.},
}
@article {pmid41034738,
year = {2025},
author = {Devkar, V and Ghose, K and D'Agostino, L and Patil, GB},
title = {Exonuclease-fused CRISPR-cas system enhances targeted genome editing for functional genomics in soybean.},
journal = {BMC plant biology},
volume = {25},
number = {1},
pages = {1283},
pmid = {41034738},
issn = {1471-2229},
mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; *Glycine max/genetics ; *Genome, Plant ; *Exonucleases/genetics/metabolism ; Genomics/methods ; },
abstract = {CRISPR/Cas technologies have revolutionized plant genome editing, yet their inherent bias toward small insertions or deletions (indels) limits their utility for dissecting regulatory elements and generating impactful allelic variants. Here, we report the development and systematic evaluation of exonuclease-fused CRISPR/Cas systems in soybean to overcome this limitation. We engineered fusions of Cas9 and Cas12a with bacteriophage T5 exonuclease and human TREX2 and assessed their editing performance at the GmWOX5 locus using Agrobacterium rhizogenes-mediated transformation and deep amplicon sequencing. While native Cas9 and Cas12a predominantly generated micro-size deletions (1-10 bp), T5-Exo fusions shifted the mutation spectrum, producing a high frequency of moderate (26-50 bp) and large (> 50 bp) deletions. TREX2 fusions preferentially enhanced the generation of small (11-25 bp) to moderate deletions (26-50 bp). Fusion of exonucleases to Cas9 substantially reduced insertion frequencies and promoted more precise deletion patterns, as observed in T5-Exo-Cas9 and TREX2-Cas9. Deletions from both exonuclease fusions were biased toward the PAM-proximal region, reflecting altered repair outcomes likely driven by directional exonuclease activity and enhanced end resection. These results demonstrate that exonuclease fusions effectively expand the CRISPR toolkit by enabling efficient, targeted generation of larger deletions, which are often required for targeting cis-regulatory elements and microRNAs.},
}
@article {pmid40974621,
year = {2025},
author = {Zheng, X and Yao, S and Yin, C and Zhao, H and Wang, J and Su, T and Li, H and Wang, J and Zhao, C},
title = {CRISPR-integrated nanoconfined interparticle catalytic hairpin assembly for enhanced dual-mode SARS-CoV-2 detection in wastewater.},
journal = {Biosensors &amp; bioelectronics},
volume = {290},
number = {},
pages = {118008},
doi = {10.1016/j.bios.2025.118008},
pmid = {40974621},
issn = {1873-4235},
mesh = {*SARS-CoV-2/isolation &amp; purification/genetics ; *Wastewater/virology ; *Biosensing Techniques/methods/instrumentation ; *COVID-19/diagnosis/virology ; *CRISPR-Cas Systems ; Limit of Detection ; Humans ; *RNA, Viral/analysis/genetics/isolation &amp; purification ; Colorimetry/methods ; Metal-Organic Frameworks/chemistry ; Nanoparticles/chemistry ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {Accurate monitoring of pathogenic viruses in wastewater is critical for early outbreak and risk assessment. This study presented a novel biosensing platform that combined an interparticle magnetic covalent organic framework (MCOF)-assisted mismatched catalytic hairpin assembly (iMMCHA) with CRISPR/Cas12a-activated colorimetric-photothermal dual-mode detection of SARS-CoV-2 RNA. The system strategically immobilized CHA reactants (H1 and mismatched H2) on separate MCOF nanoparticles, creating a spatially confined and collision-enhanced interparticle MCHA that achieved 270-fold higher local reactant concentration and 20-min faster kinetics than solution-phase CHA. Upon target recognition, the iMMCHA system generated dsDNA activators that triggered Cas12a-mediated cleavage of ssDNA linkers on magnetic bead-glucose oxidase conjugates. This cleavage event reduced the TMB-oxidizing activity of the magnetically isolated integrated enzyme system, producing inversely correlated colorimetric and photothermal signals. This iMMCHA-CRISPR dual-mode assay allowed for the rapid and sensitive detection of SARS-CoV-2 pseudovirus in sanitary wastewater samples, with detection limits of 100 and 120 copies/μL (colorimetric mode) and 100 and 140 copies/μL (photothermal mode) for S and N genes, respectively. This work established a powerful platform for aqueous environmental virus monitoring that combined the specificity of CRISPR with the signal enhancement and kinetics acceleration of nanoconfined interparticle CHA and the reliability of dual-mode detection.},
}
@article {pmid40961645,
year = {2025},
author = {He, Y and Zhang, Y and Xiang, H and Ren, K and Yin, Y and Gao, Y and Yang, Y and Zhang, W and Liu, L and Han, H and Wang, W},
title = {Magnetic bead-assisted one-pot RCA-activated CRISPR/Cas12a electrochemiluminescence biosensor for the detection of citrus Huanglongbing pathogen.},
journal = {Biosensors &amp; bioelectronics},
volume = {290},
number = {},
pages = {117986},
doi = {10.1016/j.bios.2025.117986},
pmid = {40961645},
issn = {1873-4235},
mesh = {*Biosensing Techniques/methods ; *Citrus/microbiology ; *Plant Diseases/microbiology ; Luminescent Measurements/methods ; CRISPR-Cas Systems/genetics ; Electrochemical Techniques/methods ; Limit of Detection ; Nucleic Acid Amplification Techniques/methods ; *Liberibacter/isolation &amp; purification/genetics/pathogenicity ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {Huanglongbing (HLB) poses a catastrophic threat to the global citrus industry, necessitating early detection of pathogen for disease control and minimize economic losses. Herein, we reported a one-pot electrochemiluminescence (ECL) biosensor for integrating rolling circle amplification (RCA)-activated CRISPR/Cas12a dual cleavage activity, and engineered magnetic beads-based quenched ECL emitter. Target-initiated RCA generated amplicons that activated Cas12a, simultaneously leveraging cis-cleavage for template recycling and trans-cleavage to degrade single stranded DNA attached on Ru(bpy)3[2+]-loaded magnetic beads. This dual-amplification strategy restored ECL signals, enabling ultrasensitive detection of Candidatus Liberibacter asiaticus (CLas) ribonucleotide-diphosphate reductase subunit beta gene fragments with high specificity. A linear range 10 fM-1 nM with the detection limit of 2 fM was obtained. The integrated platform eliminated multi-step incubations, and exhibited satisfactory performance in citrus leaf samples, offering a powerful tool for HLB diagnostics.},
}
@article {pmid40945113,
year = {2025},
author = {Che, R and Tang, D and Fu, B and Wen, T and Wang, Z and Feng, D and Huang, KJ and Xu, J},
title = {Smartphone-integrated tri-mode RCA-CRISPR/Cas12a biosensor with Fe3O4@Au nanozyme for on-site detection of sugarcane smut at attomolar level.},
journal = {Biosensors &amp; bioelectronics},
volume = {290},
number = {},
pages = {117985},
doi = {10.1016/j.bios.2025.117985},
pmid = {40945113},
issn = {1873-4235},
mesh = {*Biosensing Techniques/instrumentation ; *Saccharum/microbiology ; Smartphone ; Gold/chemistry ; CRISPR-Cas Systems ; Nucleic Acid Amplification Techniques ; Limit of Detection ; *Plant Diseases/microbiology ; Colorimetry ; Electrochemical Techniques ; Metal Nanoparticles/chemistry ; },
abstract = {The devastating sugarcane smut causes up to 70 % sugar yield loss and secondary infections, but field-deployable diagnostics remain challenging due to the limitations of lab-dependent methods. Herein, we report a portable CRISPR/Cas12a-powered biosensor integrated with tri-functional Fe3O4@Au nanozymes and triple-modal signal readout for precise and on-site pathogen detection. By synergizing rolling circle amplification (RCA) with CRISPR/Cas12a trans-cleavage activity, the system achieves ultrasensitive target recognition (detection limit: 32.11 aM for electrochemical mode). The Fe3O4@Au@GOD bioconjugates simultaneously enables magnetic separation, optimizes GOD-mediated colorimetric signals (visual LOD: 49.28 fM), and enhances photothermal responses (LOD: 42.17 fM) via precise biocatalyst-catalyzed TMB oxidation. A smartphone-coupled 3D-printed device integrates electrochemical, colorimetric, and photothermal detection modes, providing cross-validated results that eliminate false positives in complex matrices (recovery: 98-104 %). This field-portable platform completes detection within 2.5-4.5 h (80 % cost reduction vs. qPCR) and demonstrates high specificity against non-target pathogens. The fusion of nanozyme engineering, CRISPR amplification, and multi-modal sensing offers a transformative tool for precision agriculture.},
}
@article {pmid40939269,
year = {2025},
author = {Zhou, C and Jiang, F and Chen, W and Nugen, SR and Huang, C},
title = {Synthetic biology meets diagnostics: Engineering biosensing platforms for rapid and accurate pathogen and viral detection.},
journal = {Biosensors &amp; bioelectronics},
volume = {290},
number = {},
pages = {117946},
doi = {10.1016/j.bios.2025.117946},
pmid = {40939269},
issn = {1873-4235},
mesh = {*Biosensing Techniques/methods/instrumentation ; *Synthetic Biology/methods/instrumentation ; Humans ; *Viruses/isolation &amp; purification/genetics/pathogenicity ; *Virus Diseases/diagnosis/virology ; Nanotechnology/methods ; CRISPR-Cas Systems ; },
abstract = {The integration of synthetic biology with biosensor technologies has catalyzed a paradigm shift in the development of programmable, field-deployable diagnostic systems for precision detection of pathogens and viral threats. This review provides a comprehensive overview of current synthetic biology toolkits, such as Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR-based) systems, argonaute proteins, and modular genetic circuits. These tools were integrated into biosensors and in vitro diagnostic devices. The applications of cell-free systems, modular genetic circuits, and nanomaterial-enhanced platforms have further expanded the versatility of these tools, which include infectious disease diagnostics, public health monitoring, and food safety. Recent studies integrate synthetic biology with artificial intelligence (AI) and nanotechnology, enabling the development of automated, low-cost, and high-throughput diagnostic systems. This review provides a comprehensive overview of current technologies, emerging trends, future directions, and challenges, which offers valuable insights for advancing pathogen detection and in vitro diagnostics through synthetic biology.},
}
@article {pmid40914019,
year = {2025},
author = {Ding, Y and Zhang, J and Li, K and Wang, X and Shi, X and Zhao, C and Dai, J and Wang, Q and Yao, S and Wang, J},
title = {A cascade amplification platform integrating entropy-driven DNA nanomachine with CRISPR/Cas12a for microRNA-21 and Listeria monocytogenes detection.},
journal = {Biosensors &amp; bioelectronics},
volume = {290},
number = {},
pages = {117947},
doi = {10.1016/j.bios.2025.117947},
pmid = {40914019},
issn = {1873-4235},
mesh = {*Listeria monocytogenes/isolation &amp; purification/genetics ; CRISPR-Cas Systems/genetics ; *MicroRNAs/genetics/isolation &amp; purification ; *Biosensing Techniques/methods ; Entropy ; Nucleic Acid Amplification Techniques/methods ; Limit of Detection ; Metal Nanoparticles/chemistry ; Gold/chemistry ; Animals ; DNA/chemistry ; Swine ; Humans ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {As one of enzyme-free amplification strategies, entropy-driven catalytic (EDC) based on toehold-mediated strand displacement reaction could achieve efficient amplification without cumbersome temperature changing and expensive enzymes, which shows great potential in biological sensing. However, the limitations in reaction velocity and sensitivity need to be further improved. Herein, a cascade platform integrating entropy-driven DNA nanomachine with CRISPR/Cas12a was proposed. Benefiting from the increased local concentration of DNA on AuNPs, the reaction velocity was enhanced 2-fold compared to solution-based EDC efficiently and the signal was cascade amplified through specific recognition by the designed CRISPR/Cas12a with high sensitivity and selectivity. Impressively, utilizing the flexible design capabilities of DNA molecules, the proposed method achieved both nucleic acid and non-nucleic acid targets detection. The platform achieved a low limit of 6.1pM for microRNA-21 detection and 6 CFU/mL for Listeria monocytogenes detection. Moreover, it showed good performance in 10-fold diluted serum with 98.6-102.3 % recovery. And it has achieved good consistency with traditional plate culture methods in pork. Here, a rapid and sensitive platform based on entropy-driven DNA nanomachine coupled with CRISPR/Cas12a is proposed with great potential of application for early disease detection and food safety screening.},
}
@article {pmid40914018,
year = {2025},
author = {Deng, Z and Mao, X and Yang, Y and Wu, G and Meng, H and Hou, J and Yu, XF and Zhou, W and Mao, G and Ma, Y},
title = {Amplification-free CRISPR/Cas12a biosensor integrating AuNPs-mediated surface plasmon resonance for human papillomavirus detection and genotyping.},
journal = {Biosensors &amp; bioelectronics},
volume = {290},
number = {},
pages = {117960},
doi = {10.1016/j.bios.2025.117960},
pmid = {40914018},
issn = {1873-4235},
mesh = {Humans ; Gold/chemistry ; *Surface Plasmon Resonance/methods ; CRISPR-Cas Systems/genetics ; *Papillomavirus Infections/virology/diagnosis ; Metal Nanoparticles/chemistry ; *Papillomaviridae/genetics/isolation &amp; purification ; *DNA, Viral/genetics/isolation &amp; purification ; *Biosensing Techniques ; Limit of Detection ; Genotyping Techniques ; Female ; Alkaline Phosphatase/chemistry ; Genotype ; Human Papillomavirus Viruses ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {Screening for high-risk human papillomavirus (hrHPV) infection is essential for cervical cancer prevention. However, developing a simple, portable, and low-cost hrHPV genotyping method remains challenging, particularly in resource-limited settings. Herein, we present an innovative amplification-free, point-of-care hrHPV genotyping platform integrating CRISPR/Cas12a with alkaline phosphatase (ALP)-mediated surface plasmon effect. The platform detects HPV DNA through the Cas12a-crRNA complex recognition, activating the cleavage of ALP-labeled oligonucleotides within microwells and releasing ALP. The output signal is generated by changes in surface plasmon resonance of gold nanoparticles (AuNPs) induced by the ALP-mediated reaction of AuNPs with p-aminophenyl phosphate. This ALP-integrated CRISPR/Cas12a biosensing strategy enhances sensitivity by 10,000-fold compared to Cas12a-based detection integrating ALP-mediated p-nitrophenyl phosphate (p-NPP) hydrolysis. This approach allows the sensitive detection of HPV DNA with a detection limit of 300 aM. Moreover, integration with microplate separation allows specifically screen for the nine HPV subtypes targeted by the nine-valent HPV vaccine within 2.5 h. The platform's performance is validated using cervical swab samples, confirming its accuracy for HPV genotyping. Overall, this strategy provides a simple, portable, and cost-effective solution for multiplex nucleic acid targets detection without preamplification or instrumentation.},
}
@article {pmid40907452,
year = {2025},
author = {Hu, D and Lin, K and Xu, X and Chen, P and Wang, G and Parvin, R and Chen, X and Wang, D and Zhang, L and Ye, F},
title = {SEE-phAST: Spatially encapsulated emulsions for phenotypic antibiotic susceptibility testing via sequential digital RAA-CRISPR.},
journal = {Biosensors &amp; bioelectronics},
volume = {290},
number = {},
pages = {117937},
doi = {10.1016/j.bios.2025.117937},
pmid = {40907452},
issn = {1873-4235},
mesh = {*Anti-Bacterial Agents/pharmacology ; *Biosensing Techniques ; Emulsions/chemistry ; Microbial Sensitivity Tests ; Humans ; DNA Copy Number Variations/genetics ; CRISPR-Cas Systems/genetics ; Escherichia coli/genetics/drug effects ; Pseudomonas aeruginosa/genetics/drug effects ; Nucleic Acid Amplification Techniques ; Recombinases/chemistry ; Klebsiella pneumoniae/genetics/drug effects ; DNA, Bacterial/genetics ; },
abstract = {The escalating threat of antimicrobial resistance is exacerbated by delayed diagnostics and improper antibiotics use, underscoring an urgent demand for rapid, versatile AST tools to support evidence-based prescribing. In this study, we present an innovative, generalizable phenotypic AST approach by quantifying bacterial gDNA copy number variations (CNVs) following 0.5-h-brief culturing with antibiotic exposure, termed spatially encapsulated emulsions (SEE)-phAST. It employed gelatin-PEG biomimetic phase separation and microfluidic technology to one-step fabricate spatial-confined microcarriers that enabled on-demand separation and mixing of recombinase-aided amplification (RAA) and clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 12a (CRISPR/Cas12a) reactions in a sequentially orchestrated manner. Importantly, it retained the superiority of droplet-based digitalization framework to identify 3-4 folds CNVs while synergistically enhancing signal-to-noise ratio and detection speed through RAA pre-amplification. By utilizing a phase diagram for precise separation, coupled with channel design and rate regulation, we controllably synthesized 180-210 μm microcarriers with 90-100 μm cores, achieving a 30-min RAA amplification that boosted sensitivity from 10[11] to 10[1] aM. The artificial urinary infection samples were prepared by introducing common bacteria, specifically Klebsiella pneumoniae, Escherichia coli, and Pseudomonas aeruginosa, into urinary samples obtained from healthy individuals. We demonstrated the system's capability to classify susceptibility and resistance to three specific drugs following a 30-min pre-culture, with epigenetic changes quantified as reduced to 0.5 for inhibition and maintained at 2 for unaffected growth. This strategy presents valuable potential for AST diagnosis and versatility in other sequence recognition scenarios.},
}
@article {pmid40902593,
year = {2025},
author = {Chan, BKC and Zhang, C and Poon, CH and Lee, MHY and Chu, HY and Wang, B and Chen, SG and Yan, HHN and Leung, SY and Wong, ASL},
title = {A combined enteric neuron-gastric tumor organoid reveals metabolic vulnerabilities in gastric cancer.},
journal = {Cell stem cell},
volume = {32},
number = {10},
pages = {1595-1613.e10},
doi = {10.1016/j.stem.2025.08.006},
pmid = {40902593},
issn = {1875-9777},
mesh = {*Organoids/metabolism/pathology/drug effects ; *Stomach Neoplasms/metabolism/pathology ; Humans ; Animals ; *Neurons/metabolism/pathology/drug effects ; Mice ; Cell Line, Tumor ; Xenograft Model Antitumor Assays ; CRISPR-Cas Systems ; Lipid Metabolism/drug effects ; Acetyl-CoA Carboxylase/antagonists &amp; inhibitors/metabolism ; },
abstract = {The discrepancy between organoid and immortalized cell line cultures for cancer target discovery remains unclear. Here, our multi-tiered clustered regularly interspaced short palindromic repeats (CRISPR) screens reveal in vivo-relevant metabolic dependencies and synthetic lethal pairs that can be uncovered with tumor organoids but not cell lines or even three-dimensional (3D) spheroids. These screens identify lanosterol synthase and acetyl-coenzyme A (CoA) carboxylase inhibitors as effective treatments that impede xenografted tumor growth in mice. These lipid metabolic inhibitors exhibit nanomolar half-maximal inhibitory concentration (IC50) values across diverse human gastric cancer organoids resistant to first-line treatments. Mechanistically, gastric cancer organoids and in vivo tumors exhibit lipid metabolic adaptations not seen in two-dimensional (2D) in vitro cultures. Additionally, enteric neurons modulate lipid metabolism in tumor organoids, altering drug sensitivity by up to two orders of magnitude. A neuron-cocultured CRISPR screen further reveals that acetyl-CoA carboxylase expression determines lanosterol synthase inhibitor efficacy. These findings highlight the critical roles of organoid environment and neuronal interaction in cancer lipid reliance.},
}
@article {pmid40674704,
year = {2025},
author = {Yamaguchi, K and Koya, J and Mizuno, K and Mizukami, Y and Yoshifuji, K and Saito, Y and Tabata, M and Shingaki, S and Yuasa, M and Ito, Y and Nakashima, K and Dreval, K and Morin, RD and Chiba, K and Okada, A and Shiraishi, Y and Murakami, K and Kogure, Y and Ohshima, K and Kataoka, K},
title = {In vivo CRISPR screening reveals cooperation of KMT2D and TP53 deficiencies in B-cell lymphomagenesis.},
journal = {Blood advances},
volume = {9},
number = {19},
pages = {5040-5055},
doi = {10.1182/bloodadvances.2024015519},
pmid = {40674704},
issn = {2473-9537},
mesh = {*Tumor Suppressor Protein p53/genetics/deficiency ; Humans ; Animals ; Mice ; *Lymphoma, Large B-Cell, Diffuse/genetics/pathology ; *DNA-Binding Proteins/genetics/deficiency ; *CRISPR-Cas Systems ; *Lymphoma, B-Cell/genetics ; *Neoplasm Proteins/genetics/deficiency ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Cell Line, Tumor ; },
abstract = {Although recent genetic studies have identified numerous genetic alterations in diffuse large B-cell lymphoma (DLBCL), their biological relevance remains elusive. Here, we performed in vivo CRISPR loss-of-function screening targeting 86 genes recurrently altered in DLBCL to examine oncogenicity of single-guide RNA (sgRNA)-targeted genes, association between genotype and lineage, occurrence of second-hit alterations, and cooperability among sgRNA-targeted genes and second-hit alterations. Transplantation of the CRISPR library-transduced hematopoietic stem/progenitor cells induces various hematologic malignancies, including B-cell lymphomas in mice. Enrichment analysis of sgRNA-targeted genes demonstrates significant overrepresentation of Kmt2d, Pax5, and Trp53 in B-cell lymphomas. Whole-exome sequencing identifies recurrent second-hit driver alterations, showing significant enrichment of Trp53 alterations in sgKmt2d-targeted B-cell lymphomas. Importantly, KMT2D and TP53 mutations are found to be the most prevalent co-occurring combination in human DLBCL, which is more prominent in relapsed/refractory DLBCL. Moreover, this combination confers significantly worse prognosis independent of clinical factors. Transcriptomic sequencing identifies overexpression of Yap1, the Hippo pathway component, in double sgKmt2d-targeted/Trp53-altered B-cell lymphomas. Furthermore, chromatin accessibility analysis demonstrates enrichment of transcriptional enhanced associate domain 1 binding motifs in regions that gained accessibility and increased expression of their nearest genes in these B-cell lymphomas. Most importantly, genetic and pharmacological inhibition of YAP1 suppresses in vitro cell proliferation and in vivo tumor growth of a human KMT2D/TP53-altered DLBCL cell line and prolongs survival of mice transplanted with double sgKmt2d-targeted/Trp53-altered B-cell lymphoma cells. Our findings demonstrate the utility of in vivo CRISPR screening to integrate human cancer genomics with mouse modeling and highlight the functional interplay between KMT2D and TP53 aberrations, providing insights into therapeutic strategies in DLBCL.},
}
@article {pmid41033726,
year = {2025},
author = {Gupta, Y and Chosdol, K},
title = {Practical approaches to advanced molecular biology techniques.},
journal = {Methods in cell biology},
volume = {198},
number = {},
pages = {73-101},
doi = {10.1016/bs.mcb.2025.02.022},
pmid = {41033726},
issn = {0091-679X},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; *Molecular Biology/methods ; Single-Cell Analysis/methods ; Animals ; Chromatin Immunoprecipitation/methods ; Proteomics/methods ; },
abstract = {The field of molecular biology has undergone tremendous advancements in recent years, with the development of powerful techniques that allow for in-depth exploration of cellular processes at the molecular level. This chapter, "Advanced Molecular Biology Techniques," provides a detailed protocol of the molecular techniques. We begin with CRISPR-Cas9 genome editing, a transformative tool for precise and efficient gene manipulation, enabling targeted mutations and gene knockouts in various organisms. Gene amplification via Real-Time PCR is then discussed, highlighting its ability to quantify gene expression and detect rare genetic variants with high sensitivity. Flowcytometry follows, offering a robust platform for analyzing cellular populations based on specific markers, enabling the study of immune cells, cancer diagnostics, and cell cycle analysis. Chromatin Immunoprecipitation Sequencing (ChIP-Seq) is explored as a method for mapping protein-DNA interactions, providing insights into gene regulation and epigenetic modifications. The chapter also covers Single-cell RNA sequencing (scRNA-Seq), a groundbreaking technique for profiling gene expression at the single-cell level, allowing for the discovery of cell heterogeneity and complex biological processes. Next, we explore into proteomics through Mass Spectrometry-Based Analysis, which offers detailed proteome characterization and biomarker discovery by identifying and quantifying proteins in complex samples. Finally, Fluorescence In Situ Hybridization (FISH) is discussed as a method for visualizing the spatial localization of specific nucleic acid sequences within intact cells or tissues. Together, these advanced molecular biology techniques offer unparalleled precision and insight into the molecular mechanisms underlying health, disease, and cellular function.},
}
@article {pmid41033723,
year = {2025},
author = {Hasan, N and Palungan, J and Ullah, M},
title = {Gene editing techniques in cancer research.},
journal = {Methods in cell biology},
volume = {198},
number = {},
pages = {287-312},
doi = {10.1016/bs.mcb.2025.03.002},
pmid = {41033723},
issn = {0091-679X},
mesh = {*Gene Editing/methods ; Humans ; *Neoplasms/genetics/therapy ; *CRISPR-Cas Systems/genetics ; Zinc Finger Nucleases/genetics ; Transcription Activator-Like Effector Nucleases/genetics ; Animals ; },
abstract = {The process of editing genes has emerged as a game-changing instrument in the field of cancer research. It has the potential to provide a whole new understanding of the biology of tumors and to facilitate the creation of tailored medicines. Zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and the clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 system are the three basic methods of gene editing techniques that are discussed in this chapter. We investigate the protocol modifications that are specific to each approach, focusing on high-prevalence tumors, and we investigate the utility, efficiency, and application issues that are associated with each technique in oncology. In addition, we describe current developments in improving these methods to successfully target oncogenes and tumor suppressor genes, with the goal of driving forward advances in precision cancer therapy.},
}
@article {pmid41033719,
year = {2025},
author = {Tiwari, PC and Chaudhary, MJ and Pal, R and Nath, R},
title = {In vivo cancer modeling using mouse models.},
journal = {Methods in cell biology},
volume = {198},
number = {},
pages = {221-250},
doi = {10.1016/bs.mcb.2025.02.013},
pmid = {41033719},
issn = {0091-679X},
mesh = {Animals ; Mice ; *Disease Models, Animal ; *Neoplasms/pathology/genetics ; Humans ; Tumor Microenvironment ; Xenograft Model Antitumor Assays/methods ; CRISPR-Cas Systems ; },
abstract = {Mouse models have contributed to a better understanding of cancer biology and the development of new treatments. This chapter elaborates on the various types of mouse models applied in cancer research, such as xenograft, syngeneic, and humanized models, together with the state-of-the-art techniques of genetic engineering involved in their generation. We described the methodologies of tumor induction and engraftment procedures and these model applications in drug development, efficacy testing, and studies on immuno-oncology. Further, the chapter covers ethical considerations and regulatory requirements on the use of animals in research, essentially aligned with international guidelines and those in India. The chapter illustrates that mouse models will not become outdated in preclinical testing any time soon but continue to be relevant for the study of tumor biology and the tumor microenvironment besides their use for investigating genetic and molecular pathways in cancer. Emerging technologies, such as CRISPR/Cas9 and organoid integration, are also highlighted for their work in improving the accuracy and translational potential of models. These developments combined with initiatives on collaborative and open science that enable the sharing of data and resources, hold great promise for the future of in vivo cancer modeling. The mouse models will continue to be one of the prime movers in advancing cancer research and formulating individual medication strategies that lead to improved patient outcomes through their integration of classical approaches with modern technologies.},
}
@article {pmid41032194,
year = {2025},
author = {Naderian, R and Alibabaei, F and Paraandavaji, E and Dehghan, P and Eslami, M},
title = {Phage-Microbiota Interactions in the Gut: Implications for Health and Therapeutic Strategies.},
journal = {Probiotics and antimicrobial proteins},
volume = {},
number = {},
pages = {},
pmid = {41032194},
issn = {1867-1314},
abstract = {The diversified ecology of microorganisms, including bacteria, archaea, fungi, protozoa, and viruses known collectively as the gut microbiota, which includes bacteriophages, is crucial to human health because it affects functions like immune system regulation, vitamin production, and pathogen protection. Bacteriophages are viruses that infect bacteria and are increasingly recognized as a viable treatment option for antibiotic-resistant strains, owing to their high host specificity, which enables precise targeting of drug-resistant bacteria while sparing commensal microbiota. The complex relationships between bacteriophages and gut microbiota are examined, with emphasis on their roles in maintaining health and contributing to disease. Gut microbiota homeostasis is influenced by a number of factors, including age, nutrition, and drugs. Bacteriophages, via lytic cycles and lysogenic conversion, influence the gut microbiota composition and microbial community structure. Gaining an understanding of these processes is crucial to appreciating their contribution to the stability and variety of microbes. Recent research highlights the gut phageome's potential for therapeutic interventions by demonstrating its substantial influence on immunological responses and metabolic problems. The study of phage-microbiota interactions has been transformed by cutting-edge technologies, including high-throughput sequencing, CRISPR-Cas systems, and viral metagenomics, which allow for thorough research and the creation of new therapeutics. Even though tailored medicine and pathogen management hold great potential, obstacles such as regulatory difficulties and bacterial resistance call for additional investigation. Phage-based therapeutic strategies are rapidly advancing, ranging from genetically engineered phages and phages with modified capsid proteins designed to enhance efficacy to phage cocktails that target multiple bacterial strains.},
}
@article {pmid40803956,
year = {2025},
author = {Murata, S and Kushiyama, N and Yabu, Y and Watanabe, K and Fujii, T and Yasui, R and Nojima, D and Maeda, Y and Yoshino, T and Matsuda, Y and Tanaka, T},
title = {Establishment of genome editing techniques in the marine oleaginous diatom Fistulifera solaris for improved oil accumulation.},
journal = {Journal of bioscience and bioengineering},
volume = {140},
number = {5},
pages = {271-276},
doi = {10.1016/j.jbiosc.2025.07.008},
pmid = {40803956},
issn = {1347-4421},
mesh = {*Diatoms/genetics/metabolism ; *Gene Editing/methods ; CRISPR-Cas Systems ; Biofuels ; Lipase/genetics/metabolism ; Gene Knockout Techniques ; *Oils/metabolism ; Microalgae/genetics/metabolism ; Triglycerides/metabolism ; *Lipid Metabolism/genetics ; },
abstract = {Biofuel production using microalgae has attracted considerable attention owing to high growth rate and lipid accumulation properties. However, further enhancement in lipid productivity is required to render this economically feasible. CRISPR/Cas9, which is one of the powerful genome editing tools, is an essential technique that may solve this problem. The marine diatom Fistulifera solaris JPCC DA0580 is a promising candidate of the biofuel production, since it accumulates significant amount of lipids. However, genome editing techniques have not yet been established for F. solaris, which prevent the construction of valuable strains. In this study, CRISPR/Cas9-mediated specific gene knockout technique was established in F. solaris, through targeting adenine phosphoribosyl transferase gene (apt) and triacylglycerol (TAG) lipase gene (tgl1). Mutations in the target sequence were detected in apt- and tgl1-edited mutants. Moreover, the mutants showed distinct phenotypes, such as suppression of TAG degradation and resistance to 2-fluoroadenine. These results indicate the successful demonstration of CRISPR/Cas9-mediated genome editing in the oleaginous marine diatom F. solaris. Furthermore, oil degradation was successfully suppressed by knocking-out tgl1. The CRISPR/Cas9-mediated genome editing established in this study provides key molecular tools for both the basic biology and the future biotechnological applications of F. solaris, such as biofuel production.},
}
@article {pmid40774862,
year = {2025},
author = {Karashima, T and Oda, K and Futagami, T and Hokazono, H and Takashita, H},
title = {Ribonucleoprotein-based CRISPR/Cas9 genome co-editing in Aspergillus luchuensis mut. kawachii.},
journal = {Journal of bioscience and bioengineering},
volume = {140},
number = {5},
pages = {298-305},
doi = {10.1016/j.jbiosc.2025.07.006},
pmid = {40774862},
issn = {1347-4421},
mesh = {*CRISPR-Cas Systems/genetics ; *Ribonucleoproteins/genetics/metabolism ; *Gene Editing/methods ; *Aspergillus/genetics/metabolism ; Gene Knockout Techniques ; *Genome, Fungal ; RNA, Guide, CRISPR-Cas Systems/genetics ; Sulfate Adenylyltransferase/genetics ; },
abstract = {In this study, we established a ribonucleoprotein-based clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) genome co-editing method for the white koji fungus, Aspergillus luchuensis mut. kawachii. To introduce the single guide RNA-Cas9 ribonucleoprotein complex into protoplast cells of A. luchuensis mut. kawachii, we investigated the conditions for protoplast preparation using Yatalase -Plus-. Subsequently, we employed the ribonucleoprotein-based method to knockout the ATP sulfurylase-encoding sC gene, which imparts selenate resistance in the model strain NBRC 4308 and the industrial strain No. 8046. Furthermore, we explored genome co-editing by simultaneously targeting sC along with either the orotidine 5'-phosphate decarboxylase-encoding pyrG gene or the transcriptional activator of protease genes-encoding prtR gene in NBRC 4308. The transformants were selected in medium containing selenate, resulting in the successful generation of pyrG- and prtR-knockout strains. Similarly, transformants were selected on medium containing selenate, resulting in the successful generation of prtR-knockout strain in No. 8046. These results demonstrate that the ribonucleoprotein-based genome co-editing method is applicable not only to the model strain but also to industrial strains, making it a promising approach for manipulating A. luchuensis mut. kawachii.},
}
@article {pmid40634141,
year = {2025},
author = {Shan, L and Verstrepen, KJ and Wang, Q and Dai, Z},
title = {A homologous recombination-proficient Yarrowia lipolytica chassis for multiplex genome manipulation.},
journal = {Trends in biotechnology},
volume = {43},
number = {10},
pages = {2627-2645},
doi = {10.1016/j.tibtech.2025.06.009},
pmid = {40634141},
issn = {1879-3096},
mesh = {*Yarrowia/genetics ; *Homologous Recombination/genetics ; *Gene Editing/methods ; DNA End-Joining Repair/genetics ; *Genome, Fungal/genetics ; CRISPR-Cas Systems ; },
abstract = {Homologous recombination (HR) greatly facilitates precise genome editing. However, most organisms prefer error-prone non-homologous end joining (NHEJ) for DNA double-strand break (DSB) repair. Here, the NHEJ-proficient Yarrowia lipolytica was transformed into a HR-proficient strain by systematic engineering of recombination machinery, regulating the multiinvasion-induced rearrangement (MIR) process, and expressing cognate single-stranded DNA-annealing protein (SSAP)-single-stranded DNA-binding protein (SSB) pairs. These strategies improved HR efficiency by 38.9, 1.6, and 1.2-fold compared with the NHEJ-deficient strain for multifragment multisite integration, and multi- and single-fragment single-site integration, respectively. Moreover, HR efficiency remained high at 58% even with 50-base pair (bp) homology arms (HAs) and reached 11% for simultaneously integrating two mega-DNA fragments (18.0 kb and 13.5 kb) at two genome sites. This strain also enabled simultaneous editing, repression, and activation of multiple genes, while cellular robustness parameters showed marked increases over the NHEJ-deficient strain. Our work provides a HR-proficient Y. lipolytica chassis allowing efficient and precise genome editing of this increasingly important microbe.},
}
@article {pmid40605271,
year = {2025},
author = {Stigzelius, V and Cavallo, AL and Chandode, RK and Nitsch, R},
title = {Peeling back the layers of immunogenicity in Cas9-based genomic medicine.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {33},
number = {10},
pages = {4714-4730},
doi = {10.1016/j.ymthe.2025.06.045},
pmid = {40605271},
issn = {1525-0024},
mesh = {*CRISPR-Cas Systems/immunology ; Humans ; *Gene Editing/methods ; *Genetic Therapy/methods ; Animals ; *CRISPR-Associated Protein 9/immunology/genetics ; *Genomics/methods ; },
abstract = {The CRISPR-Cas9 genome editing system is rewriting the treatment of genetic disorders, offering unprecedented potential for detrimental and previously untreatable diseases. As this technology advances toward wider utilization in clinical applications, the immunogenicity of Cas9 nuclease has emerged as a potential challenge for in vivo therapies. Immune recognition of CRISPR-Cas9 components can trigger both innate and adaptive responses. The complex interactions between Cas9, delivery vectors, and host immune reactivity play a crucial role in determining the safety and efficacy of CRISPR-based treatments. Recent advances in mitigating Cas9 immunogenicity include epitope engineering, optimized delivery systems, and nucleic acid modifications. These strategies, explored across various tissue contexts and delivery methods, show promise in enhancing the tolerability of CRISPR-based therapies. However, pre-existing immunity to Cas9 and the potential for long-term adaptive immune responses remain important considerations. Addressing these immunological challenges requires an integrated approach, combining insights from immunology with innovative engineering solutions. As the field progresses, overcoming Cas9 immunogenicity will be crucial for realizing the full therapeutic potential of the CRISPR-Cas9 system in diverse clinical applications.},
}
@article {pmid40368676,
year = {2025},
author = {Zhang, S and Xu, D and Li, F and Wang, J},
title = {CRISPR-based non-nucleic acid detection.},
journal = {Trends in biotechnology},
volume = {43},
number = {10},
pages = {2494-2508},
doi = {10.1016/j.tibtech.2025.04.012},
pmid = {40368676},
issn = {1879-3096},
mesh = {*CRISPR-Cas Systems/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; *Nucleic Acids/genetics/analysis ; Gene Editing/methods ; Humans ; },
abstract = {Characterization of clustered regularly interspaced short palindromic repeat (CRISPR)-associated (Cas) trans-cleavage activities has initiated the era of next-generation CRISPR diagnostics. By using the trans-cleavage reaction for signal output, CRISPR systems have been engineered to detect non-nucleic acids (NNAs), including ions, inorganic small molecules, organic compounds, proteins, and bacteria. Diverse strategies are being used to specifically recognize NNAs and regulate Cas trans-cleavage activities, via generation or depletion of output signals. In this review, we introduce the principles and advantages of CRISPR-based NNA detection. We then classify CRISPR-based NNA detection strategies into three classes: the generation or depletion of free activators, synthesis of crRNAs, and reconstruction of active Cas effectors. Finally, we discuss the challenges and potential strategies to advance both clinical and nonclinical applications of CRISPR-based NNA detection.},
}
@article {pmid41032193,
year = {2025},
author = {Yetiman, AE and Horzum, M and Kanbur, E and Çadir, M and Bahar, D and Gürbüz, &#x15e; and Karaman, MZ and Fidan, &#xd6; and Kaya, M and Yetiman, S and Doğan, M and Akbulut, M},
title = {Pangenome Analysis and Genome-Guided Probiotic Evaluation of Cyclic Dipeptides Producing Levilactobacillus brevis DY55bre Strain from a Lactic Acid Fermented Shalgam to Assess Its Metabolic, Probiotic Potentials, and Cytotoxic Effects on Colorectal Cancer Cells.},
journal = {Probiotics and antimicrobial proteins},
volume = {},
number = {},
pages = {},
pmid = {41032193},
issn = {1867-1314},
support = {FBA-2025-14779//Bilimsel Ara&#x15f;t&#x131;rma Projeleri, Erciyes &#xdc;niversitesi/ ; },
abstract = {This study investigates the genetic, metabolic, and probiotic characteristics of Levilactobacillus brevis DY55bre, a strain isolated from the traditional Turkish fermented beverage, shalgam. Whole-genome sequencing revealed a circular genome of 2.485 Mb with a GC content of 45.72%, predicted 2791 genes, and multiple CRISPR-Cas systems. Pangenome analysis demonstrated an open structure, with 18.9% core genes and 103 strain-specific genes, highlighting its genetic diversity. The DY55bre exhibits heterofermentative carbohydrate metabolism due to the presence of the araBAD operon and the lack of 1-phosphofructokinase (pfK) and fructose-1,6-bisphosphate aldolase enzymes. Probiotic evaluation revealed firm survival under simulated gastrointestinal conditions, including resistance to acidic pH (as low as 3.0) and bile salts (up to 1%), along with significant adhesion to intestinal epithelial cell lines (HT29;59.3%, Caco-2;87%, and DLD-1;60.8%). The strain exhibited high auto-aggregation (84.55%) and cell surface hydrophobicity (56.69%), essential for gut colonization. Safety assessments confirmed its non-hemolytic nature and absence of horizontally acquired antibiotic resistance genes. Notably, GC-MS analysis identified bioactive cyclic dipeptides, Cyclo(D-Phe-L-Pro) and Cyclo(L-Leu-L-Pro), which demonstrated cytotoxic effects against colorectal cancer cell lines, with IC50 values of 7.71 mg/mL for HT29 and 3.19 mg/mL for DLD-1. The cell-free supernatant exhibited antimicrobial activity against pathogens, likely due to the synergistic effects of cyclic dipeptides, organic acids, and other metabolites. Antioxidant assays revealed significant ABTS[+] (76.63%) and DPPH (34.25%) radical scavenging activities, while cholesterol assimilation tests showed a 27.29% reduction. These findings position the DY55bre as a promising candidate for functional foods, nutraceuticals, and therapeutic applications, warranting further in vivo validation.},
}
@article {pmid41032156,
year = {2025},
author = {Meshram, V and Jadhav, SK and Chandrawanshi, NK},
title = {Strain improvement of Cordyceps militaris for optimized bioactive metabolite biosynthesis: current progress and prospective approaches.},
journal = {Antonie van Leeuwenhoek},
volume = {118},
number = {11},
pages = {162},
pmid = {41032156},
issn = {1572-9699},
support = {201610136180//University Grants Commission/ ; },
mesh = {*Cordyceps/metabolism/genetics ; *Metabolic Engineering/methods ; Gene Editing ; CRISPR-Cas Systems ; Biosynthetic Pathways ; },
abstract = {Cordyceps militaris is a rare and highly valued medicinal fungus that has attracted considerable attention due to its production of diverse bioactive compounds, including nucleosides such as cordycepin, polysaccharides, lovastatin, carotenoids, etc., all of which exhibit significant nutritional and therapeutic potential. However, the large-scale utilization of C. militaris is constrained by several critical challenges. A major limitation is the progressive degeneration of strains over successive subcultures, which adversely affects fruiting body formation and metabolite biosynthesis. Moreover, genetic instability during long-term culture, contamination risks in large-scale production, and the lack of standardized cultivation and extraction protocols often result in variable product quality. The absence of efficient genetic transformation systems and the low success rate of genome editing approaches further complicate efforts in molecular strain improvement. This review provides a comprehensive overview of the principal bioactive compounds produced by C. militaris and critically evaluates the current challenges and limitations associated with both conventional and advanced strain improvement strategies. These include conventional approaches such as mutagenesis and protoplast fusion, as well as genome-editing technologies like CRISPR/Cas9, which are employed to enhance the biosynthesis of target metabolites. Moreover, the integration of metabolic engineering frameworks offers significant potential for rational strain design, optimization of bioprocesses, and the discovery of novel therapeutic agents.},
}
@article {pmid41032013,
year = {2025},
author = {Huang, X and Li, H and Du, J and Xie, W and Liu, Y and Lin, Y and Xing, H},
title = {HUH endonuclease-mediated DNA-protein conjugates: sequence-specific tools and cellular applications.},
journal = {Chemical communications (Cambridge, England)},
volume = {},
number = {},
pages = {},
doi = {10.1039/d5cc04628a},
pmid = {41032013},
issn = {1364-548X},
abstract = {This highlight review article summarizes recent advances in employing HUH endonucleases as self-labeling protein tags for the sequence-specific covalent conjugation of unmodified ssDNA and examines their applications in cellular studies via engineered DNA-protein conjugates. We outline the structural basis and catalytic mechanism of the conserved HUH and Y motifs, which enable high selectivity, bioorthogonality, and robust conjugation under physiological conditions. Recent applications demonstrate the versatility of HUH-based DNA-protein conjugates in programmable cellular interface engineering, targeted therapeutic delivery, and enhancement of genome editing systems such as CRISPR-Cas. In the perspective section, we further highlight two emerging directions: computational tools such as the HUHgle platform for predictive substrate design, and directed evolution strategies extending HUH reactivity toward RNA substrates. Together, these advancements establish HUH endonucleases as powerful, programmable tools for generating DNA-protein conjugates that enable innovations in chemical biology, synthetic biology, and therapeutics.},
}
@article {pmid41030550,
year = {2025},
author = {Yang, ZQ and Li, MJ and Ahmad, F and Jin, CZ and Li, T and Jin, FJ and Shin, KS and Jin, L},
title = {Application of the transposon-associated TnpB system of CRISPR-Cas in bacteria: Deinococcus.},
journal = {Frontiers in microbiology},
volume = {16},
number = {},
pages = {1604032},
pmid = {41030550},
issn = {1664-302X},
abstract = {Deinococcus radiodurans is one of the most radioresistant organisms found on Earth to date, showing extreme resistance to damage factors such as UV, drought, and mutagens, and is of great interest to scientists around the world. It was determined that the TnpB protein from D. radiodurans ISDra2 functions as an RNA-guided endonuclease, serving as a functional ancestor for the widely used CRISPR-Cas endonucleases. The CRISPR-Cas system is an "acquired immune system" found in most Bacteria and Archaea, and used in a wide range of biological and medical research fields. Cas12f is the smallest RNA-directed nuclease that is currently known and possesses unique characteristics. There has been extensive research conducted on the origin, classification, and mechanism of action of CRISPR-Cas12f, as well as its application in the field of gene editing. TnpB, as the protein closest to Cas12f in the evolutionary tree, has the potential to be used as a new micro-editing tool. Systematic studies have been conducted on it to develop smaller volumes of precision gene editing and treatment tools. In this review, the research progress, mechanism, and application of TnpB protein in D. radiodurans were reviewed. In addition, the classification of CRISPR-Cas system and the application and function of CRISPR-Cas12f in gene editing are also introduced and summarized.},
}
@article {pmid41028589,
year = {2026},
author = {Bhargava, CN and Karuppannasamy, A and Ramasamy, A},
title = {CRISPR/Cas9-Mediated Genome Editing in the Management of Oriental Fruit Fly, Bactrocera dorsalis (Hendel) (Tephritidae: Diptera).},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2966},
number = {},
pages = {259-270},
pmid = {41028589},
issn = {1940-6029},
mesh = {Animals ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; *Tephritidae/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; Genome, Insect ; Pest Control, Biological/methods ; },
abstract = {The oriental fruit fly, Bactrocera dorsalis (Hendel), is a highly invasive polyphagous pest that causes significant damage to horticultural crops of global importance. Traditional management practices have not been effective in controlling this pest, and therefore, there is a need for alternative management strategies. CRISPR/Cas9-driven genome editing has been successfully used in a wide range of insects to induce site-specific, off-target minimized mutations that result in loss of function. This technique can be used to develop precision-guided sterile insect technique (pgSIT) and gene drive programs, which can be used for area-wide suppression of the pest. This chapter provides a brief overview of the workflow for RNP-based genome editing, which can be used to validate and establish gene function for large-scale gene drive programs aimed at combating this pest. The RNP, or ribonucleoprotein complex, comprises the sgRNA and Cas9 protein, which are microinjected into the G0 stage embryos for heritable editing of the target gene(s).},
}
@article {pmid41028581,
year = {2026},
author = {Ashokkumar, S and Ponnurangan, V and Krish, KK and Loganathan, A and Eswaran, K and Vaikuntavasan, P and Duraialagaraja, S and Shanmugam, V},
title = {CRISPR-Mediated Gene Editing for Inducing Thermosensitive Genic Male Sterility and Sheath Blight Resistance in Rice.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2966},
number = {},
pages = {127-140},
pmid = {41028581},
issn = {1940-6029},
mesh = {*Gene Editing/methods ; *Oryza/genetics/microbiology ; *CRISPR-Cas Systems ; *Disease Resistance/genetics ; *Plant Infertility/genetics ; *Plant Diseases/genetics/microbiology ; Plants, Genetically Modified/genetics ; },
abstract = {Recent advances in genome editing enable the researchers to focus more and more on the ability to manipulate genomes at specific sites. Efficient methods for genome editing further promote gene discovery and functional gene analyses in model plants as well as the introduction of novel desired agricultural traits in important species. CRISPR/Cas9 technology enables precise genetic modification through the creation of double-strand breaks in a target region and the generation of desired alterations during the repair process. In this chapter, we describe the cloning strategy, transformation protocols, triparental mating procedure, and characterization of genome-edited genetic male sterile mutants and sheath blight disease-resistant mutant plants.},
}
@article {pmid41028476,
year = {2026},
author = {Raschmanová, H and Weninger, A and Kovar, K},
title = {Engineering Pichia pastoris Strains Using CRISPR/Cas9 Technologies: The Basic Protocol.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2697},
number = {},
pages = {361-371},
pmid = {41028476},
issn = {1940-6029},
mesh = {*CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; *Saccharomycetales/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; *Metabolic Engineering/methods ; *Pichia/genetics ; Genome, Fungal ; },
abstract = {The CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats, CRISPR-associated protein 9) system has become a commonly used tool for genome editing and metabolic engineering. For Komagataella phaffii, commercialized as Pichia pastoris, the CRISPR/Cas9 protocol for genome editing was established in 2016 and since then has been employed to facilitate genetic modifications such as markerless gene disruptions and deletions as well as to enhance the efficiency of homologous recombination.In this chapter, we describe a robust basic protocol for CRISPR-based genome editing, demonstrating near 100% targeting efficiency for gene inactivation via a frameshift mutation. As described in other chapters of this volume, CRISPR/Cas9 technologies for use in P. pastoris have been further optimized for various specific purposes.},
}
@article {pmid41028475,
year = {2026},
author = {Smirnov, K and Rieder, L and Glieder, A},
title = {High-Throughput Generation of Pichia pastoris Knock-Out Strains by Using CRISPR/Cas9.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2697},
number = {},
pages = {345-360},
pmid = {41028475},
issn = {1940-6029},
mesh = {*CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; Plasmids/genetics ; *Gene Knockout Techniques/methods ; *Saccharomycetales/genetics ; Genetic Vectors/genetics ; },
abstract = {The CRISPR/Cas9 (CRISPR is an acronym for clustered regularly interspaced short palindromic repeats) system is a powerful molecular biological tool simplifying the process of genome engineering. Originally demonstrated to function in human and mouse cells, the portfolio of organisms that can be engineered by the new and groundbreaking technology was soon expanded. In the past years, CRISPR/Cas9 tools for use in Komagataella phaffii were reported to allow the generation of K. phaffii mutant strains in less than 2 weeks. In addition, the K. phaffii tailored system uses episomal vectors for the expression of the CRISPR/Cas9 elements, which allows the recycling of the plasmid after the CRISPR editing to obtain empty mutant strains. This means that the engineered strains do not carry the expression cassette of the resistance marker and CRISPR/Cas9 plasmid in their genome and are therefore a superb starting point for further investigations.In this chapter, we describe a pipeline for the high-throughput generation of K. phaffii mutant strains with interrupted open reading frames of genes, by using the CRISPR/Cas9 system in combination with error-prone repair of the double-strand break by NHEJ. The pipeline we developed consists of four steps: (a) CRISPR/Cas9 plasmids assembly, (b) transformation of K. phaffii, (c) screening for mutant strains, and (d) plasmid elimination and is due to the detailed description of every step being easily reproducible. To intensify and simplify the research work, most of the described procedures can be performed in a 96-well format.},
}
@article {pmid41028463,
year = {2026},
author = {Pichler, C and Weiss, F and Glieder, A},
title = {Autonomously Replicating Sequence-Bearing Plasmids Utilized in Pichia pastoris.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2697},
number = {},
pages = {191-203},
pmid = {41028463},
issn = {1940-6029},
mesh = {*Plasmids/genetics ; *Saccharomycetales/genetics ; *DNA Replication ; Transformation, Genetic ; Saccharomyces cerevisiae/genetics ; },
abstract = {Plasmids are a common tool in biotechnology to deliver recombinant DNA into microbial cells for the production of enzymes, pharmaceutical proteins, chemicals, or metabolites. Therefore, a stable plasmid system that provides reliable gene expression over generations is essential for the successful utilization of single-cell organisms in research and production applications. Most Komagataella phaffii expression clones are generated by the integration of linear plasmids into the genome, as circular episomal plasmids are not stable under non-selective conditions. The low rate of homology-directed specific integration and the large variation among transformants of random integration limit the organism's application in enzyme engineering approaches or comparative studies where high transformation rates and uniform expression levels are desired. In the yeast Saccharomyces cerevisiae, the problem of circular plasmid stability and partition to the daughter cells during mitosis has been solved by combining centromeric sequences or elements of the 2-micron plasmid with an autonomously replicating sequence (ARS) that serves as an origin of replication. Similar attempts have not yet been successful or widely adapted in K. phaffii; hence, permanent selection pressure is required to maintain episomal plasmids in K. phaffii. There are no reports so far about functional 2-micron plasmids for P. pastoris, and CEN/ARS plasmids for P. pastoris are usually rather large and do not provide the high transformation rates as known for episomal plasmids of S. cerevisiae expression systems. However, the availability of a broad set of resistance, auxotrophic, and carbon source utilization markers facilitates reliable plasmid selection in small-scale screening applications and recently also proved to be successful for bioreactor-scale expression. This allows the combined advantages of high transformation rates and low clonal variability of ARS plasmids to be exploited. This article describes the successful utilization of ARS1-containing plasmids in K. phaffii, including antibiotic-free selection, complementation of knockout strains, or even for the application of CRISPR/Cas by transient gRNA and CAS9 gene expression in K. phaffii.},
}
@article {pmid41028441,
year = {2025},
author = {Kannan, S and Tennyson, J},
title = {Regeneration of Transgenic Nicotiana benthamiana Raised from the Genome-Edited Protoplast.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2973},
number = {},
pages = {261-274},
pmid = {41028441},
issn = {1940-6029},
mesh = {*Nicotiana/genetics/growth &amp; development ; *Protoplasts/metabolism ; *Gene Editing/methods ; *Plants, Genetically Modified/genetics ; CRISPR-Cas Systems ; *Genome, Plant ; Transfection/methods ; RNA, Guide, CRISPR-Cas Systems/genetics ; *Regeneration/genetics ; },
abstract = {Plant genome editing is an emerging technique that has revolutionized plant genome engineering which helps to edit the plant genome precisely for the development of traits in many crops. Specifically, with clustered regularly interspaced short palindromic sequence (CRISPR)-CRISPR-associated protein (Cas) system, a progressive improvement in genome editing has been achieved with protoplast. Though protoplast isolation, transfection, and regeneration are available for many plants, regeneration of protoplast for many plants remains major challenge. In this methodology chapter, we outlined the construction of sgRNA for genome editing, transfection, and regeneration of transgenic N. benthamiana from the genome-edited protoplast and assay for gene targeting.},
}
@article {pmid41028420,
year = {2025},
author = {Andrew-Peter-Leon, MT and Pillai, MA and Kumar, KK and Sumithra, V},
title = {Agrobacterium-Mediated Genetic Transformation and Genome Editing Using CRISPR-Cas9 Constructs in Rice.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2973},
number = {},
pages = {27-49},
pmid = {41028420},
issn = {1940-6029},
mesh = {*Oryza/genetics ; *Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; *Transformation, Genetic ; *Agrobacterium/genetics ; Plants, Genetically Modified/genetics ; *Genome, Plant ; },
abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated (Cas)9 has become an essential tool in every molecular plant breeding laboratory. CRISPR-Cas9 causes highly precise knock-out mutations in plants that can be exploited in crop improvement programmes. However, poor response to Agrobacterium-mediated genetic transformation in recalcitrant rice genotypes is a major limiting factor. This protocol describes a detailed procedure for genome editing with CRISPR-Cas9 in recalcitrant rice genotypes that otherwise show a poor response to tissue culture. With this method, high transformation efficiency can be achieved in relatively a short period.},
}
@article {pmid41028383,
year = {2026},
author = {Yang, H and Bao, A and Tran, LP and Cao, D},
title = {CRISPR/Cas9-Based Gene Editing in Soybean.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2977},
number = {},
pages = {251-267},
pmid = {41028383},
issn = {1940-6029},
mesh = {*Glycine max/genetics ; *Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; Plants, Genetically Modified/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; Plasmids/genetics ; Agrobacterium/genetics ; Transformation, Genetic ; },
abstract = {CRISPR/Cas9 (clustered regularly interspaced short palindromic repeat/CRISPR-associated Cas9)-based gene editing is a robust tool for functional genomics research and breeding programs in various crops. In soybean (Glycine max), a number of laboratories have obtained mutants by the CRISPR/Cas9 system; however, there has not yet been a detailed method for the CRISPR/Cas9-based gene editing in soybean. Here, we describe the procedures for constructing the CRISPR/Cas9 plasmid suitable for soybean gene editing and the modified protocols for Agrobacterium-mediated soybean transformation and regeneration from cotyledonary node explants containing the Cas9/sgRNA (single-guide RNA) transgenes.},
}
@article {pmid41028112,
year = {2025},
author = {Najar, IN and Sharma, P and Das, R and Mondal, K and Singh, AK and Radha, A and Sharma, V and Sharma, S and Thakur, N and Gandhi, SG and Kumar, V},
title = {Unveiling the probiotic potential of the genus Geobacillus through comparative genomics and in silico analysis.},
journal = {Scientific reports},
volume = {15},
number = {1},
pages = {33748},
pmid = {41028112},
issn = {2045-2322},
mesh = {*Probiotics ; *Geobacillus/genetics ; *Genome, Bacterial ; *Genomics/methods ; Computer Simulation ; CRISPR-Cas Systems ; Humans ; Prophages/genetics ; },
abstract = {Pursuing new probiotic targets has surged, driven by next-generation sequencing, facilitating a thorough exploration of bacterial traits. The genus Geobacillus stands out as a promising candidate for probiotics. The study explored the genetic attributes of the genus Geobacillus for their resilience to gastrointestinal conditions, nutrient production, and immunomodulatory compound creation, revealing potential probiotic traits. Predictive analyses of genomic elements like prophages, CRISPR-Cas systems, insertion sequences, genomic islands, antibiotic resistance genes, and CAZymes were conducted to assess safety. Comparative genomic analysis was performed using 18 published Geobacillus genomes and a few Lactobacillus and Bifidobacterium genomes as controls. Genes associated with probiotic traits, such as adhesion, stress tolerance (acid/bile, osmotic, oxidative), immune modulation, and molecular chaperones, were uniformly detected in Geobacillus. Mobile genetic elements (such as plasmids, prophages, and insertion sequences), virulence factors, toxins, and antibiotic resistance genes were absent, while CRISPR-Cas systems and CAZymes were present. The pan-genome comprised 25,284 protein-coding genes. Comparative genomic analysis revealed an open pan-genome for Geobacillus. Pan-genome exhibited variability, particularly in genes linked to environmental interaction and secondary metabolite synthesis. Geobacillus appears potentially safe and well-suited for the gut habitat. However, further in vitro studies are essential to confirm its probiotic potential.},
}
@article {pmid41027434,
year = {2025},
author = {Jin, S and Zhu, Z and Li, Y and Zhang, S and Liu, Y and Li, D and Li, Y and Luo, Y and Cheng, Z and Zhao, KT and Gao, Q and Yang, G and Li, H and Liang, R and Zhang, R and Qiu, JL and Zhang, YE and Liu, JG and Gao, C},
title = {Functional RNA splitting drove the evolutionary emergence of type V CRISPR-Cas systems from transposons.},
journal = {Cell},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.cell.2025.09.004},
pmid = {41027434},
issn = {1097-4172},
abstract = {Transposon-encoded TnpB nucleases gave rise to type V CRISPR-Cas12 effectors through multiple independent domestication events. These systems use different RNA molecules as guides for DNA targeting: transposon-derived right-end RNAs (reRNAs or omega RNAs) for TnpB and CRISPR RNAs for type V CRISPR-Cas systems. However, the molecular mechanisms bridging transposon activity and CRISPR immunity remain unclear. We identify TranCs (transposon-CRISPR intermediates) derived from distinct IS605- or IS607-TnpB lineages. TranCs utilize both CRISPR RNAs and reRNAs to direct DNA cleavage. The cryoelectron microscopy (cryo-EM) structure of LaTranC from Lawsonibacter sp. closely resembles that of the ISDra2 TnpB complex; however, unlike a single-molecule reRNA, the LaTranC guide RNA is functionally split into a tracrRNA and crRNA. An engineered RNA split of ISDra2 TnpB enabled activity with a CRISPR array. These findings indicate that functional RNA splitting was the primary molecular event driving the emergence of diverse type V CRISPR-Cas systems from transposons.},
}
@article {pmid41026506,
year = {2025},
author = {Demissie, HA and Das, S and Thompson, JR and Lucks, JB},
title = {An Integrated Nucleic Acid Sequence-Based Amplification (NASBA) and CRISPR-Cas13a-Based Platform for Accurate and Sensitive Detection of Cucumber Mosaic Virus.},
journal = {ACS synthetic biology},
volume = {},
number = {},
pages = {},
doi = {10.1021/acssynbio.5c00406},
pmid = {41026506},
issn = {2161-5063},
abstract = {Cucumber mosaic virus (CMV) is a highly prevalent ssRNA viral crop pathogen that contributes to substantial losses in agricultural productivity worldwide. The first step in managing the impact of this pathogen is an accurate and timely diagnosis. However, current sensing strategies are hampered by several limitations, including insufficient sensitivity, off-target effects, and the need for complex instrumentation. To address these challenges, we refined a highly specific and sensitive system that pairs nucleic acid sequence-based amplification (NASBA) with clustered regularly interspaced short palindromic repeats (CRISPR)-Cas13a to selectively amplify and detect crop pathogens. To configure this system for CMV biosensing, we first screened guide RNAs and successfully validated designs that detect attomolar concentrations of purified CMV fragments. We then developed a simplified reaction assembly workflow toward optimizing the system for downstream point-of-use utility. Using this workflow, we demonstrated minimal matrix effects when detecting purified CMV fragments in a range of plant lysate backgrounds and showed high test specificity to CMV in the presence of common nontarget viral crop pathogens. We also showed that the NASBA-Cas13a system effectively detects the viral target in infected plant samples, as validated by reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Finally, we optimized the system for lyophilization and long-term storage, toward preparing it for point-of-use settings. This work expands the suite of CMV diagnostic tools, offering a sensitive, specific, and user-friendly biosensing strategy. Through modular design, this assay has the potential to be reconfigured for the detection of a range of crop viruses, enhancing viral surveillance and improving infection management.},
}
@article {pmid41026406,
year = {2025},
author = {Rahman, MU and Shah, JA and Khan, MN and Bilal, H and Zhu, D and Du, Z and Mu, DS},
title = {Innovative Approaches to Combat Antimicrobial Resistance: A Review of Emerging Therapies and Technologies.},
journal = {Probiotics and antimicrobial proteins},
volume = {},
number = {},
pages = {},
pmid = {41026406},
issn = {1867-1314},
abstract = {The threat of antimicrobial resistance (AMR) presents a challenge in infectious diseases, leading to higher illness and deaths worldwide. No new antibiotic has been introduced, leaving healthcare systems vulnerable to resistant pathogens. Researchers are exploring innovative approaches to overcome this growing resistance crisis. One promising strategy is synergistic therapy using combined drugs to enhance efficacy and reduce resistance. Other approaches focus on targeting the specific enzymes or proteins responsible for resistance mechanisms, thereby neutralizing the defense strategies of microorganisms. Advances in drug delivery systems have also shown promise in improving the effectiveness of existing antimicrobial agents. Biotechnological breakthroughs, such as bacteriophages and antibodies, have seen partial clinical implementation, while newer approaches like antimicrobial peptides (AMPs), lysins, and probiotics are still under development. Emerging technologies such as CRISPR-Cas and engineered phages demonstrate significant potential in preclinical studies, offering precision targeting of resistance genes and pathogen-specific lysis, respectively. However, their translational success hinges on overcoming delivery challenges, scalability, and regulatory hurdles. Additionally, physicochemical methods that disrupt microbial activity are being explored as alternative treatments. While innovative therapies like phage-derived lysins and CRISPR-Cas systems show promise in preclinical models, their clinical impact remains to be validated through large-scale trials. Their integration into mainstream medicine will depend on addressing practical challenges such as manufacturing consistency, cost considerations, and real-world efficacy assessments. These efforts are crucial for addressing the growing threat of AMR and advancing more effective, sustainable infection control strategies in clinical settings.},
}
@article {pmid41025805,
year = {2025},
author = {Nadar, S and Brown, JC and Coe, LSY and Koukoulidis, NM and Czyż, EM and Czyż, DM},
title = {Antimicrobial resistance and One Health in the high school biology curriculum.},
journal = {Journal of microbiology &amp; biology education},
volume = {},
number = {},
pages = {e0014525},
doi = {10.1128/jmbe.00145-25},
pmid = {41025805},
issn = {1935-7877},
abstract = {Antimicrobial resistance (AMR) is the ability of a microbial organism to resist treatment designed to kill it. It poses a significant global threat to public health, affecting humans, animals, and the environment, in a concept collectively referred to as One Health. While one of the major mitigators of this pressing issue is education, the high school curriculum in the United States does not cover any aspects of AMR. As such, to address this challenge, we developed and delivered a one-week-long unit on AMR within a One Health framework into a high school biology curriculum. The unit aimed to enhance students' understanding of AMR and its implications across the One Health sectors. A survey was designed and administered to measure current knowledge, awareness, interest, and motivation. Through a combination of lectures developed using Universal Design of Learning principles, interactive discussions using team-based learning (TBL) with the help of content experts, hands-on laboratory exercise, and poster presentations, biology students explored the mechanisms of resistance and novel mitigation strategies. Pre- and post-assessments revealed a marked improvement in students' knowledge and comprehension of AMR and therapeutic strategies, such as silver nanoparticles, bacteriocins, bacteriophages, CRISPR-Cas, and immunotherapy. This research study provides a detailed overview of the curriculum design, instructional strategies, and assessment outcomes, offering a replicable model for broadly integrating AMR education into high school curricula. We found that the AMR mitigation strategies lesson, delivered through TBL, significantly enhanced students' understanding of novel therapeutic strategies and fostered high levels of engagement throughout the AMR and One Health unit.},
}
@article {pmid40961329,
year = {2025},
author = {Joseph, RA and Haley, RM and Padilla, MS and Ricciardi, AS and Yamagata, HM and Mitchell, MJ},
title = {Cas9 Protein Outperforms mRNA in Lipid Nanoparticle-Mediated CFTR Repair.},
journal = {Nano letters},
volume = {25},
number = {39},
pages = {14348-14355},
doi = {10.1021/acs.nanolett.5c03548},
pmid = {40961329},
issn = {1530-6992},
mesh = {*Cystic Fibrosis Transmembrane Conductance Regulator/genetics/metabolism ; *Cystic Fibrosis/genetics/therapy ; *CRISPR-Cas Systems/genetics ; Gene Editing/methods ; *CRISPR-Associated Protein 9/genetics ; Humans ; *Nanoparticles/chemistry ; Animals ; *RNA, Messenger/genetics/chemistry/administration &amp; dosage ; RNA, Guide, CRISPR-Cas Systems/genetics ; Mice ; *Lipids/chemistry ; Liposomes ; },
abstract = {Lipid nanoparticles (LNPs) are currently one of the most clinically advanced delivery systems for nucleic acid cargo and hold great potential for clinical applications in gene editing and the treatment of genetic diseases. LNP-mediated delivery of Cas9 with single guide RNA (sgRNA) and homology-directed repair DNA template (ssDNA) enables efficient and precise editing in vitro and in vivo. Comparative analysis of LNP delivery of Cas9 as protein or mRNA for relevant clinical targets, such as cystic fibrosis (CF), which is caused by mutations in the CFTR gene, is imperative in the design of corrective therapeutics for genetic diseases. Here, we show that delivery of Cas9 protein LNPs outperforms Cas9 mRNA LNPs when evaluated for in vivo lung editing as well as corrective CRISPR/Cas9 editing and functional recovery of the CFTR protein. These results demonstrate the ability to optimize the use of CRISPR/Cas9 LNPs for cystic fibrosis applications.},
}
@article {pmid40938323,
year = {2025},
author = {Martin-Vicente, A and Nywening, AV and Xie, J and Thorn, HI and Guruceaga, X and Fortwendel, JR},
title = {Genetic analysis of common triazole resistance mechanisms in a collection of Aspergillus lentulus clinical isolates from the United States.},
journal = {Antimicrobial agents and chemotherapy},
volume = {69},
number = {10},
pages = {e0069025},
doi = {10.1128/aac.00690-25},
pmid = {40938323},
issn = {1098-6596},
support = {R01AI143197/NH/NIH HHS/United States ; R01AI158442/NH/NIH HHS/United States ; R21AI178048/NH/NIH HHS/United States ; },
mesh = {*Triazoles/pharmacology ; *Antifungal Agents/pharmacology ; *Aspergillus/drug effects/genetics/isolation &amp; purification ; *Drug Resistance, Fungal/genetics ; Microbial Sensitivity Tests ; United States ; Fungal Proteins/genetics/metabolism ; Aspergillosis/microbiology/drug therapy ; Humans ; Cytochrome P-450 Enzyme System/genetics/metabolism ; Aspergillus fumigatus/drug effects/genetics ; CRISPR-Cas Systems/genetics ; },
abstract = {Aspergillus fumigatus continues to be the leading cause of invasive aspergillosis. However, the number of cases by drug-resistant cryptic species has increased in recent years. Aspergillus lentulus is a sibling species of Aspergillus section Fumigati that can only be distinguished from A. fumigatus by molecular methods. The clinical importance of this species resides in its low susceptibility to triazoles and intrinsic resistance to amphotericin B, making invasive aspergillosis treatments extremely challenging and producing high mortality rates. In this study, we investigate known molecular mechanisms important for triazole resistance in A. fumigatus in a collection of 25 clinical A. lentulus isolates from the United States. Using CRISPR-Cas9 gene editing technology, we performed cyp51A and hmg1 allele replacements between susceptible and resistant isolates. Phenotypic characterization of the resulting mutants, together with mRNA expression analyzes of cyp51A, cyp51B, and the putative ABC efflux pump, abcC, suggests that triazole resistance in our A. lentulus isolates is independent of the mechanisms studied.},
}
@article {pmid40692527,
year = {2025},
author = {Kohso, A and Inaba, H and Kanemaki, MT and Gabazza, EC and Toyoda, H and Hirayama, M and Goto, H},
title = {Aurora-A Promotes Cell-Cycle Progression From Quiescence Through Primary Cilia Disassembly.},
journal = {Cancer science},
volume = {116},
number = {10},
pages = {2763-2773},
doi = {10.1111/cas.70153},
pmid = {40692527},
issn = {1349-7006},
support = {JP22K06219//Japan Society for the Promotion of Science/ ; JP23H04925//Japan Society for the Promotion of Science/ ; JP23K06674//Japan Society for the Promotion of Science/ ; JP23K07651//Japan Society for the Promotion of Science/ ; //Takeda Science Foundation/ ; },
mesh = {Humans ; *Cilia/metabolism ; HCT116 Cells ; *Aurora Kinase A/metabolism/genetics ; *Cell Cycle ; CRISPR-Cas Systems ; Apoptosis/genetics ; Cell Proliferation ; },
abstract = {Aurora-A (AurA) is a member of the mitotic kinase family and is highly expressed in various tumors. Inhibition of AurA generally leads to fetal mitotic errors. We previously reported that AurA inhibition induces G0/G1 cell cycle arrest in noncancerous cells by promoting the reassembly of primary cilia. However, the mechanisms by which AurA regulates cell cycle progression beyond mitosis remain largely unknown. In this study, we generated noncancerous RPE1 and cancerous HCT116 cell lines expressing endogenous AurA tagged with a minimal auxin-inducible degron (mAID) using CRISPR/Cas9-based gene editing. This system enabled specific and rapid depletion of endogenous AurA protein. By combining this approach with cell synchronization in RPE1 cells, we investigated AurA function specifically in the transition from quiescence to the proliferative cell cycle. Targeted degradation of AurA not only delayed cell cycle progression but also impaired the disassembly of primary cilia at the G0/G1 transition in RPE1 cells. Since this delay in cell cycle progression was rescued by forced deciliation via the knockout of IFT20, AurA facilitates deciliation, which in turn accelerates the transition from quiescence to the proliferative phase of the cell cycle in RPE1 cells. AurA depletion for 4 days increased apoptotic markers in HCT116 cells but not in RPE1 cells. Notably, forced deciliation in RPE1 cells partially enhanced apoptosis induced by AurA depletion. These results suggest that the ability to assemble primary cilia may serve as a protective mechanism against cell death following AurA inhibition.},
}
@article {pmid40675820,
year = {2025},
author = {Turn, RE and Hilgendorf, KI and Johnson, CT and Han, K and Aziz-Zanjani, MO and Swails Bollinger, S and Domizi, P and Cheng, R and Rabiee, A and Zhu, Y and Jiang, Z and Asthana, A and Demeter, J and Svensson, KJ and Bassik, MC and Jackson, PK},
title = {A genome-wide, CRISPR-based screen reveals new requirements for translation initiation and ubiquitination in driving adipogenic fate change.},
journal = {Genes &amp; development},
volume = {39},
number = {19-20},
pages = {1241-1264},
doi = {10.1101/gad.352779.125},
pmid = {40675820},
issn = {1549-5477},
support = {P30 DK116074/DK/NIDDK NIH HHS/United States ; R01 DK125260/DK/NIDDK NIH HHS/United States ; },
mesh = {Animals ; Mice ; *Ubiquitination/genetics ; *Adipogenesis/genetics ; 3T3-L1 Cells ; Lipogenesis/genetics ; Adipocytes/cytology/metabolism ; *Peptide Chain Initiation, Translational/genetics ; CRISPR-Cas Systems/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Mitosis/genetics ; },
abstract = {In response to excess nutrients, white adipose tissue expands by both generating new adipocytes and upregulating lipogenesis in existing adipocytes. Here, we performed a genome-wide functional CRISPR screen to identify regulators of adipogenesis in the mouse 3T3-L1 preadipocyte model. In this pooled screening strategy, we used FACS to isolate populations based on lipid content, gating for fluorescence intensity of lipophilic fluorescent BODIPY dye. Additionally, we categorized whether the gene functions primarily during mitotic clonal expansion, lipogenesis, or both. We found that translation initiation and ubiquitin-dependent protein stability regulators drive both adipogenic fate change and lipogenesis. We further supported these findings with proteomics, demonstrating that essential changes in protein reprogramming can drive or inhibit 3T3-L1 adipogenesis independent of transcription. Furthermore, we demonstrated that specific branches of the hypusination pathway, a conserved regulator of translation initiation, are critical for translating adipogenic inducers of mitotic clonal expansion and that the neddylation/ubiquitin pathway modulates insulin sensitivity during lipogenesis.},
}
@article {pmid40671314,
year = {2025},
author = {Kumar, P and Bisht, NC},
title = {High-level production of health-beneficial glucoraphanin by multiplex editing of AOP2 gene family in mustard.},
journal = {Plant biotechnology journal},
volume = {23},
number = {10},
pages = {4668-4680},
doi = {10.1111/pbi.70171},
pmid = {40671314},
issn = {1467-7652},
support = {BT/PR23893/GET/119/81/2017//Department of Biotechnology, Ministry of Science and Technology, India/ ; BT/PR53959/PBN/18/17/2024//Department of Biotechnology, Ministry of Science and Technology, India/ ; },
mesh = {Sulfoxides ; *Glucosinolates/metabolism/biosynthesis ; Oximes ; *Imidoesters/metabolism ; *Mustard Plant/genetics/metabolism ; *Gene Editing/methods ; CRISPR-Cas Systems ; Plants, Genetically Modified/metabolism ; *Plant Proteins/genetics/metabolism ; *Isothiocyanates/metabolism ; },
abstract = {Intake of glucosinolates through the consumption of cruciferous vegetables has been associated with numerous health benefits. In recent decades, glucosinolate glucoraphanin has gained a lot of attention, as its hydrolysis product (sulforaphane) is known to possess numerous health-promoting benefits, including anti-cancer and chemopreventive activities. However, due to the low availability of glucoraphanin in most of the cultivated Brassica crops (except broccoli), there is an increasing interest in many laboratories around the world to manipulate the glucosinolate profile for human benefit. Here, we report the high-level production of health-beneficial glucoraphanin by CRISPR/Cas9 editing of the ALKENYL HYDROXALKYL PRODUCING 2 (BjuAOP2) gene family, displaying distinct expression profiles in the allotetraploid mustard, Brassica juncea. Multiplex editing of five BjuAOP2 homologues, using four gRNAs, provided glucoraphanin accumulation up to 41.60, 75.10, 59.21 and 27.64 μmoles/g dry weight in sprouts, microgreens, seeds and leaves, respectively, of the transgene-free BjuAOP2-edited lines, while providing a significant reduction of the anti-nutritional and goitrogenic alkenyl glucosinolates including progoitrin. The glucoraphanin enhancement in BjuAOP2-edited lines was found to be dose-dependent, wherein loss-of-function mutations in BjuAOP2.A09 and BjuAOP2.B01 homologues had a more prominent effect. The transgene-free BjuAOP2-edited lines were stable for high glucoraphanin and performed at par with the wild-type plants for various seed quality and yield parameters when tested under containment conditions in the field. The development of high-glucoraphanin mustard will help its adoption as a global superfood with health-promoting benefits and as a bioactive source of high-value sulforaphane for industrial production.},
}
@article {pmid40652545,
year = {2025},
author = {Zhu, M and Yan, L and Zhan, Z and Chen, H and Wang, D and Xu, M and Zheng, Z and Zhang, Y and Yang, N and Wu, J and Zhan, H and Tian, Y and Xiong, L and He, Y},
title = {Transgene-Killer-CRISPR version 2 (TKC2) eliminates occasional transgene escape by coupling with a RUBY reporter.},
journal = {Plant biotechnology journal},
volume = {23},
number = {10},
pages = {4621-4632},
doi = {10.1111/pbi.70257},
pmid = {40652545},
issn = {1467-7652},
support = {32200335//National Natural Science Foundation of China/ ; U21A20207//National Natural Science Foundation of China/ ; Y2023QC39//Youth innovation Program of Chinese Academy of Agricultural Sciences/ ; CARS-01//Earmarked Fund for China Agriculture Research System/ ; YBXM2405//Nanfan special project of CAAS/ ; YBXM2446//Nanfan special project of CAAS/ ; B23CJ0208//Hainan Seed Industry Laboratory/ ; },
mesh = {*Transgenes/genetics ; Plants, Genetically Modified/genetics ; *Gene Editing/methods ; Genes, Reporter/genetics ; *CRISPR-Cas Systems/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; Oryza/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; },
abstract = {A critical step in generating gene-edited plants is the removal of CRISPR-related transgenes from T0 plants and their progenies, a process that is generally time-consuming and labour-intensive. We previously reported a Transgene Killer CRISPR (TKC) technology that enables self-elimination of transgenes after the targeted gene has been edited. However, we observed that a small number of T1 plants generated by TKC still retained the transgenes. Herein, we first integrated Cas9 and guide RNA (gRNA) with the RUBY reporter gene (RUBY-CRISPR) to monitor the Cas9/sgRNA expression and track the presence or absence of transgenes in the T0 generation and its progenies. We then combined the RUBY-CRISPR unit with several TKC variants to develop four RUBY-TKC (TKC2) systems including TKC2.1, TKC2.2, TKC2.3 and TKC2.4, to facilitate the elimination of escaped transgene plants. Compared to non-TKC, TKC alone and RUBY-CRISPR, our TKC2s were much more efficient in the generation of transgene-free edited progenies by up to 100% in the T0 generation. TKC2s not only omit the need for screening of the plants with transgenes in the T0 generation, but also enable visualisation of the escape of plants with transgenes in the following progenies. The TKC2 systems developed here provide straightforward yet highly effective approaches for the generation of transgene-free edited plants for either rice functional genomics or genetic improvement, with potential applications in gene editing of other crops.},
}
@article {pmid39970341,
year = {2025},
author = {Fan, F and Wu, MY and Zhang, HQ and Li, G and Luo, C},
title = {Rapid and Simple Detection of Anilinopyrimidine Resistance in Botrytis cinerea by Combining Recombinase Polymerase Amplification with the CRISPR/Cas12a Assay.},
journal = {Plant disease},
volume = {109},
number = {9},
pages = {1831-1838},
doi = {10.1094/PDIS-11-24-2346-SR},
pmid = {39970341},
issn = {0191-2917},
mesh = {*Botrytis/drug effects/genetics ; *Drug Resistance, Fungal/genetics ; *CRISPR-Cas Systems/genetics ; *Fungicides, Industrial/pharmacology ; *Nucleic Acid Amplification Techniques/methods ; Plant Diseases/microbiology ; *Pyrimidines/pharmacology ; Mutation ; Recombinases/genetics/metabolism ; },
abstract = {Anilinopyrimidine (AP) fungicides have been widely adopted to control Botrytis cinerea since the 1990s. As a high-risk pathogen for the development of fungicide resistance, B. cinerea developed resistance to AP fungicides soon after their application. To ensure the proper use of these fungicides, it is necessary to establish a rapid and simple method for resistance detection. Our previous study demonstrated that the E407K mutation in Bcmdl1 was the major mutation conferring AP resistance in China. Based on the combination of recombinase polymerase amplification (RPA) and CRISPR/Cas12a nucleic acid detection assay (RPA/Cas12a detection assay), a simple method for the rapid detection of AP resistance was established by specifically identifying this resistance-related mutation. The new detection assay could precisely identify the E407K mutants from other mutants and wild-type isolates within 50 min, relying solely on a water/metal bath and a UV flashlight. Moreover, this assay could detect genomic DNA at a concentration as low as 1.8 × 10[6] fg/μl, which is comparable with conventional PCR, indicating its high sensitivity. High specificity among different species was also observed in this assay. Above all, this assay was compatible with a 2-min DNA extraction method, implying its feasibility for field application. In conclusion, the RPA/Cas12a detection assay developed in this study is rapid and simple, making it an ideal method for AP resistance detection in local agencies and other points of care. Instant information on resistance monitoring can provide important guidance on resistance management.},
}
@article {pmid41025245,
year = {2025},
author = {Shin, K and Kim, ET},
title = {Efficient CRISPR-based genome editing for inducible degron systems to enable temporal control of protein function in large double-stranded DNA virus genomes.},
journal = {Journal of microbiology (Seoul, Korea)},
volume = {63},
number = {9},
pages = {e2504008},
doi = {10.71150/jm.2504008},
pmid = {41025245},
issn = {1976-3794},
support = {RS-2024-00352590//Ministry of Science and ICT/ ; RS-2023-00270936//Ministry of Education/ ; //Korea Health Industry Development Institute/ ; RS-2022-KH129726//Ministry of Health and Welfare/ ; RS-2024-00438990//Ministry of Health and Welfare/ ; },
mesh = {*CRISPR-Cas Systems ; *Gene Editing/methods ; Humans ; *Cytomegalovirus/genetics ; *Genome, Viral ; *Viral Proteins/genetics/metabolism ; Indoleacetic Acids/metabolism/pharmacology ; Green Fluorescent Proteins/genetics ; Recombinational DNA Repair ; Degrons ; },
abstract = {CRISPR-Cas9-based gene editing enables precise genetic modifications. However, its application to human cytomegalovirus (HCMV) remains challenging due to the large size of the viral genome and the essential roles of key regulatory genes. Here, we establish an optimized CRISPR-Cas9 system for precise labeling and functional analysis of HCMV immediate early (IE) genes. By integrating a multifunctional cassette encoding an auxin-inducible degron (AID), a self-cleaving peptide (P2A), and GFP into the viral genome via homology-directed repair (HDR), we achieved efficient knock-ins without reliance on bacterial artificial chromosome (BAC) cloning, a labor-intensive and time-consuming approach. We optimized delivery strategies, donor template designs, and component ratios to enhance HDR efficiency, significantly improving knock-in success rates. This system enables real-time fluorescent tracking and inducible protein degradation, allowing temporal control of essential viral proteins through auxin-mediated depletion. Our approach provides a powerful tool for dissecting the dynamic roles of viral proteins throughout the HCMV life cycle, facilitating a deeper understanding of viral pathogenesis and potential therapeutic targets.},
}
@article {pmid41025215,
year = {2025},
author = {Park, J and Sipe, GO and Tang, X and Ojha, P and Fernandes, G and Leow, YN and Zhang, C and Osako, Y and Natesan, A and Drummond, GT and Jaenisch, R and Sur, M},
title = {Astrocytic modulation of population encoding in mouse visual cortex via GABA transporter 3 revealed by multiplexed CRISPR/Cas9 gene editing.},
journal = {eLife},
volume = {14},
number = {},
pages = {},
doi = {10.7554/eLife.107298},
pmid = {41025215},
issn = {2050-084X},
support = {R01DA049005/NH/NIH HHS/United States ; R01MH126351/NH/NIH HHS/United States ; R01NS130361/NH/NIH HHS/United States ; R01MH133066/NH/NIH HHS/United States ; Multidisciplinary University Research Initiative W911NF2110328//United States Department of Defense/ ; F32EY022264/NH/NIH HHS/United States ; Autism Research Initiative Bridge to Independence award//Simons Foundation/ ; Picower Postdoctoral Fellowship//JPB Foundation/ ; },
mesh = {Animals ; *Visual Cortex/physiology ; *Astrocytes/physiology/metabolism ; *GABA Plasma Membrane Transport Proteins/metabolism/genetics ; Mice ; *CRISPR-Cas Systems ; *Gene Editing ; Mice, Knockout ; Neurons/physiology ; },
abstract = {Astrocytes, which are increasingly recognized as pivotal constituents of brain circuits governing a wide range of functions, express GABA transporter 3 (Gat3), an astrocyte-specific GABA transporter responsible for maintenance of extra-synaptic GABA levels. Here, we examined the functional role of Gat3 in astrocyte-mediated modulation of neuronal activity and information encoding. First, we developed a multiplexed CRISPR construct applicable for effective genetic ablation of Gat3 in the visual cortex of adult mice. Using in vivo two-photon calcium imaging of visual cortex neurons in Gat3 knockout mice, we observed changes in spontaneous and visually driven single neuronal response properties such as response magnitudes and trial-to-trial variability. Gat3 knockout exerted a pronounced influence on population-level neuronal activity, altering the response dynamics of neuronal populations and impairing their ability to accurately represent stimulus information. These findings demonstrate that Gat3 in astrocytes profoundly shapes the sensory information encoding capacity of neurons and networks within the visual cortex.},
}
@article {pmid41024337,
year = {2025},
author = {Zhang, L and Fu, J and Long, T and Zhang, C and Fan, F and Lang, Z and Zhu, JK},
title = {A Modular and Customizable CRISPR/Cas Toolkit for Epigenome Editing of Cis-regulatory Modules.},
journal = {Advanced science (Weinheim, Baden-Wurttemberg, Germany)},
volume = {},
number = {},
pages = {e03917},
doi = {10.1002/advs.202503917},
pmid = {41024337},
issn = {2198-3844},
support = {KQTD20240729102038044//Shenzhen Science and Technology Program/ ; 32188102//National Natural Science Foundation of China/ ; },
abstract = {Epigenome and cis-regulome, comprising cis-regulatory elements (CREs) and modules (CRMs), jointly define the architecture of gene regulation. However, the causal mechanisms by which epigenetic marks influence CRM function remain elusive. To address this, modular epigenome editing frameworks, exemplified by dead Cas9-coupled DNA demethylation (dCd) and DNA methylation (dCm) platforms, are developed for programmable dissection and engineering of CRM activity. The dCd system modulates methylation levels and transcriptional output at CRMs in situ or ex situ, in accordance with CRM-specific methylation responsiveness, and alters co-transcriptional RNA processing to yield predictable phenotypic outcomes in plants. These findings underscore the reliability of targeted DNA demethylation. In parallel, the dCm system reconstitutes methylation-dependent and -sensitive CRMs of diverse origins in Saccharomyces cerevisiae, a species devoid of native DNA methylation, enabling causal dissection of epigenetic regulation and revealing cross-species portability. This system further uncovers crosstalk between DNA methylation and chromatin modifications, and enables logic-gated control of endogenous genes through CRM engineering. Incorporation of optogenetic and temperature-sensitive anti-CRISPR inhibitors confers tunable, reversible regulation, proposing dCm as a foundation for input-responsive synthetic epigenome editors. Together, these frameworks provide a versatile platform to decode and reprogram cis-regulatory epigenetic logic, with broad applications in trait design and synthetic biology.},
}
@article {pmid41023727,
year = {2025},
author = {Zhao, XY and Gao, C and Zhao, WW and Zhou, ZH and Zhuang, TC and Guo, C and Ji, MH},
title = {Development of a single-tube, dual-target CRISPR Cas12a/Cas13a system for rapid screening of coinfection with respiratory syncytial virus and rhinovirus.},
journal = {Virology journal},
volume = {22},
number = {1},
pages = {311},
pmid = {41023727},
issn = {1743-422X},
support = {Jiangsu Education Department (2023) No.11//"Nursing Science" Funded by the 4th Priority Discipline Development Program of Jiangsu Higher Education Institutions/ ; Jiangsu Education Department (2023) No.11//"Nursing Science" Funded by the 4th Priority Discipline Development Program of Jiangsu Higher Education Institutions/ ; Jiangsu Education Department (2023) No.11//"Nursing Science" Funded by the 4th Priority Discipline Development Program of Jiangsu Higher Education Institutions/ ; Jiangsu Education Department (2023) No.11//"Nursing Science" Funded by the 4th Priority Discipline Development Program of Jiangsu Higher Education Institutions/ ; Jiangsu Education Department (2023) No.11//"Nursing Science" Funded by the 4th Priority Discipline Development Program of Jiangsu Higher Education Institutions/ ; Jiangsu Education Department (2023) No.11//"Nursing Science" Funded by the 4th Priority Discipline Development Program of Jiangsu Higher Education Institutions/ ; Ym2023064//Jiangsu Provincial Health Commission Public Health Research Project/ ; Ym2023064//Jiangsu Provincial Health Commission Public Health Research Project/ ; Ym2023064//Jiangsu Provincial Health Commission Public Health Research Project/ ; Ym2023064//Jiangsu Provincial Health Commission Public Health Research Project/ ; Ym2023064//Jiangsu Provincial Health Commission Public Health Research Project/ ; Ym2023064//Jiangsu Provincial Health Commission Public Health Research Project/ ; NJYFKT202409//Nanjing Preventive Medicine Research Project/ ; NJYFKT202409//Nanjing Preventive Medicine Research Project/ ; NJYFKT202409//Nanjing Preventive Medicine Research Project/ ; NJYFKT202409//Nanjing Preventive Medicine Research Project/ ; NJYFKT202409//Nanjing Preventive Medicine Research Project/ ; NJYFKT202409//Nanjing Preventive Medicine Research Project/ ; },
mesh = {Humans ; *Coinfection/diagnosis/virology ; *Respiratory Syncytial Virus Infections/diagnosis/virology ; *Rhinovirus/genetics/isolation &amp; purification ; *CRISPR-Cas Systems ; Sensitivity and Specificity ; *Respiratory Syncytial Virus, Human/genetics/isolation &amp; purification ; *Picornaviridae Infections/diagnosis/virology ; *Molecular Diagnostic Techniques/methods ; *Respiratory Tract Infections/diagnosis/virology ; CRISPR-Associated Proteins/genetics ; Infant ; Bacterial Proteins ; Endodeoxyribonucleases ; },
abstract = {BACKGROUND: Respiratory syncytial virus (RSV) and human rhinovirus (HRV) are leading causes of respiratory infections in children, with increasing reports of coinfections leading to severe complications. Current CRISPR-based detection systems, such as Cas12a and Cas13a, are limited in multiplex detection due to the lack of specific reporter cleavage mechanisms. This study aims to develop a rapid, sensitive, and single-tube dual-gene detection method for RSV and HRV using the orthogonal trans-cleavage activities of CRISPR-Cas12a/13a combined with reverse transcription-recombinase polymerase amplification (RT-RPA).<br><br>METHODS: We designed a novel detection system leveraging RT-RPA for amplification and the distinct cleavage activities of Cas12a and Cas13a for simultaneous dual-gene detection.<br><br>RESULTS: The reaction components were optimized to complete detection within 30&#xa0;min, achieving sensitivities of 10 copies/&#xb5;L for RSV and 10[2] copies/&#xb5;L for HRV. Clinical validation was performed on 543 respiratory infection samples, confirming high accuracy and specificity.<br><br>CONCLUSIONS: The RT-RPA-CRISPR-Cas12a/13a system provides a rapid, sensitive, and efficient solution for RSV and HRV coinfection detection. This method supports early diagnosis and improved clinical management, offering significant potential for public health applications in preventing severe respiratory complications in children.},
}
@article {pmid41023259,
year = {2025},
author = {Ravenel, K and Poirier, W and Razafimandimby, B and Bouchara, JP and Gastebois, A and Giraud, S},
title = {Optimization of the Genome Editing CRISPR-Cas9 Technology in Scedosporium apiospermum.},
journal = {Mycopathologia},
volume = {190},
number = {6},
pages = {94},
pmid = {41023259},
issn = {1573-0832},
mesh = {*CRISPR-Cas Systems ; *Gene Editing/methods ; *Scedosporium/genetics ; },
abstract = {Scedosporium species are opportunistic pathogens causing a large variety of human infections. To date, there is limited information on the pathogenic mechanisms of these fungi, partly because of the limited number of genetic tools available. Here, the CRISPR-Cas9 technology, which provided promising results for functional genomic studies in filamentous fungi, was optimized for Scedosporium species using in vitro assembled Cas9 ribonucleoprotein (RNP) complexes. In these fungi, functional genomic studies are particularly complex in a wild-type strain, because of the high frequency of non-homologous recombination. Prior disruption of the KU70 gene encoding one of the components of the non-homologous end joining system is required, which necessitates the use of a first selection marker. The cleavage of the target gene at each end using a dual RNA-guided Cas9 complex, followed by recombination with a repair template containing the hygromycin resistance gene, allowed disruption of the target gene in the ΔKU70 mutant. Four genes encoding dioxygenases, catalyzing the critical ring-opening step in aromatic hydrocarbons, were successfully disrupted, and the optimum efficiency was observed using 5 μg of the HygR repair cassette. Alternatively, in the wild-type strain, the exclusive use of two Cas9 RNP complexes was enough to achieve an efficient deletion method; one dioxygenase gene was successfully deleted in up to 20% of the obtained colonies. These last experimental conditions path the way to multiple gene deletions and complementation experiments, which cannot be reached using our first procedure since only two selection markers are available for Scedosporium species.},
}
@article {pmid41022734,
year = {2025},
author = {Fu, C and Saddawi-Konefka, R and Chinai, JM and Kim, SY and Kammula, AV and Perera, JJ and Jiang, A and Tiwari, P and Kistler, EN and Tang, S and Luna, SM and Colvin, KJ and Dubrot, J and Anderson, S and Fetterman, RA and Chuong, CL and Lane-Reticker, SK and Cheruiyot, CK and Muscato, AJ and Alipour, Z and Adkins, DR and Griffin, GK and Bernstein, BE and Egloff, AM and Yates, KB and Chernock, RD and Gutkind, JS and Uppaluri, R and Manguso, RT},
title = {In vivo CRISPR screening in head and neck cancer reveals Uchl5 as an immunotherapy target.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {8572},
pmid = {41022734},
issn = {2041-1723},
support = {U01DE029188//U.S. Department of Health &amp; Human Services | NIH | National Institute of Dental and Craniofacial Research (NIDCR)/ ; },
mesh = {Animals ; Humans ; *Ubiquitin Thiolesterase/genetics/metabolism/immunology ; Mice ; *Head and Neck Neoplasms/genetics/immunology/therapy/pathology ; *Immunotherapy/methods ; *Squamous Cell Carcinoma of Head and Neck/genetics/immunology/therapy/pathology ; Epithelial-Mesenchymal Transition/genetics/immunology ; Cell Line, Tumor ; CD8-Positive T-Lymphocytes/immunology ; CRISPR-Cas Systems ; Extracellular Matrix/metabolism ; Immune Checkpoint Inhibitors/pharmacology/therapeutic use ; Tumor Escape/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats ; Gene Expression Regulation, Neoplastic ; Female ; },
abstract = {Recurrent/metastatic head and neck squamous cell carcinoma (HNSCC) is an aggressive malignancy with a significant unmet need for enhancing immunotherapy response given current modest efficacy. Here, we perform an in vivo CRISPR screen in an HNSCC mouse model to identify immune evasion genes. We identify several regulators of immune checkpoint blockade (ICB) response, including the ubiquitin C-terminal hydrolase 5 (UCHL5). Loss of Uchl5 in tumors increases CD8[+] T cell infiltration and improved ICB responses. Uchl5 deficiency attenuates extracellular matrix (ECM) production and epithelial-mesenchymal-transition (EMT) transcriptional programs, which contribute to stromal desmoplasia, a histologic finding we describe as associated with reduced anti-PD1 response in human HNSCCs. COL17A1, a collagen highly and specifically expressed in HNSCC, mediates in part Uchl5-mediated immune evasion. Our findings suggest an unappreciated role for UCHL5 in promoting EMT in HNSCC and highlight ECM modulation as a strategy to improve immunotherapy responses.},
}
@article {pmid41022133,
year = {2025},
author = {Jin, X and Ouyang, C and Sun, T and Li, C and Gu, J and An, B and Wang, Z},
title = {A CRISPR/Cas9 mutant resource for OsSm RNA-binding genes in rice.},
journal = {The Plant journal : for cell and molecular biology},
volume = {124},
number = {1},
pages = {e70475},
doi = {10.1111/tpj.70475},
pmid = {41022133},
issn = {1365-313X},
support = {2021CX02N173//Guangdong Pearl River Talents Program/ ; 2025B03J0025//Guangzhou Science and Technology Plan Project/ ; 2024KJ31//the Provincial Rural Revitalization Strategy Special Project of Guangdong in 2024/ ; ZDYF2024XDNY179//Hainan Province Science and Technology Special Fund/ ; 32100252//National Natural Science Foundation of China/ ; 32100294//National Natural Science Foundation of China/ ; 32171292//National Natural Science Foundation of China/ ; 211207157080997//Zhanjiang plan for navigation/ ; QT2024-017//Young Talent Support Project of Guangzhou Association for Science and Technology/ ; 2023A1515010428//Guangdong Basic and Applied Basic Research Foundation/ ; },
mesh = {*Oryza/genetics/growth &amp; development/metabolism ; *CRISPR-Cas Systems/genetics ; *Plant Proteins/genetics/metabolism ; Mutation ; Gene Expression Regulation, Plant ; *RNA-Binding Proteins/genetics/metabolism ; Genes, Plant ; RNA Splicing ; },
abstract = {Pre-mRNA, produced by eukaryotic DNA transcription, undergoes splicing by the spliceosome, which removes introns and joins exons to form mRNA. The spliceosome is a large and highly dynamic molecular machine. Its core components include five small nuclear ribonucleoproteins (snRNPs) and the various spliceosome-related proteins. The conserved Smith (Sm) complex and the Sm-like proteins (LSm) serve as primary components of the snRNPs. Sm proteins are involved in processes such as pre-mRNA splicing and mRNA degradation, which can regulate gene expression, thereby influencing plant growth, development, and stress responses. While 25 Sm proteins have been identified in rice, their specific roles in regulating rice growth and development remain unclear. In this study, we employed the CRISPR/Cas9 system to edit 15 OsSm genes, and 13 mutants were obtained, with mutation rates ranging from 20.83 to 83.87%. In comparison to the wild type (WT), the mutants exhibited dwarfism, reduced tiller numbers, lower seed-setting rates or sterility, and increased susceptibility to diseases. One Sm mutant, ossmf-2, exhibited dwarfism, delayed flowering, and small grains. Through transcriptome analysis, three target genes, OsMRG702, OsRGG2, and OsLA1, were identified. Mutations of the OsSmF protein may lead to the abnormal splicing of these genes and finally lead to the inhibition of growth and development. Our study first edited the OsSm genes and generated a mutant library in rice. Most of the mutants exhibited abnormal growth and development, underscoring the essential roles of OsSm proteins in rice physiology. Furthermore, this work addresses a critical gap in the functional characterization of Sm proteins in rice. The resulting mutant collection offers valuable germplasm resources and lays a theoretical foundation for elucidating the molecular regulatory networks involving spliceosomal components and their target genes in the control of crop growth, development, and reproduction.},
}
@article {pmid41021290,
year = {2025},
author = {Johnson, KA and Cooper, C and Philippe, C and Catchpole, RJ and Mitchell, S and Terns, MP},
title = {A Phage Variable Region Encodes Anti-CRISPR Proteins Inhibiting All Streptococcus thermophilus CRISPR Immune Systems.},
journal = {The CRISPR journal},
volume = {},
number = {},
pages = {},
doi = {10.1177/25731599251369720},
pmid = {41021290},
issn = {2573-1602},
abstract = {Bacteria and archaea utilize CRISPR-Cas systems to defend against invading mobile genetic elements (MGEs) such as phages and plasmids. In turn, MGEs have evolved anti-CRISPR (Acr) proteins to counteract these defenses. While several type II-A Acrs have been identified in Streptococcus thermophilus (Sth) phages, a more comprehensive understanding of Acr diversity in Sth phages has yet to be explored. Guided by the genomic context of known Acrs, we systematically screened uncharacterized phage proteins and identified several novel Acrs that inhibit type I-E, type II-A or type III-A Sth CRISPR-Cas systems. These acr genes are clustered within a variable phage genomic region, indicating a hotspot for anti-defense activity. We also identified neighboring proteins with predicted enzymatic or structural domains that may modulate phage-host interactions through Acr-independent mechanisms. Together, our findings expand the known repertoire of Sth Acrs and highlight the phage variable region as a key reservoir of immune-modulating factors.},
}
@article {pmid41021273,
year = {2025},
author = {Sgodda, M and Gebel, E and Dignas, L and Alfken, S and Eggenschwiler, R and Stalke, A and Dröge, C and Pfister, ED and Baumann, U and Luedde, T and Esposito, I and Keitel, V and Cantz, T},
title = {iPSC-based hepatic organoids reveal a heterozygous MYO5B variant as driver of intrahepatic cholestasis.},
journal = {Hepatology communications},
volume = {9},
number = {10},
pages = {},
doi = {10.1097/HC9.0000000000000812},
pmid = {41021273},
issn = {2471-254X},
mesh = {Humans ; *Cholestasis, Intrahepatic/genetics/metabolism ; *Organoids/metabolism ; ATP Binding Cassette Transporter, Subfamily B, Member 11/genetics/metabolism ; *Induced Pluripotent Stem Cells ; Heterozygote ; *Myosin Type V/genetics ; *Myosin Heavy Chains/genetics ; ATP Binding Cassette Transporter, Subfamily B/genetics/metabolism ; Liver/metabolism ; Multidrug Resistance-Associated Protein 2 ; },
abstract = {BACKGROUND: Hereditary intrahepatic cholestasis is caused by variants of various genes involved in enterohepatic bile circulation, metabolization, and conjugation. Originally classified into 3 groups, the number of contributing genes is still increasing, underlining the need for a deeper understanding of the molecular interaction during intrahepatic cholestasis.<br><br>METHODS: In the present study, we investigate the interplay of heterozygous variants in 3 cholestasis-associated genes (ABCB11, ABCB4, and MYO5B) by exploiting iPSC-based hepatic organoids from a patient suffering from recurrent intrahepatic cholestasis.<br><br>RESULTS: Functional characterization of MRP2-mediated cholyl-lysyl-fluorescein (CLF) and BSEP-mediated Tauro-nor-THCA-24-DBD transport demonstrated a marked reduction of transport in MYO5B-deficient organoids, in comparison to unaffected control organoids. Moreover, iPSC-based organoids derived from the patient carrying 3 heterozygous variants in ABCB11, ABCB4, and MYO5B also exhibited absence of BSEP-mediated Tauro-nor-THCA-24-DBD transport, but functional MRP2-mediated CLF-transport. Interestingly, CRISPR/Cas9-mediated correction of the mutated ABCB11 allele could not restore the impaired BSEP function, suggesting the heterozygous MYO5B variant as the main driver of the transport deficiency. In fact, CRISPR/Cas-mediated correction of the MYO5B variant finally resulted in a restoration of the BSEP-mediated Tauro-nor-THCA-24-DBD transport.<br><br>CONCLUSIONS: iPSC-based organoids serve as an authentic model for functional assessment of the hepatobiliary transport with fluorescent substrates. This allows the characterization of variants of uncertain significance and other variants in cholestasis-associated genes and revealed that a heterozygous MYO5B variant increases the susceptibility to defective hepatobiliary BSEP-mediated transport.},
}
@article {pmid41021067,
year = {2025},
author = {Zou, S and Chen, W and Cao, Y and Liu, X and Wang, J and Wang, Y and Zhou, S},
title = {Lethal endotoxin (ccdB) based counterselection improved the efficiency of sequential gene editing in Escherichia coli.},
journal = {Biotechnology letters},
volume = {47},
number = {5},
pages = {118},
pmid = {41021067},
issn = {1573-6776},
support = {Guike AA24206048//Science and Technology Major Project of Guangxi/ ; AA24206050//Science and Technology Major Project of Guangxi/ ; 4301/00960//Hubei University of Technology High-Level Talent Research Startup Fund Program/ ; },
mesh = {*Gene Editing/methods ; *Escherichia coli/genetics ; Plasmids/genetics ; CRISPR-Cas Systems ; *Endotoxins/genetics ; *Escherichia coli Proteins/genetics ; },
abstract = {The CRISPR/Cas9-based technology has been used for sequential gene editing in E. coli. The plasmids carrying the sgRNA and/or Cas9 genes need to be cured after each round of editing. Curing of these plasmids, particularly the sgRNA plasmid, limits the efficiency of sequential gene editing. In this study, a lethal endotoxin (ccdB) based counterselection was established for improving the overall efficiency of sequential gene editing in E. coli. This approach was validated for sequential editing (deletion) of cstA and ppsA genes in HBUT-P2 strain (W derivative). The experimental results showed that the transformation efficiency of sgRNA plasmid (pTargetF-tcr-PL-ccdB-N20) reached 10[8]-10[9] cfu/μg-DNA, resulting in a 100% and 93.75% recombination rate for cstA and ppsA gene, respectively. Upon completion of cstA gene editing, the sgRNA plasmid (pTargetF-tcr-PL-ccdB-N20 (cstA)) was effectively cured through ccdB based counterselection at 42 °C, with a 43.75% efficiency. At the end of sequential editing of ppsA gene, both Cas9 (25A) and sgRNA (pTargetF-tcr-PL-ccdB-N20 (ppsA)) plasmids were cured simultaneously through the sacB and ccdB based counterselections by incubating the cells on LB-sucrose (5%) plate at 42 °C, achieving a curing rate of 100% for Cas9 plasmid (25A), 37.5% for sgRNA plasmid (pTargetF-tcr-PL-ccdB-N20 (ppsA)), and 37.5% for both Cas9 and sgRNA plasmids. Moreover, this approach was further validated through efficient site-specific insertion of the csc operon into the slmA gene in DH5α (K12 derivative) and S322 (C derivative) strains. These results demonstrated that the endotoxin (ccdB) based counterselection improved the transformation efficiency of sgRNA plasmid, the recombination rate of the editing target gene, the curing rate of sgRNA plasmid, and the overall efficiency of sequential gene editing.},
}
@article {pmid41020501,
year = {2025},
author = {Yu, L and Jin, Y and Chen, J and Zhu, Z and Su, S and Wilkerson, EM and Gongora, J and Cloer, EW and Major, MB and Liu, P},
title = {Stable Cas9 expression regulates cell growth by facilitating mTORC2 activation.},
journal = {Nucleic acids research},
volume = {53},
number = {18},
pages = {},
pmid = {41020501},
issn = {1362-4962},
support = {//Mary Kay Ash Foundation/ ; //University of North Carolina/ ; //Chapel Hill University Cancer Research/ ; },
mesh = {Humans ; Ribosomal Proteins/metabolism/genetics ; *Mechanistic Target of Rapamycin Complex 2/metabolism/genetics ; *CRISPR-Associated Protein 9/genetics/metabolism ; Cell Line, Tumor ; *Cell Proliferation/genetics ; Signal Transduction ; *CRISPR-Cas Systems ; Adaptor Proteins, Signal Transducing/metabolism/genetics ; Repressor Proteins/metabolism/genetics ; Gene Editing ; Phosphatidylinositol 3-Kinases/metabolism ; },
abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR), widely used for gene editing, relies on bacterial endonucleases like Cas9 to study gene functions and develop therapies. However, its potential effects on mammalian cellular behavior remain unclear. Here, we systematically profiled effects of stable Cas9 expression on growth of 32 cell lines spanning 9 cancer types and non-cancerous cells, finding growth alterations in a subset. To investigate mechanisms, we established the SpCas9 interactome in DU145 and MDA-MB-231 cells, both showing Cas9-enhanced growth, and identified ribosomal proteins as the top shared interactors. RNA-seq analysis revealed that Cas9 expression in DU145 cells activated PI3K signaling. Mechanistic studies showed that ribosomal proteins, including RPL26 and RPL23a, bind to Sin1, a core mTORC2 component, leading to mTORC2 activation. Notably, SpCas9 interacts with both RPL26/RPL23a and Sin1, acting as a scaffold to stabilize their association and enhance mTORC2 activation, even in the absence of growth factors. Our study systematically characterizes Cas9's effects on cell growth regulation and uncovers a novel Cas9-ribosome-mTORC2 signaling axis that promotes cell growth. These findings underscore the need to consider unintended cellular effects in CRISPR applications and highlight the importance of engineering safer Cas9 variants for biomedical research and clinical therapies.},
}
@article {pmid41020266,
year = {2025},
author = {Back, F and Sandoval, A and Vu, LM and Hong, VM and Bhaskara, A and Rodriguez, SR and O'Brien, JT and Kolber, BJ and Kroener, S and Ploski, JE},
title = {Adeno-associated viral vector resource for the RNA-targeting Cas13d: A comparison of high-fidelity variants, DjCas13d and hfCas13d.},
journal = {Molecular therapy. Methods &amp; clinical development},
volume = {33},
number = {4},
pages = {101565},
pmid = {41020266},
issn = {2329-0501},
abstract = {RNA-targeting CRISPR-Cas systems have emerged as alternatives to RNA-interference technology to knock down specific RNA transcripts. In particular, Cas13d derived from Ruminococcus flavefaciens (CasRx, RfxCas13d) has generated interest due to its superior knockdown efficiencies; however, accumulating evidence indicates that CasRx is prone to inducing transcriptome alterations due to its tendency to cleave bystander RNAs. High-fidelity Cas13d (hfCas13d) derived from CasRx and DjCas13d, an ortholog of Cas13d derived from Ruminococcus sp. UBA7013 (gut metagenome), are two recently identified variants that are superior to CasRx, as they both show a reduced tendency to cleave bystander RNAs. In this study, we created a resource of adeno-associated viral (AAV) vectors designed to deliver Cas13d, including hfCas13d and DjCas13d. We directly compared hfCas13d and DjCas13d for their on- and off-target potential in 293FT and neuro 2A cells. Specifically, we examined their ability to knockdown several endogenous and ectopically expressed transcripts using several different guide RNAs (gRNAs), and we examined knockdown specificity using a combination of reporter assays, RNA integrity analysis, and RNA sequencing (RNA-seq). We report that while both of these enzymes exhibit generally similar levels of knockdown potential, with DjCas13d sometimes outperforming hfCas13d, hfCas13d consistently caused significantly fewer transcriptome alterations when targeting highly expressed genes compared to DjCas13d.},
}
@article {pmid40974028,
year = {2025},
author = {Teng, J and Chen, Y and Zhang, W and Xu, H and Ke, L and Xu, H and Wang, J},
title = {An RCA-CRISPR-Enhanced SERS Platform for Ultrasensitive and Single-Nucleotide-Resolved Detection of Exosomal miRNA-21 in Early Lung Cancer.},
journal = {Analytical chemistry},
volume = {97},
number = {38},
pages = {21098-21105},
doi = {10.1021/acs.analchem.5c04448},
pmid = {40974028},
issn = {1520-6882},
mesh = {*Lung Neoplasms/diagnosis/genetics ; Humans ; *MicroRNAs/genetics/analysis/blood ; *Exosomes/genetics/chemistry ; *Spectrum Analysis, Raman/methods ; *Nucleic Acid Amplification Techniques/methods ; Limit of Detection ; CRISPR-Cas Systems ; Biomarkers, Tumor/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {Exosomal miRNA-21 has emerged as a promising biomarker for early-stage lung cancer due to its close association with tumor progression and its stability in circulation. However, its low abundance, short sequence length, and high-sequence similarity present significant detection challenges. To address this, we developed an ultrasensitive surface-enhanced Raman scattering (SERS) platform that integrates rolling circle amplification (RCA) with clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein 12a (Cas12a) for the detection of exosomal miRNA-21. RCA provides target-dependent amplification with stringent sequence discrimination via padlock probe ligation, while the CRISPR/Cas12a system facilitates robust signal generation through trans-cleavage activity. The final SERS readout enables molecular-level sensitivity by detecting nanotag-labeled cleavage events. The assay achieved a limit of detection as low as 0.62 aM and effectively discriminated miRNA-21 from multiple single- and multinucleotide variants. As a proof of concept, we applied this method to the detection of exosomal miRNA-21 extracted from the serum of 20 early-stage lung cancer patients and 20 healthy controls, achieving 100% sensitivity and 100% specificity (AUC = 1.0) in this preliminary cohort. These findings demonstrate the strong potential of the RCA-CRISPR-SERS platform for noninvasive early-stage lung cancer diagnosis based on exosomal miRNA-21 detection.},
}
@article {pmid40955638,
year = {2025},
author = {Sun, F and Mao, R and Li, J and Wang, X and Hou, P and Zhou, H},
title = {Organic Photoelectrochemical Transistor/Visual Sensing Platform Based on CS/MCS Schottky Heterojunction and CRISPR/Cas12a-Driven Triple-Modal Synergistic Signal Amplification.},
journal = {Analytical chemistry},
volume = {97},
number = {38},
pages = {21079-21088},
doi = {10.1021/acs.analchem.5c04321},
pmid = {40955638},
issn = {1520-6882},
mesh = {*MicroRNAs/analysis/genetics ; *Transistors, Electronic ; *Electrochemical Techniques/methods ; Humans ; *CRISPR-Cas Systems ; *Biosensing Techniques/methods ; DNA, Catalytic/chemistry/metabolism ; Photochemical Processes ; G-Quadruplexes ; Colorimetry ; Limit of Detection ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {Developing novel signal amplification and transduction technologies is the key to overcoming the bottlenecks of high-sensitivity and on-site detection in nucleic acid analysis. In this study, a dual-mode sensing platform based on organic electrochemical transistors (OPECT) and colorimetry was established to achieve ultrasensitive detection of miRNA-21. 1D/3D Co9S8/Mn0.3Cd0.7S Schottky heterojunction was synthesized as the photoactive material, which significantly enhanced the photoelectric conversion efficiency. The sensing and detection system cleverly integrated a quadruple signal amplification mechanism. The target triggered the catalytic hairpin assembly (CHA) reaction, generating H1 and H2 long chains. These chains activated the CRISPR/Cas12a system, which carried out nondiscriminatory cleavage to block the tandem strand displacement reaction (TSDR). This triggered the hybrid chain reaction (HCR) and formation of G-quadruplex/hemin DNAzyme (GQH DNAzyme), realizing cascade signal amplification. Under the catalysis of GQH DNAzyme, the detection had dual-signal outputs. It catalyzed the oxidation of 4-CN to form a deposition layer, inhibiting electron transport and achieving cascade signal amplification for OPECT. It catalyzed the H2O2-mediated TMB colorimetric reaction to complete the visual colorimetric analysis. Through triple-modal synergistic signal amplification of biological, chemical, and electronic modalities, this biosensing platform reduced the detection limits to as low as 36.5 aM and 3.8 fM, respectively. It provided a new solution for the accurate analysis of miRNA markers in the early diagnosis of cancer.},
}
@article {pmid40916664,
year = {2025},
author = {Barker, EN and Ashiri, M and Saville, JT and Hemming, R and Furletti, N and Dhume, SH and Yu, S and Anjos, E and Wu, X and Fresnoza, A and Merz, DC and Jackson, M and Del Bigio, MR and Siddiqui, TJ and Fuller, M and Mark, BL and Triggs-Raine, B},
title = {Generation of mice with combined Hexa Gly269Ser KI or KO and Neu3 KO alleles to create new models of GM2 gangliosidoses.},
journal = {Biology open},
volume = {14},
number = {9},
pages = {},
doi = {10.1242/bio.062045},
pmid = {40916664},
issn = {2046-6390},
support = {/CAPMC/CIHR/Canada ; //Research Manitoba/ ; /AS/Autism Speaks/United States ; //Natural Sciences and Engineering Council of Canada/ ; },
mesh = {Animals ; *Disease Models, Animal ; Mice ; *Alleles ; Mice, Knockout ; *Gangliosidoses, GM2/genetics/metabolism/pathology ; Phenotype ; CRISPR-Cas Systems ; *beta-Hexosaminidase alpha Chain/genetics ; Gene Knock-In Techniques ; Mutation ; Humans ; N-Acetylgalactosaminyltransferases ; },
abstract = {The GM2 gangliosidoses are lysosomal storage disorders exhibiting a spectrum of neurological phenotypes ranging from childhood death to debilitating adult-onset neurological impairment. To date, no mouse model harbouring a specific human mutation causing GM2 gangliosidosis has been created. We used CRISPR/Cas9 to generate knockin (KI) mice with the common adult-onset Hexa Gly269Ser variant as well as knockout (KO) mice with Hexa mutations expected to cause complete HexA deficiency. We also created Neu3 KO alleles that combined with Hexa KO or KI alleles were expected to create acute and chronic models of GM2 gangliosidosis, respectively. However, both models accumulated GM2 ganglioside throughout the brain when compared to controls (CON), and exhibited progressive loss of reflexes, gait abnormalities, and premature death by 24 weeks of age. Although survival and behavioural phenotypes did not differ between KO and KI models, the KI model had substantial Hexa mRNA and evidence of GM2 turnover. This KI model will be useful for developing gene editing to correct the variant causing the Gly269Ser substitution and its novel biochemical phenotype suggests it may be suitable for testing therapies that treat partial β-hexosaminidase A deficiency.},
}
@article {pmid39433694,
year = {2025},
author = {Farrokhi, S and Eslahi, A and Alizadeh, F and Kerachian, MA and Mojarrad, M},
title = {Assessment the Efficacy of the CRISPR System for Inducing Mutations in the AIMP2 Gene to Create a Cell Line Model of HLD17 Disease.},
journal = {Molecular biotechnology},
volume = {67},
number = {10},
pages = {3922-3929},
pmid = {39433694},
issn = {1559-0305},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; HEK293 Cells ; *Gene Editing/methods ; *Mutation ; Plasmids/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; },
abstract = {Hypomyelinating leukodystrophy-17 is a neurodevelopmental disorder caused by autosomal recessive mutations in the AIMP2 gene, resulting in a lack of myelin deposition during brain development, leading to variable neurological symptoms. Research on brain function in these disorders is challenging due to the lack of access to brain tissue. To overcome this problem, researchers have utilized different cell and animal models. The CRISPR-Cas9 system is considered the most optimal and effective method for genetic modification and developing cell models. We studied the efficacy of the CRISPR-Cas9 technology in inducing mutations in the AIMP2 gene in HEK293 cell lines. The study involved transfecting HEK293 cells with recombinant PX458 plasmids targeting spCas-9 and AIMP2 sgRNA. The cells were evaluated using fluorescent microscopy and enriched using serial dilution. The CRISPR/Cas9 plasmids were validated through PCR and Sanger sequencing. After serial dilution, AS-PCR, Sanger sequencing, and TIDE program analysis showed the construct successfully induces an indel mutation in HEK cells. Our findings demonstrated the great efficacy of the CRISPR system and produced a construct for inducing mutations in the AIMP2 gene, which can be utilized to edit the AIMP2 gene in nerve cells and create a cellular model of the HLD17 disease.},
}
@article {pmid39377911,
year = {2025},
author = {Bhoomika, S and Salunkhe, SR and Sakthi, AR and Saraswathi, T and Manonmani, S and Raveendran, M and Sudha, M},
title = {CRISPR-Cas9: Unraveling Genetic Secrets to Enhance Floral and Fruit Traits in Tomato.},
journal = {Molecular biotechnology},
volume = {67},
number = {10},
pages = {3786-3799},
pmid = {39377911},
issn = {1559-0305},
mesh = {*Solanum lycopersicum/genetics/growth &amp; development ; *Fruit/genetics/growth &amp; development ; *CRISPR-Cas Systems ; *Flowers/genetics/growth &amp; development ; Gene Editing/methods ; Plants, Genetically Modified/genetics ; Gene Expression Regulation, Plant ; Plant Proteins/genetics/metabolism ; Plant Breeding/methods ; },
abstract = {Tomato, a globally consumed vegetable, possesses vast genetic diversity, making it suitable for genetic manipulation using various genetic improvement techniques. Tomatoes are grown extensively for their market value and health benefits, primarily contributed by enhanced yield and nutritional value respectively, influenced by floral and fruit traits. Floral morphology is maintained by genes involved in meristem size control, regulation of inflorescence transition, and pollen development. SP (SELF-PRUNING) and SP5G (SELF-PRUNING 5G) determine growth habit and flowering time. RIN (RIPENING INHIBITOR) and PG (POLYGALACTURONASE) are responsible for the shelf life of fruits. In addition to this, nutrition-enriched tomatoes have been developed in recent times. In this review, we comprehensively discuss the major genes influencing floral morphology, flowering time, fruit size, fruit shape, shelf life, and nutritional value, ultimately resulting in enhanced yield. Additionally, we address the advances in CRISPR/Cas9 applied for the genetic improvement of tomatoes along with prospects of areas in which research development in terms of tomato genetic improvement has to be advanced.},
}
@article {pmid41020188,
year = {2025},
author = {Shalaby, KE and Hmila, I and Uddin, SMN and Zawia, NH and El-Agnaf, OMA and Aouida, M},
title = {Enhanced Cellular Uptake of Compact Cas Proteins: A Comparative Study of Cas12f and Cas9 in Human Cells.},
journal = {Engineering in life sciences},
volume = {25},
number = {9},
pages = {e70042},
pmid = {41020188},
issn = {1618-0240},
abstract = {The clinical translation of CRISPR genome-editing therapies is often hindered by inefficient delivery of the CRISPR-Cas RNA-protein complex into target cells. The most widely used CRISPR-Cas9 system poses a significant challenge for efficient delivery into cells due to its large size (∼1.4 kDa). Recently reported compact Cas proteins, such as Cas12f (552 Da), Cas12k (639 Da), and Cas12m (596 Da) represent attractive alternatives as cargoes for delivery. In this brief research report, we employ efficient delivery vectors to evaluate the efficiency of cellular uptake of a compact Cas protein (Cas12f) compared to the widely used larger Cas9 in human cells. Our findings demonstrate that compact Cas proteins may facilitate more efficient cellular penetration and delivery, making them a promising alternative for the development of CRISPR-based therapies. Practical Application: Our study demonstrates that compact Cas proteins significantly enhance cellular uptake compared to larger Cas proteins. This improved uptake efficiency suggests that compact Cas proteins could be more effective for clinical application, where size constraints and delivery efficiency are critical challenges. Combined with the optimization and refinement of the editing efficiencies of compact Cas systems, our study provokes further exploration of compact Cas proteins in various therapeutic contexts to advance the development of more efficient CRISPR-based therapies.},
}
@article {pmid41019300,
year = {2025},
author = {Bilger, R and Drepper, F and Knapp, B and Berndt, T and Landerer, H and Putzer, H and Huesgen, PF and Hess, WR},
title = {Involvement of RNase J in CRISPR RNA maturation in the cyanobacterium Synechocystis sp. PCC 6803.},
journal = {microLife},
volume = {6},
number = {},
pages = {uqaf022},
pmid = {41019300},
issn = {2633-6693},
abstract = {Many bacteria and archaea use CRISPR-Cas systems, which provide RNA-based, adaptive, and inheritable immune defenses against invading viruses and other foreign genetic elements. The proper processing of CRISPR guide RNAs (crRNAs) is a crucial step in the maturation of the defense complexes and is frequently performed by specialized ribonucleases encoded by cas genes. However, some systems employ enzymes associated with degradosome or housekeeping functions, such as RNase III or the endoribonuclease RNase E. Here, the endo- and 5´-exoribonuclease RNase J was identified as an additional enzyme involved in crRNA maturation, acting jointly with RNase E in the crRNA maturation of a type III-Bv CRISPR-Cas system, and possibly together with a further RNase in the cyanobacterium Synechocystis sp. PCC 6803. Co-IP experiments revealed a small set of proteins that were co-enriched with RNase J, among them the exoribonuclease polyribonucleotide nucleotidyltransferase (PNPase). Despite a measured, strong 3' exonucleolytic activity of the recombinant enzyme, PNPase was not confirmed to contribute to crRNA maturation. However, the co-IP results indicate that PNPase in Synechocystis is an enzyme that can recruit either RNase E or RNase J, together with additional proteins.},
}
@article {pmid41018953,
year = {2025},
author = {Ajmal, H and Nandi, S and Kebabci, N and Ryan, CJ},
title = {Benchmarking genetic interaction scoring methods for identifying synthetic lethality from combinatorial CRISPR screens.},
journal = {NAR genomics and bioinformatics},
volume = {7},
number = {3},
pages = {lqaf129},
pmid = {41018953},
issn = {2631-9268},
mesh = {*Synthetic Lethal Mutations ; Benchmarking ; Humans ; Algorithms ; *CRISPR-Cas Systems ; *Epistasis, Genetic ; *Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {Synthetic lethality (SL) is an extreme form of negative genetic interaction, where simultaneous disruption of two non-essential genes causes cell death. SL can be exploited to develop cancer therapies that target tumour cells with specific mutations, potentially limiting toxicity. Pooled combinatorial CRISPR screens, where two genes are simultaneously perturbed and the resulting impacts on fitness estimated, are now widely used for the identification of SL targets in cancer. Various scoring methods have been developed to infer SL genetic interactions from these screens, but there has been no systematic comparison of these approaches. Here, we performed a comprehensive analysis of five scoring methods for SL detection using five combinatorial CRISPR datasets. We assessed the performance of each algorithm on each screen dataset using two different benchmarks of paralog SL. We find that no single method performs best across all screens but identify two methods that perform well across most datasets. Of these two scores, Gemini-Sensitive has an available R package that can be applied to most screen designs, making it a reasonable first choice.},
}
@article {pmid41017815,
year = {2025},
author = {Alipanahi, R and Safari, L and Khanteymoori, A},
title = {Advancing CRISPR with deep learning: A comprehensive review of models and databases.},
journal = {Molecular therapy. Nucleic acids},
volume = {36},
number = {4},
pages = {102691},
pmid = {41017815},
issn = {2162-2531},
abstract = {CRISPR is considered a powerful tool for targeted genome editing. However, off-target effects remain a significant challenge in the CRISPR field, hindering its broader clinical application. To enhance the development of gene-editing therapies, it is essential to predict the efficiency of CRISPR-based genome editing experiments, before trying them on clinical cases. Machine learning (ML) and deep learning (DL) tools are projected to become the leading methods for predicting CRISPR on-target and off-target activity. Current prediction accuracy is limited by the amount of available training data. As more sequence features are identified and incorporated in DL tools, predictions of them are expected to better align with experimental results. Hence, the increasing focus on ML/DL approaches to predict off-target sites necessitates large and easily searchable databases. In this review, we will take a closer look at available CRISPR databases.},
}
@article {pmid41017813,
year = {2025},
author = {Sharma, S and Pokharel, YR},
title = {Measles and rubella: From global health challenges to advancements in molecular diagnostics in the elimination era.},
journal = {Molecular therapy. Nucleic acids},
volume = {36},
number = {4},
pages = {102698},
pmid = {41017813},
issn = {2162-2531},
abstract = {Measles and rubella are highly contagious viral infections with significant public health implications, particularly in low- and middle-income countries. Despite the availability of effective vaccines, these diseases continue to cause periodic outbreaks, contributing to substantial global morbidity, mortality, and economic burden. Immunization programs have drastically abridged disease incidence; however, gaps in vaccination coverage and surveillance systems deter complete elimination. The economic impact of outbreaks includes direct healthcare costs and indirect societal losses, emphasizing the need for robust disease control strategies. Accurate and timely diagnosis is pivotal to measles and rubella elimination efforts. Current diagnostic approaches range from conventional RT-PCR (including multiplex and real-time formats), ELISA, and plaque reduction neutralization test (PRNT), to emerging methods such as isothermal amplification loop-mediated isothermal amplification, recombinase polymerase amplification), CRISPR-Cas systems, next-generation sequencing (NGS), microfluidics, and lateral flow assays. Despite their sensitivity, many of these methods require complex infrastructure and skilled personnel, limiting their utility in field settings. To bridge diagnostic gaps, there is an urgent need for rapid, affordable, and field-deployable nucleic acid-based diagnostics that are simple to use with minimal training. Innovations like CRISPR-Cas and microfluidic platforms hold promise for decentralized testing and real-time surveillance, potentially transforming global measles and rubella elimination programs for the future.},
}
@article {pmid41017535,
year = {2025},
author = {Han, B and Xie, X and Zhao, Y and Zhang, J and Yang, X and Jiang, Y and Zhang, W and Zhang, X},
title = {Recent development and applications of emerging biosensing technologies and on-site analytical devices for food adulteration detection: a critical review.},
journal = {Critical reviews in food science and nutrition},
volume = {},
number = {},
pages = {1-20},
doi = {10.1080/10408398.2025.2564216},
pmid = {41017535},
issn = {1549-7852},
abstract = {The increasing incidence of food adulteration poses a significant challenge to global health and food safety. Although current detection methods can effectively complete food adulteration detection, they usually require complex pre-preparation processes and professional technicians to some extent. Therefore, the development of rapid and on-site detection technologies for food adulteration is imperative. Recently, biosensing technologies and portable devices have been developed for efficient and precise food adulteration detection. In this review, the strengths and weaknesses of conventional food adulteration detection methods were compared. The recent development of emerging biosensing technologies (i.e., antibody-based biosensors, aptamer-based biosensors, molecular imprinted polymers (MIPs)-based biosensors, and clustered regularly interspaced short palindromic repeats-associated proteins (CRISPR/Cas) systems-based biosensors) and portable analytical devices (e.g., lateral flow assays (LFAs), microfluidic devices, handheld Raman, and nanopore-based devices) for food adulteration detection has been comprehensively summarized and discussed. Remarkably, the challenges and opportunities in this field have been proposed.},
}
@article {pmid41016988,
year = {2025},
author = {Zeng, J and Luo, J and Zeng, Y},
title = {Cancer gene therapy: historical perspectives, current applications, and future directions.},
journal = {Functional &amp; integrative genomics},
volume = {25},
number = {1},
pages = {200},
pmid = {41016988},
issn = {1438-7948},
support = {S202413705064//Innovation and Entrepreneurship Training Program for College Students/ ; 82104084//National Natural Science Foundation of China/ ; 2024qnGzn12 and 2024kjTzn08//CMC Excellent-talent Program of Chengdu Medical College/ ; },
mesh = {Humans ; *Genetic Therapy/methods/trends/history ; *Neoplasms/therapy/genetics ; Gene Editing ; CRISPR-Cas Systems ; Genetic Vectors/genetics ; Animals ; Oncolytic Virotherapy ; },
abstract = {Gene therapy has emerged as a transformative approach in cancer treatment, leveraging genetic modifications to target malignancies with enhanced precision. Early efforts faced challenges such as inefficient vector delivery (< 5% tumor transduction rates with first-generation adenoviruses), immune responses (neutralizing antibodies in ~ 30% of patients), and limited clinical efficacy (< 10% objective response rates in 1990s trials). However, advancements in viral and non-viral vectors (e.g., AAVs achieving > 50% transduction efficiency in solid tumors), alongside CRISPR-Cas9 (90% target gene knockout rates in preclinical models) and RNA interference technologies, have revolutionized the field. Presently, gene therapy strategies, including tumor suppressor gene restoration, oncogene silencing, and immune modulation, demonstrate promising clinical outcomes. Despite persistent hurdles like off-target effects and high costs, emerging innovations in personalized gene editing, oncolytic viruses, and combination therapies signal a paradigm shift in oncology. This review explores the evolution of gene therapy for cancer, highlighting key milestones, current applications, and future directions that could unlock its full therapeutic potential.},
}
@article {pmid41016806,
year = {2025},
author = {Watanabe, T},
title = {[Exploration and Functional Analysis of Epstein-Barr Virus Pathogenic Factors Using a Multidimensional Approach].},
journal = {Uirusu},
volume = {75},
number = {1},
pages = {73-86},
doi = {10.2222/jsv.75.73},
pmid = {41016806},
issn = {0042-6857},
mesh = {*Herpesvirus 4, Human/genetics/pathogenicity/physiology ; Humans ; Animals ; Mice ; *Epstein-Barr Virus Infections/virology ; Genome, Viral/genetics ; CRISPR-Cas Systems ; Disease Models, Animal ; Chromosomes, Artificial, Bacterial ; Gene Editing ; Virus Latency/genetics ; },
abstract = {Epstein-Barr virus (EBV), a member of the herpesvirus family, infects more than 90% of adults and establishes a lifelong latent infection. In addition to its involvement in a wide range of malignancies such as lymphomas, nasopharyngeal carcinoma, and gastric cancer, recent evidence has shown its potential association with autoimmune diseases, positioning EBV as an interdisciplinary research model linking virology, oncology, and immunology. Historically, EBV research has been hindered by technical limitations in viral culture systems and animal models. However, recent advances-including whole-genome cloning using bacterial artificial chromosomes (BACs), gene editing via CRISPR/Cas9, and the development of in vivo models such as humanized mice-have accelerated the elucidation of EBV' s unique life cycle and tumorigenic mechanisms. In this review, we discuss the evolution of techniques for generating recombinant EBVs and in vivo modeling, both essential for functional genetic analysis, and highlight our contributions to the advancement of these tools and their application in researching EBV-associated tumorigenesis.},
}
@article {pmid41016566,
year = {2025},
author = {Hussen, BM and Abdullah, SR and Hidayat, HJ and Glassy, MC and Safarzadeh, A and Komaki, A and Samsami, M and Taheri, M},
title = {CRISPR/Cas as a Tool to Overcome Drug Resistance in Cancer: From Challenge to Opportunity.},
journal = {Molecular and cellular probes},
volume = {},
number = {},
pages = {102052},
doi = {10.1016/j.mcp.2025.102052},
pmid = {41016566},
issn = {1096-1194},
abstract = {Drug resistance remains a significant challenge in cancer therapy, often resulting in treatment failure, tumor progression, and metastasis. The underlying resistance mechanisms-including genetic mutations, epigenetic alterations, and modifications in drug efflux pathways-are complex and not yet fully understood. This review explores the application of CRISPR-Cas gene editing technology in understanding and overcoming drug resistance in cancer. It focuses on how CRISPR can identify and target resistance-associated genes to restore drug sensitivity. CRISPR-based approaches enable precise genetic modifications that offer new insights into the molecular basis of drug resistance. The technology has shown promise in dissecting resistance mechanisms and developing targeted therapeutic strategies. Nevertheless, key limitations such as inefficient delivery systems, off-target effects, and limited specificity hinder clinical translation. Current efforts focus on improving guide RNA design, creating more effective delivery vectors, and integrating CRISPR with existing treatments. CRISPR-Cas technology holds significant potential to address drug resistance in cancer by enabling targeted genetic interventions. Continued advancements are required to enhance its safety, specificity, and delivery, paving the way for its integration into future clinical applications.},
}
@article {pmid41015553,
year = {2025},
author = {Zhang, Y and Shi, Q and Xie, H and Xie, B and Li, L and Wu, W and Xie, H and Xiao, Z and Xie, D and Lai, R},
title = {Construction and phenotypic analysis of p2rx2 knockout zebrafish lines.},
journal = {Zhong nan da xue xue bao. Yi xue ban = Journal of Central South University. Medical sciences},
volume = {50},
number = {6},
pages = {919-930},
pmid = {41015553},
issn = {1672-7347},
support = {2023JJ30753//the Natural Science Foundation of Hunan Province/ ; 2023SK4030//the Innovative Construction Foundation of Hunan Province/ ; kq2208326//the Natural Science Foundation of Changsha/ ; },
mesh = {Animals ; *Zebrafish/genetics ; *Receptors, Purinergic P2X2/genetics/deficiency ; CRISPR-Cas Systems/genetics ; *Gene Knockout Techniques ; Phenotype ; *Zebrafish Proteins/genetics ; Disease Models, Animal ; },
abstract = {OBJECTIVES: The purinergic receptor P2X2 (P2RX2) encodes an ATP-gated ion channel permeable to Na[+], K[+], and especially Ca[2+]. Loss-of-function mutations in P2RX2 are known to cause autosomal dominant nonsyndromic deafness 41 (DFNA41), which manifests as high-frequency hearing loss, accelerated presbycusis, and increased susceptibility to noise-induced damage. Zebrafish, owing to their small size, rapid development, high fecundity, transparent embryos, and high gene conservation with humans, provide an ideal model for studying human diseases and developmental mechanisms. This study aims to generate a p2rx2 knockout zebrafish model using CRISPR/Cas9 gene editing system to investigate the effect of p2rx2 deficiency on the auditory system, providing a basis for understanding P2RX2-related hearing loss and developing gene therapy strategies.<br><br>METHODS: Two CRISPR targets (sgRNA1 and sgRNA2) spaced 47 bp apart were designed within the zebrafish p2rx2 gene. Synthesized sgRNAs and Cas9 protein were microinjected into single-cell stage T&#xfc;bingen (TU)-strain zebrafish embryos. PCR and gel electrophoresis verified editing efficiency at 36 hours post-fertilization (hpf). Surviving embryos were raised to adulthood (F0), tail-clipped, genotyped, and screened for positive mosaics. F1 heterozygotes were generated by outcrossing, and F2 homozygous mutants were obtained by intercrossing. Polymerase chain reaction (PCR) combined with sequencing verified mutation type and heritability. At 5 days post-fertilization (dpf), YO-PRO-1 staining was used to examine hair cell morphology and count in lateral line neuromasts and the otolith region. Auditory evoked potential (AEP) thresholds at 600, 800, 1 000, and 2 000 Hz were measured in nine 4-month-old wild type and mutant zebrafish per group.<br><br>RESULTS: A stable p2rx2 knockout zebrafish line was successfully established. Sequencing revealed a 66 bp insertion at the first target site introducing a premature stop codon (TAA), leading to early termination of protein translation and loss of function. Embryos developed normally with no gross malformations. At 5 dpf, mutants exhibited significantly reduced hair cell density in the otolith region compared with wild type, although lateral line neuromasts were unaffected. AEP testing showed significantly elevated auditory thresholds at all 4 frequencies in homozygous mutants compared with wild type (all P<0.001), indicating reduced hearing sensitivity.<br><br>CONCLUSIONS: We successfully generated a p2rx2 loss-of-function zebrafish model using CRISPR/Cas9 technology. p2rx2 deficiency caused hair cell defects in the otolith region and increased auditory thresholds across frequencies, indicating its key role in maintaining zebrafish auditory hair cell function and hearing perception. The phenotype's restriction to the otolith region suggests tissue-specific roles of p2rx2 in sensory organs. This model provides a valuable tool for elucidating the molecular mechanisms of P2RX2-related hearing loss and for screening otoprotective drugs and developing gene therapies.},
}
@article {pmid41013258,
year = {2025},
author = {Shelenkov, A and Slavokhotova, A and Yunusova, M and Kulikov, V and Mikhaylova, Y and Akimkin, V},
title = {Genomic typing, antimicrobial resistance gene, virulence factor and plasmid replicon database for the important pathogenic bacteria Staphylococcus aureus.},
journal = {BMC genomic data},
volume = {26},
number = {1},
pages = {65},
pmid = {41013258},
issn = {2730-6844},
mesh = {*Virulence Factors/genetics ; *Plasmids/genetics ; *Staphylococcus aureus/genetics/classification/pathogenicity/drug effects ; *Replicon ; *Drug Resistance, Bacterial/genetics ; Genome, Bacterial ; *Databases, Genetic ; Humans ; Genomics ; Staphylococcal Infections/microbiology ; Anti-Bacterial Agents/pharmacology ; },
abstract = {BACKGROUND: Bacterial infections pose a global health threat across clinical and community settings. Over the past decade, the alarming expansion of antimicrobial resistance (AMR) has progressively narrowed therapeutic options, particularly for healthcare-associated infections. This critical situation has been formally recognized by the World Health Organization as a major public health concern. Epidemiological studies have demonstrated that the dissemination of AMR is frequently mediated by specific high-risk bacterial lineages, often designated as "global clones" or "clonal complexes." Consequently, surveillance of these epidemic clones and elucidation of their pathogenic mechanisms and AMR acquisition pathways have become essential research priorities. The advent of whole genome sequencing has revolutionized these investigations, enabling comprehensive epidemiological tracking and detailed analysis of mobile genetic elements responsible for resistance gene transfer. However, despite the exponential increase in available bacterial genome sequences, significant challenges persist. Current genomic datasets often suffer from uneven representation of clinically relevant strains and inconsistent availability of accompanying metadata. These limitations create substantial obstacles for large-scale comparative studies and hinder effective surveillance efforts.<br><br>DESCRIPTION: This database represents a comprehensive genomic analysis of 98,950&#xa0;Staphylococcus aureus&#xa0;isolates, a high-priority bacterial pathogen of global clinical significance. We provide detailed isolate characterization through several established typing schemes including multilocus sequence typing (MLST), clonal complex (CC) assignments,&#xa0;spa&#xa0;typing results, and core genome MLST (cgMLST) profiles. The dataset also documents the presence of CRISPR-Cas systems in these isolates. Beyond fundamental typing data, our resource incorporates the distribution of antimicrobial resistance determinants, virulence factors, and plasmid replicons. These systematically curated genomic features offer researchers valuable insights into isolate epidemiology, resistance mechanisms, and horizontal gene transfer patterns in this highly concerning pathogen.<br><br>CONCLUSION: This database is freely available under CC BY-NC-SA at https://doi.org/10.5281/zenodo.14833440 . The data provided enables researchers to identify optimal reference isolates for various genomic studies, supporting critical investigations into&#xa0;S. aureus&#xa0;epidemiology and antimicrobial resistance evolution. This resource will ultimately inform the development of more effective prevention and control measures against this high-priority pathogen.},
}
@article {pmid41013219,
year = {2025},
author = {Zhao, C and Xia, J and Liang, B and Lin, S and Song, Y and Hong, D and Gu, J},
title = {Large-scale screening of genes responsible for silique length and seed size in Brassica Napus via pooled CRISPR library.},
journal = {BMC genomics},
volume = {26},
number = {1},
pages = {829},
pmid = {41013219},
issn = {1471-2164},
support = {2022ZD04008//Biological Breeding-National Science and Technology Major Project/ ; 2022ZD04008//Biological Breeding-National Science and Technology Major Project/ ; XGKJ2024020003//Natural Science Foundation of Xiaogan Municipality/ ; },
mesh = {*Brassica napus/genetics/growth &amp; development ; *Seeds/genetics/growth &amp; development/anatomy &amp; histology ; *CRISPR-Cas Systems ; Gene Editing ; Gene Library ; *Genes, Plant ; Phenotype ; Mutation ; Gene Expression Regulation, Plant ; },
abstract = {BACKGROUND: Enhancing rapeseed (Brassica napus, B. napus) yield is critical for ensuring global vegetable oil security. However, yield is heavily influenced by silique development and seed size, the enhancement of which is limited by scarce genetic resources. The CRISPR/Cas9 system has emerged as a powerful tool for constructing genome-wide mutant libraries, even in polyploid crops with complex genomes.<br><br>RESULTS: The transcriptome-wide association study (TWAS) data, tissue-specific expression profiles data and reported genes were integrated to identify candidate genes regulating silique development and seed size. We constructed a sgRNA library targeting these genes and generated a CRISPR/Cas9 editing mutant library through genetic transformation. Specifically, 6124 sgRNAs were designed for 1739 candidate genes with &#x2266;&#x2009;4 orthologues. 681 T0 plants were obtained through genetic transformation, which harbor 453 sgRNAs. Of 408 T0 plants analyzed, 151 (37.00%) exhibited successful gene editing events, targeting 84 candidate genes. Ten homozygous mutant plants were isolated and preliminary phenotypic analysis was performed in mutants targeting the BnaHRDs. The results suggest that mutations in BnaHRD.A03 and BnaHRD.C03 may modulate plant height (PH), main inflorescence length (MIL), silique length (SL), effective silique number per plant (ENS), seed number per silique (SNPS), and thousand-seed weight (TSW).<br><br>CONCLUSIONS: This study harnessed the CRISPR/Cas9 technology to establish a preliminary library of gene-edited mutants in B. napus, thereby laying a robust foundation for the future screening of candidate genes pertaining to silique development and seed size. Furthermore, this study provides a methodological framework for rapid functional gene discovery in B. napus through CRISPR-based approaches.},
}
@article {pmid41012695,
year = {2025},
author = {Madirov, A and Iksat, N and Masalimov, Z},
title = {Tomato Bushy Stunt Virus (TBSV): From a Plant Pathogen to a Multifunctional Biotechnology Platform.},
journal = {Viruses},
volume = {17},
number = {9},
pages = {},
pmid = {41012695},
issn = {1999-4915},
support = {No. BR21882269//Science Committee of the Ministry of Science and Higher Education of the Republic of Kazakhstan/ ; },
mesh = {*Biotechnology/methods ; *Tombusvirus/genetics/physiology ; Genetic Vectors ; Gene Editing ; *Plant Diseases/virology ; CRISPR-Cas Systems ; Gene Silencing ; Nanotechnology ; },
abstract = {Plant viruses have evolved from being viewed exclusively as pathogens into versatile and powerful tools for modern biotechnology. Among them, Tomato bushy stunt virus (TBSV) holds a special place due to its well-studied molecular biology and unique structural properties. This review systematizes the knowledge on TBSV's dual role as a multifunctional platform. On one hand, we cover its application as a viral vector for the highly efficient expression of recombinant proteins in plants, as well as a tool for functional genomics, including Virus-Induced Gene Silencing (VIGS) and the delivery of CRISPR/Cas9 gene-editing components. On the other hand, we provide a detailed analysis of the use of the stable and monodisperse TBSV virion in nanobiotechnology. Its capsid serves as an ideal scaffold for creating next-generation vaccine candidates, platforms for targeted drug delivery to tumor cells, and as a building block for the programmable self-assembly of complex nanoarchitectures. In conclusion, key challenges limiting the widespread adoption of the platform are discussed, including the genetic instability of vectors and difficulties in scalable purification, along with promising strategies to overcome them.},
}
@article {pmid41012691,
year = {2025},
author = {Zhao, X and Jiang, G and Ruan, Q and Qu, Y and Yang, X and Shi, Y and Wang, D and Zhou, J and Liu, J and Hou, L},
title = {Rapid Visual Detection of Senecavirus A Based on RPA-CRISPR/Cas12a System with Canonical or Suboptimal PAM.},
journal = {Viruses},
volume = {17},
number = {9},
pages = {},
pmid = {41012691},
issn = {1999-4915},
support = {National Key Research and Development Program of China (2023YFD1800501)//Lei Hou/ ; Introduction Program of High-Level Innovation and Entrepreneurship Talents in Jiangsu Province//Jue Liu/ ; the 111 Project D18007 (D18007)//Jue Liu/ ; Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD)//Jue Liu/ ; },
mesh = {Animals ; *CRISPR-Cas Systems ; Swine ; *Picornaviridae/genetics/isolation &amp; purification ; *Swine Diseases/virology/diagnosis ; *Nucleic Acid Amplification Techniques/methods ; Sensitivity and Specificity ; *Picornaviridae Infections/diagnosis/veterinary/virology ; Recombinases/metabolism/genetics ; },
abstract = {Senecavirus A (SVA) is an emerging pathogen responsible for vesicular lesions and neonatal mortality in swine. In the absence of effective vaccines or therapeutics, early and accurate diagnosis is essential for controlling SVA outbreaks. Although nucleic acid-based detection methods are commonly employed, there remains a pressing need for rapid, convenient, highly sensitive, and specific diagnostic tools. Here, we developed a two-pot assay combining recombinase polymerase amplification (RPA) with CRISPR/Cas12a containing crRNA targeting canonical protospacer adjacent motifs (PAMs) for simple, rapid, and visual identification of SVA in clinical samples. Subsequently, we successfully streamlined this system into a one-pot assay by selecting a specially designed crRNA targeting suboptimal PAM and integrating RPA amplification reagents and CRISPR/Cas12a detection components into a single reaction system in one tube. The developed methods exhibited diagnostic specificity, showing no cross-reactivity with four major swine viruses, while showing remarkable sensitivity with a lower detection limit of just two copies. Clinical validation in field samples using these two methods revealed perfect agreement (100% concordance) with conventional quantitative PCR (qPCR) results (sample size, n = 28), with both assays completing detection within 30 min. These results demonstrate that both the one-pot and two-pot RPA-CRISPR/Cas12a assays offer a reliable and efficient method for detecting SVA in this pilot study. Despite the limited sample size, the assays combine rapid reaction time with high sensitivity and specificity, showing great potential for future diagnostic applications.},
}
@article {pmid41012605,
year = {2025},
author = {Lee, MF and Tham, SK and Poh, CL},
title = {Antiviral Strategies Targeting Enteroviruses: Current Advances and Future Directions.},
journal = {Viruses},
volume = {17},
number = {9},
pages = {},
doi = {10.3390/v17091178},
pmid = {41012605},
issn = {1999-4915},
mesh = {*Antiviral Agents/pharmacology/therapeutic use ; Humans ; *Enterovirus Infections/drug therapy/virology ; *Enterovirus/drug effects/genetics ; Animals ; Virus Replication/drug effects ; },
abstract = {Enteroviruses, a diverse genus within the Picornaviridae family, are responsible for a wide range of human infections, including hand, foot, and mouth disease, respiratory disease, aseptic meningitis, encephalitis, myocarditis, and acute flaccid paralysis. Despite their substantial global health burden and the frequent emergence of outbreaks, no specific antiviral therapies are currently approved for clinical use against non-polio enteroviruses. This review provides a comprehensive overview of the current landscape of antiviral strategies targeting enteroviruses, including direct-acting antivirals such as capsid binders, protease inhibitors, and viral RNA polymerase inhibitors. We also examine the potential of host-targeting agents that interfere with virus-host interactions essential for replication. Emerging strategies such as immunotherapeutic approaches, RNA interference, CRISPR-based antivirals, and peptide-based antivirals are also explored. Furthermore, we address key challenges, including viral diversity, drug resistance, and limitations in preclinical models. By highlighting recent advances and ongoing efforts in antiviral development, this review aims to guide future research and accelerate the discovery of effective therapies against enterovirus infections.},
}
@article {pmid41011478,
year = {2025},
author = {Cortés, M and Olate, P and Rodriguez, R and Diaz, R and Martínez, A and Hernández, G and Sepulveda, N and Paz, EA and Quiñones, J},
title = {Human Microbiome as an Immunoregulatory Axis: Mechanisms, Dysbiosis, and Therapeutic Modulation.},
journal = {Microorganisms},
volume = {13},
number = {9},
pages = {},
doi = {10.3390/microorganisms13092147},
pmid = {41011478},
issn = {2076-2607},
support = {N&#xb0; 21231033//Agencia Nacional de Investigaci&#xf3;n y Desarrollo/ ; },
abstract = {The human microbiome plays a central role in modulating the immune system and maintaining immunophysiological homeostasis, contributing to the prevention of immune-mediated diseases. In particular, the gut microbiota is a key ecosystem for immune system maturation, especially in early life. This review aimed to analyze the molecular and cellular mechanisms linking the microbiome to immune and neuronal functions, as well as the impact of dysbiosis and emerging therapeutic strategies targeting the microbiome. The analysis was based on scientific databases, prioritizing studies published since 2000, with special emphasis on the past decade. The microbiome influences immune signaling through microorganism-associated molecular patterns (MAMPs) and pattern recognition receptors (PRRs), including Toll-like receptors (TLRs). Additionally, microbial metabolites-such as short-chain fatty acids (SCFAs), tryptophan derivatives, and secondary bile acids-exert significant immunomodulatory effects. The intestinal epithelial barrier is also described as an active immunological interface contributing to systemic regulation. The literature highlights innovative therapies, including fecal microbiota transplantation (FMT), probiotics, and microbiome editing with CRISPR-Cas technologies. These strategies aim to restore microbial balance and improve immune outcomes. The growing body of evidence positions the microbiome as a valuable clinical and diagnostic target, with significant potential for application in personalized medicine.},
}
@article {pmid41011338,
year = {2025},
author = {Meng, S and Zhao, Z and Huang, L and Peng, X and Chen, H and Tang, X},
title = {CRISPR/Cas Technology for the Diagnosis of Animal Infectious Diseases.},
journal = {Microorganisms},
volume = {13},
number = {9},
pages = {},
doi = {10.3390/microorganisms13092006},
pmid = {41011338},
issn = {2076-2607},
support = {2023174004//Beihai Science and Technology Program/ ; AB241484035//Guangxi Key R&amp;D Program Project/ ; YCSW2024140//Innovation Project of Guangxi Graduate Education/ ; },
abstract = {Increasingly complex epidemics of animal infectious diseases have emerged as a major risk to livestock production and human health. However, current detection methods for animal infectious diseases suffer from shortcomings such as insufficient sensitivity, complicated operation, and reliance on skilled personnel, highlighting the urgent need for novel sensing platforms. CRISPR/Cas systems are adaptive immune systems found in many prokaryotes. Owing to their ability to precisely and reliably target and cleave nucleic acids, the CRISPR/Cas-based nucleic acid detection technology is considered a promising new detection method. When leveraged with a pre-amplification step and established readout methods, CRISPR/Cas-based sensing platforms can achieve a high sensitivity of single-base resolution or attomolar levels on-site. In this review, we first outline the history, working principles, and nucleic acid detection platforms derived from various CRISPR/Cas systems. Next, we evaluate the advantages and limitations of different nucleic acid pre-amplification methods integrated with CRISPR/Cas systems, followed by a discussion of readout methods employed in CRISPR/Cas-based sensing platforms. Additionally, we highlight recent applications of CRISPR/Cas-based sensing platforms in identifying animal infectious diseases. Finally, we address the challenges and prospects of CRISPR/Cas-based sensing platforms for the early and accurate diagnosis of animal infectious diseases.},
}
@article {pmid41010035,
year = {2025},
author = {Angelis, KJ and Holá, M and Vágnerová, R and Vaculíková, J and Paleček, JJ},
title = {The Phenotype of Physcomitrium patens SMC6 Mutant with Interrupted Hinge Interactions.},
journal = {Genes},
volume = {16},
number = {9},
pages = {},
doi = {10.3390/genes16091091},
pmid = {41010035},
issn = {2073-4425},
support = {GA20-05095S//Czech Science Foundation/ ; GA23-05284S)//Czech Science Foundation/ ; },
mesh = {*Bryopsida/genetics/metabolism ; *Cell Cycle Proteins/genetics/metabolism ; *Plant Proteins/genetics/metabolism/chemistry ; Phenotype ; DNA Breaks, Double-Stranded ; CRISPR-Cas Systems ; Mutation ; *Chromosomal Proteins, Non-Histone/genetics ; },
abstract = {Background/Objectives: The Structural Maintenance of Chromosomes (SMC) proteins form essential heterocomplexes for the preservation of DNA structure and its functions, and hence cell viability. The SMC5/6 dimer is assembled by direct interactions of ATP heads via the kleisin NSE4 bridge and by SMC hinges. The structure might be interrupted by a single point mutation within a conserved motif of the SMC6-hinge. We describe the phenomena associated with the impairment of the SMC5/6 complex with morphology, repair of DNA double strand breaks (DSB), mutagenesis, recombination and gene targeting (GT) in the moss Physcomitrium patens (P. patens). Methods: Using CRISPR/Cas9-directed oligonucleotide replacement, we have introduced two close G to R point mutations in the hinge domain of SMC6 of P. patens and show that both mutations are not toxic and allow viability of mutant lines. Results: The G514R mutation fully prevents the interaction of SMC6 not only with SMC5, but also with NSE5 and NSE6, while the mutation at G517R has no effect. The Ppsmc6_G514R line has aberrant morphology, spontaneous and bleomycin-induced mutagenesis, and maintenance of the number of rDNA copies. The most unique feature is the interference with gene targeting (GT), which is completely abolished. In contrast, the Ppsmc6_G517R line is close to WT in many aspects. Surprisingly, both mutations have no direct effect on the rate of DSB repair in dividing and differentiated cells. Conclusions: Abolished interactions of SMC6 with SMC5 and NSE5,6 partners, which allow DSB repair, but impair other repair and recombination functions, suggests also regulatory role for SMC6.},
}
@article {pmid41009844,
year = {2025},
author = {Haval, M and Unakal, C and Ghagane, SC and Pandit, BR and Daniel, E and Siewdass, P and Ekimeri, K and Rajamanickam, V and Justiz-Vaillant, A and Lootawan, KA and Oliveira, FM and Bashetti, N and Naqvi, TA and Shettar, A and Bhasme, P},
title = {Biofilms Exposed: Innovative Imaging and Therapeutic Platforms for Persistent Infections.},
journal = {Antibiotics (Basel, Switzerland)},
volume = {14},
number = {9},
pages = {},
doi = {10.3390/antibiotics14090865},
pmid = {41009844},
issn = {2079-6382},
abstract = {Biofilms constitute a significant challenge in the therapy of infectious diseases, offering remarkable resistance to both pharmacological treatments and immunological elimination. This resilience is orchestrated through the regulation of extracellular polymeric molecules, metabolic dormancy, and quorum sensing, enabling biofilms to persist in both clinical and industrial environments. The resulting resistance exacerbates chronic infections and contributes to mounting economic burdens. This review examines the molecular and structural complexities that drive biofilm persistence and critically outlines the limitations of conventional diagnostic and therapeutic approaches. We emphasize advanced technologies such as super-resolution microscopy, microfluidics, and AI-driven modeling that are reshaping our understanding of biofilm dynamics and heterogeneity. Further, we highlight recent progress in biofilm-targeted therapies, including CRISPR-Cas-modified bacteriophages, quorum-sensing antagonists, enzyme-functionalized nanocarriers, and intelligent drug-delivery systems responsive to biofilm-specific cues. We also explore the utility of in vivo and ex vivo models that replicate clinical biofilm complexity and promote translational applicability. Finally, we discuss emerging interventions grounded in synthetic biology, such as engineered probiotic gene circuits and self-regulating microbial consortia, which offer innovative alternatives to conventional antimicrobials. Collectively, these interdisciplinary strategies mark a paradigm shift from reactive antibiotic therapy to precision-guided biofilm management. By integrating cutting-edge technologies with systems biology principles, this review proposes a comprehensive framework for disrupting biofilm architecture and redefining infection treatment in the post-antibiotic era.},
}
@article {pmid41009658,
year = {2025},
author = {Zhang, H and Teng, C and Lyu, S and Fan, Y},
title = {High-Frequency Generation of Homozygous/Biallelic Mutants via CRISPR/Cas9 Driven by AtKu70/80 Promoters.},
journal = {International journal of molecular sciences},
volume = {26},
number = {18},
pages = {},
doi = {10.3390/ijms26189094},
pmid = {41009658},
issn = {1422-0067},
support = {ZR2023MC070//Natural Science Foundation of Shandong province/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; *Promoter Regions, Genetic ; *Arabidopsis/genetics ; *Gene Editing/methods ; *Ku Autoantigen/genetics ; Homozygote ; *Arabidopsis Proteins/genetics ; Plants, Genetically Modified/genetics ; *Mutation ; Glycine max/genetics ; Alleles ; DNA End-Joining Repair ; },
abstract = {CRISPR/Cas9 gene editing technology is widely used in plant gene editing to verify gene function or improve agronomic traits. In the CRISPR/Cas9 system, Cas9 expression hinges on promoter choice, and CRISPR/Cas9 driven by a strong promoter or cell division-specific promoter has a higher editing efficiency. The CRISPR/Cas9 mechanism involves the CAS9 enzyme, which, directed by guide RNA, cleaves target double-stranded DNA and subsequently induces insertions or deletions (InDels) through the non-homologous end joining (NHEJ) repair pathway. The Ku protein plays a central role in the NHEJ repair process. It remains unclear whether driving Cas9 with promoters of AtKu70 and AtKu80, which are subunits of the Ku protein, will enhance gene editing efficiency. In this study, the promoters of AtKu70 and AtKu80 were cloned and used to drive Cas9 in the CRISPR/Cas9 system. Four different genes, GmRj7, GmNNL1, AtPDS3, and AtBRI1, were designed for soybean hairy root transformation and Arabidopsis transformation. The results showed that the CRISPR/Cas9 systems driven by the promoters of AtKu70 and AtKu80 achieved higher homozygous/biallelic mutation efficiencies than the CRISPR/Cas9 system driven by the 35S promoter in hairy root transformation by Rhizobium rhizogenes and stable genetic transformation with Rhizobium tumefaciens.},
}
@article {pmid41006228,
year = {2025},
author = {Maric, M and Segura-Bayona, S and Kuthethur, R and Takaki, T and Borel, V and Stanage, TH and Ivanov, MP and Parnandi, N and Hewitt, G and Millar, R and Fonseca, CS and Patel, H and Llorian, M and Warchal, S and Howell, M and Chaudhuri, AR and Kotsantis, P and Boulton, SJ},
title = {EXO1 as a therapeutic target for Fanconi Anaemia, ZRSR2 and BRCA1-A complex deficient cancers.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {8476},
pmid = {41006228},
issn = {2041-1723},
mesh = {*Exodeoxyribonucleases/genetics/metabolism ; Humans ; *BRCA1 Protein/genetics/metabolism/deficiency ; *Fanconi Anemia/genetics/metabolism ; DNA Repair/genetics ; *Neoplasms/genetics/metabolism ; DNA Replication/genetics ; *DNA Repair Enzymes/genetics/metabolism ; DNA Damage ; Cell Line, Tumor ; Cisplatin/pharmacology ; Synthetic Lethal Mutations ; Poly(ADP-ribose) Polymerase Inhibitors/pharmacology ; CRISPR-Cas Systems ; },
abstract = {Exonuclease EXO1 performs multiple roles in DNA replication and DNA damage repair (DDR). However, EXO1 loss is well-tolerated, suggesting the existence of compensatory mechanisms that could be exploited in DDR-deficient cancers. Using CRISPR screening, we find EXO1 loss as synthetic lethal with many DDR genes somatically inactivated in cancers, including Fanconi Anaemia (FA) pathway and BRCA1-A complex genes. We also identify the spliceosome factor and tumour suppressor ZRSR2 as synthetic lethal with loss of EXO1 and show that ZRSR2-deficient cells are attenuated for FA pathway activation, exhibiting cisplatin sensitivity and radial chromosome formation. Furthermore, FA or ZRSR2 deficiencies depend on EXO1 nuclease activity and can be potentiated in combination with PARP inhibitors or ionizing radiation. Finally, we uncover dysregulated replication-coupled repair as the driver of synthetic lethality between EXO1 and FA pathway attributable to defective fork reversal, elevated replication fork speeds, post-replicative single stranded DNA exposure and DNA damage. These findings implicate EXO1 as a synthetic lethal vulnerability and promising drug target in a broad spectrum of DDR-deficient cancers unaddressed by current therapies.},
}
@article {pmid41006213,
year = {2025},
author = {Chen, PR and Qin, PP and Wang, YN and Liu, PF and Zhang, XY and Qian, T and Ye, BC and Yin, BC},
title = {De novo design of hypercompact transcript degraders by engineering substrate-specific toxins and Cas6-CBS system.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {8446},
pmid = {41006213},
issn = {2041-1723},
mesh = {Escherichia coli/genetics/metabolism ; Humans ; *Escherichia coli Proteins/genetics/metabolism ; *Endoribonucleases/metabolism/genetics ; CRISPR-Cas Systems ; RNA Stability ; Protein Engineering/methods ; Binding Sites ; HEK293 Cells ; Bacterial Toxins/genetics/metabolism ; *CRISPR-Associated Proteins/metabolism/genetics ; DNA-Binding Proteins ; },
abstract = {Artificial assembly of small functional proteins provides effective strategies for development of compact RNA degradation systems, which overcome the challenges associated with delivery. Here, we excavate and evolve three small toxin endoribonucleases with simple RNA cleavage motifs (barnase, MqsR, and MaZF), and integrate catalytically dead Cas6 (dCas6) along with its cognate stem-loop RNA (Cas6 binding site, termed CBS) from Escherichia coli (E. coli) to create hypercompact transcript degraders (317 ~ 430 amino acids), named STAR (small toxin- and dEcCas6-CBS-based RNA degraders). We experimentally find that CBS can be fine-tuned for EcCas6 processing but exhibits high conservatism in EcCas6 and dEcCas6 binding, laying a foundation for the design of CBS guides to effectively recruit dEcCas6-toxins. STAR exhibits high-efficiency knockdown of both cytoplasmic and nuclear transcripts in the tested mammalian cells, with significantly reduced off-target activities compared to established CRISPR and RNA interference (RNAi) technologies. Moreover, the small size of STAR enables delivery via a single adeno-associated virus (AAV) for ease of multiplex RNA knockdown, including effective silencing of the oncogenic RNA MYC in human cancer cells. Together, STAR unlocks new territory for employing toxin to design miniature, efficacious and safer RNA degraders.},
}
@article {pmid41005481,
year = {2025},
author = {Gong, Z and Lu, T and Ruan, Z and Zhang, R and Zhu, S and Xia, Z and Zhong, J and Wang, G and Li, Y and He, Q and Liu, R and Che, J},
title = {A multiplexed TSA/CRISPR-mediated one-pot system for rapid detection of high-risk animal-derived infectious diseases.},
journal = {Journal of microbiological methods},
volume = {238},
number = {},
pages = {107277},
doi = {10.1016/j.mimet.2025.107277},
pmid = {41005481},
issn = {1872-8359},
abstract = {The importance of rapid and convenient pathogen detection has been emphasized by the alarming threat of the Coronavirus Disease 2019 (COVID-19) pandemic since 2019. Point-of-care testing (POCT) provides rapid diagnostic results directly at the sampling site. However, isothermal amplification-based POCT faces technical challenges including primer design complexity and false-positive rates. To address these limitations, we developed the Thermostatic Step Amplification (TSA)/Clustered regularly interspaced short palindromic repeats (CRISPR) One-Pot System (TCOPS). This sensitive, rapid, and efficient platform specifically detects Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), Mpox virus (MPXV) and Rabíes virus (RV) through integrated amplification and CRISPR-based detection. Our integrated TCOPS overcomes the technical challenges through single-tube reactions combining thermostatic amplification and CRISPR detection, reducing contamination while maintaining high accuracy for field applications. TCOPS enables single-tube CRISPR detection of high-risk viruses, with 10 copies/μL sensitivity shown using cloned DNA template for RV. In evaluations against Quantitative Polymerase Chain Reaction (qPCR) using 50 clinical samples, TCOPS incorporating freeze-dried reagents and a newly developed miniature fluorescence system (Q max) demonstrated >90 % sensitivity and 100 % specificity. Combined with the portable Q max device and its lyophilized reagent kit, TCOPS enables simple, rapid detection of multiple zoonotic viruses (SARS-CoV-2, MPXV, and RV) at the point of care. This integrated system achieves high sensitivity and specificity while establishing a practical, field-deployable prototype for next-generation POCT applications in resource-limited settings.},
}
@article {pmid41004579,
year = {2025},
author = {Gao, Q and Gao, Y and Cao, Y and Xu, H and Ma, Y and Zu, M and Yang, Q and Yang, K and Zhu, Z and Liu, C and Shi, X and Reis, RL and Kundu, SC and Ma, S and Han, H and Xiao, B},
title = {Reinforced plant-derived lipid nanoparticles for oral precise epigenome editing in colonic diseases.},
journal = {Science advances},
volume = {11},
number = {39},
pages = {eadw9275},
pmid = {41004579},
issn = {2375-2548},
mesh = {*Nanoparticles/chemistry/administration &amp; dosage ; *Gene Editing/methods ; Animals ; Administration, Oral ; Humans ; Mice ; *Lipids/chemistry ; CRISPR-Cas Systems ; *Epigenome ; *Colonic Diseases/genetics/therapy ; Ribonucleoproteins/genetics ; Colitis/genetics ; *Epigenesis, Genetic ; Histone Demethylases/genetics ; Epigenome Editing ; Liposomes ; },
abstract = {The clinical application of CRISPR-Cas9 remains limited by delivery challenges, particularly for oral administration. Lysine-specific demethylase 1 (Lsd1) plays a key role in colonic inflammation and tumorigenesis. Here, we developed an oral genome-editing platform (TPGS-RNP@LNP), where Lsd1-targeting ribonucleoproteins (RNPs) were encapsulated in mulberry leaf lipid nanoparticles (LNPs) and formulated with d-α-tocopherol polyethylene glycol succinate (TPGS). TPGS reinforced the lipid bilayer of LNPs, enhanced gastrointestinal stability, and facilitated colonic mucus penetration. Upon the galactose receptor-mediated endocytosis of TPGS-RNP@LNPs by macrophages, their fusion with the endosomal membrane and the presence of nuclear localization signals ensured the nuclear delivery of RNPs. TPGS-RNP@LNPs achieved 59.7% Lsd1 editing efficiency in macrophages, surpassing the commercial CRISPRMAX (43.0%). Oral TPGS-RNP@LNPs promoted H3K4 methylation to modulate epigenetic states, achieving inflammation mitigation, epithelial barrier restoration, and retardation of colitis and its associated tumorigenesis. As an LNP-based oral RNP delivery system, TPGS-RNP@LNPs provide a promising platform for precise treatment of colorectal diseases.},
}
@article {pmid41004518,
year = {2025},
author = {Wilbie, D and Eising, S and Amo-Addae, V and Walther, J and Bosman, E and Lei, Z and de Jong, OG and Molenaar, JJ and Mastrobattista, E},
title = {Anti-cancer compound screening identifies Aurora Kinase A inhibition as a means to favor CRISPR/Cas9 gene correction over knock-out.},
journal = {PloS one},
volume = {20},
number = {9},
pages = {e0332617},
pmid = {41004518},
issn = {1932-6203},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Aurora Kinase A/antagonists &amp; inhibitors/genetics ; HEK293 Cells ; *Antineoplastic Agents/pharmacology ; Gene Knockout Techniques ; *Protein Kinase Inhibitors/pharmacology ; Gene Editing ; Pyrimidines/pharmacology ; Drug Screening Assays, Antitumor ; Recombinational DNA Repair/drug effects ; Cell Line, Tumor ; Azepines ; },
abstract = {CRISPR gene therapy holds the potential to cure a variety of genetic diseases by causing a targeted DNA break, which is repaired by host DNA damage responses. One option to introduce precise gene corrections is via the homology-directed repair (HDR) pathway. The problem in utilizing this pathway is that CRISPR-induced double stranded DNA breaks are more likely to be erroneously repaired by the non-homologous end joining (NHEJ) pathway, which may introduce random insertions or deletions at the cut site. We screened a small library of oncological drug compounds to steer the DNA repair process towards preferential HDR activation. We included forty compounds in the screen based on their mechanism of action. After optimizing the toxicity and adding these compounds during gene editing, nine showed a potential benefit for HDR activation. Three were shown to be beneficial after validation: rucaparib, belinostat and alisertib. The Aurora Kinase A inhibitor alisertib in particular led to an over 4-fold increase in preferential gene correction over gene knock-out in two cell models (HEK293T and Hepa 1-6) at sub-micromolar dosages on the eGFP locus, prompting further validation. On the long term this pathway did show cytotoxicity especially in the HEK293T cells, indicating further mechanistic investigation is needed, but this toxicity was less pronounced in primary hepatocytes.},
}
@article {pmid41004222,
year = {2025},
author = {Taweechai, S and Totañes, FIG and Westhead, D and Herrera-Arozamena, C and Foster, R and McConkey, GA},
title = {Validated antimalarial drug target discovery using genome-scale metabolic modeling.},
journal = {Antimicrobial agents and chemotherapy},
volume = {},
number = {},
pages = {e0045925},
doi = {10.1128/aac.00459-25},
pmid = {41004222},
issn = {1098-6596},
abstract = {Given the rapid resistance of Plasmodium falciparum to antimalarial drugs, there is a continual need for new treatments. A genome-scale metabolic (GSM) model was developed with integrated metabolomics and constraint-based, experimental flux-balance data to predict genes essential for P. falciparum growth as drug targets. We selected the highly ranked P. falciparum UMP-CMP kinase (UCK) to test its necessity and the ability to inhibit parasite growth in the presence of inhibitors. Conditional deletion mutants using the DiCre recombinase system, generated by CRISPR-Cas genome editing, exhibited defective asexual growth and stage-specific developmental arrest. Based on in silico and in vitro screening, inhibitors were identified that are selective for P. falciparum UCK and exhibit antiparasitic activity. This study, for the first time, shows assertions from a GSM model identifying novel, validated "druggable" targets. These findings show a role for GSM models in antimalarial drug discovery and identify P. falciparum UCK as a novel, valid malaria drug target.},
}
@article {pmid41003754,
year = {2025},
author = {Zubair, A and Ali, M and Ahmad, F and Althobaiti, SA},
title = {Unlocking the role of transcription activator-like effector nuclease (TALENs) and zinc finger nuclease (ZFN) in the treatment of HIV.},
journal = {Molecular biology reports},
volume = {52},
number = {1},
pages = {948},
pmid = {41003754},
issn = {1573-4978},
mesh = {Humans ; *Transcription Activator-Like Effector Nucleases/genetics/metabolism/therapeutic use ; *Zinc Finger Nucleases/genetics/metabolism/therapeutic use ; *HIV Infections/therapy/genetics ; Gene Editing/methods ; HIV-1/genetics ; CRISPR-Cas Systems ; Receptors, CCR5/genetics ; Zinc Fingers ; },
abstract = {Some nucleases may be programmed to break just certain portions of DNA; examples of such enzymes include zinc-finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs). Insertions and deletions are used by cellular machinery to repair damaged DNA. By specifically targeting long terminal repeats (LTRs), zinc-finger nucleases (ZFNs) efficiently and accurately remove HIV-1 proviral DNA from inactive human T cells, offering a new and different way to eradicate HIV-1 infections. This paper examines the potential, evaluates the current situation, and draws attention to the challenges surrounding the use of TALENs and ZFNs as therapeutic tools for the treatment of HIV infection, to mitigate the adverse off-target effects that result from their extended expression. There is less off-target editing and higher success in targeting HIV escape mutations using TALENs and ZFNs than with CRISPR/Cas-9. The use of ZFNs and TALEN has resulted in changes to many host genes. These include the entrance receptors CCR5 and CXCR4, as well as the proviral integration protein LEDGF/p75. One of the viral targets is the big terminal repeats of proviral DNA. The advancement of gene therapy from the laboratory to the clinic is hindered by the need to reduce immunogenicity, cytotoxicity, and off-target editing while simultaneously enhancing cleavage efficiency and dispersion. However, TALENs technology and breakthroughs in ZFNs are making cleavage more efficient and selective. The strategy for treating HIV might be drastically changed, and maybe even eradicated, by the creation of synthetic nucleases like ZFNs and TALENs. This review explores the current developments about ZFNs and TALENs for the treatment of HIV.},
}
@article {pmid41003718,
year = {2025},
author = {Behera, B and Singh, R and Sharma, K and Rai, A and Singh, S and Balan, B},
title = {A comprehensive review of advanced strategies to combat antimicrobial resistance.},
journal = {Archives of microbiology},
volume = {207},
number = {11},
pages = {281},
pmid = {41003718},
issn = {1432-072X},
mesh = {Humans ; *Anti-Bacterial Agents/pharmacology/therapeutic use ; Gene Editing ; *Drug Resistance, Bacterial ; *Bacteria/drug effects/genetics ; CRISPR-Cas Systems ; Antimicrobial Peptides/pharmacology/therapeutic use ; Phage Therapy ; *Bacterial Infections/drug therapy/microbiology/therapy ; },
abstract = {Antimicrobial Resistance (AMR) is a growing global issue, as many first-line antibiotics are becoming less effective due to their overuse and misuse. Recent advances in novel antibiotic derivatives reveal mechanisms designed to counteract AMR. Even though conventional antimicrobial therapy has failed, no new antibiotic class has been developed in the past decade. Consequently, various innovative alternative tactics have been discovered to counteract drug-resistant pathogens. The article reviews novel approaches in combating AMR, which include antimicrobial peptides, phage therapy, CRISPR-Cas gene editing, nanomaterial-based antimicrobials, immunomodulatory agents, innovative physicochemical strategies, and combination therapy. Collectively, these approaches utilize cutting edge technologies that mark a shift from the traditional paradigm of antibiotics to integrated next-generation therapeutics. AMR remains a serious issue despite all of the noted advancements, and hence, a collaborative and multidisciplinary action involving researchers, healthcare professionals, policymakers, and pharmaceutical sector is urgently required. The emergence and burden of AMR can be better tackled by inventiveness, cooperation, and proactive approaches.},
}
@article {pmid41002431,
year = {2025},
author = {Vivarelli, R and Vantaggiato, C and Bassi, MT and Santorelli, FM and Marchese, M},
title = {Wings of Discovery: Using Drosophila to Decode Hereditary Spastic Paraplegia and Ataxias.},
journal = {Cells},
volume = {14},
number = {18},
pages = {},
doi = {10.3390/cells14181466},
pmid = {41002431},
issn = {2073-4409},
support = {GJC21131//Telethon Foundation/ ; Ricerca Corrente 2024-2025//Italian Minister of Health/ ; },
mesh = {Animals ; *Spastic Paraplegia, Hereditary/genetics/pathology ; Disease Models, Animal ; Humans ; *Drosophila melanogaster/genetics ; CRISPR-Cas Systems ; },
abstract = {Hereditary spastic paraplegia (HSP) and hereditary ataxias (HA) are clinically and genetically heterogeneous neurodegenerative disorders that primarily affect motor coordination and neural integrity. Despite distinct pathological features, such as pyramidal tract degeneration in HSP and spinocerebellar pathway involvement in HA, these conditions share overlapping genetic pathways and mechanisms. The fruit fly Drosophila melanogaster has emerged as a powerful model organism for investigating the molecular basis of rare diseases, including HSP and HA. Its genetic tractability, rapid life cycle, and high degree of gene conservation with humans make it a cost-effective and ethically viable platform for disease modelling. In this review, we provide a comprehensive overview of Drosophila-based models for HSP and HA. We highlight the use of advanced genetic tools, including RNA interference, CRISPR/Cas9, and the GAL4/UAS system, as well as behavioral and neuroanatomical assays to model disease features. Furthermore, we discuss the application of genetic "avatars" and high-throughput drug screening platforms to test therapeutic candidates. Collectively, these models have deepened our understanding of the pathophysiology of HSP and HA, offering valuable insights for the development of targeted therapies and approaches to personalized medicine.},
}
@article {pmid41002335,
year = {2025},
author = {Jayakumar, S and Vengadassalapathy, S and Venkadassalapathy, S and Durairajan, S and Vijayaraj, R and Govindan, L},
title = {Advancements and Applications of Split Technology in CRISPR/Cas12a: Transforming Molecular Diagnostics and Biosensing.},
journal = {Biosensors},
volume = {15},
number = {9},
pages = {},
doi = {10.3390/bios15090595},
pmid = {41002335},
issn = {2079-6374},
mesh = {*Biosensing Techniques ; *CRISPR-Cas Systems ; Humans ; *Pathology, Molecular ; *Molecular Diagnostic Techniques ; },
abstract = {The rapid evolution of CRISPR technology has revolutionized molecular biology, and among the various systems, CRISPR/Cas12a stands out for its high specificity and efficient collateral cleavage activity. This review article focuses on the recent advancements and applications of split technology within the CRISPR/Cas12a framework, highlighting its transformative role in molecular diagnostics and biosensing. Split technology innovatively divides functional nucleic acid components into modular segments that are activated by specific targets, significantly enhancing the specificity and sensitivity of biosensors. This design addresses the inherent limitations of traditional sensor systems, enabling the direct detection of ultrashort nucleic acids and improved discrimination of single-nucleotide variants, thereby facilitating the simultaneous detection of multiple biomolecules. The versatility of split-enabled biosensors extends beyond genetic testing, making them valuable tools in diagnostics, therapeutics, and environmental science. Despite challenges such as crRNA degradation and reassembly kinetics, ongoing research and engineering solutions continue to enhance the stability and performance of these systems. This review synthesizes the foundational principles, recent advancements, and potential applications of split technology while also identifying challenges and opportunities for future exploration. Ultimately, our insights provide a comprehensive resource to leverage the full potential of CRISPR/Cas12a-based split technology in advancing biosensing methodologies and clinical applications.},
}
@article {pmid41002137,
year = {2025},
author = {Zhang, S and Ji, Z and Cheng, X and Ma, Y and Feng, M and Cai, D and Bai, T},
title = {TMTP1-Modified Small Extracellular Vesicles Target BRAF Mutation in Anaplastic Thyroid Cancer Reversing Vemurafenib Resistance With CRISPR/Cas9 Delivery.},
journal = {Journal of extracellular vesicles},
volume = {14},
number = {9},
pages = {e70170},
doi = {10.1002/jev2.70170},
pmid = {41002137},
issn = {2001-3078},
mesh = {Humans ; *Thyroid Carcinoma, Anaplastic/genetics/drug therapy/metabolism ; *Vemurafenib/pharmacology ; *Extracellular Vesicles/metabolism ; *CRISPR-Cas Systems ; *Drug Resistance, Neoplasm/genetics/drug effects ; *Proto-Oncogene Proteins B-raf/genetics ; Cell Line, Tumor ; Animals ; *Thyroid Neoplasms/genetics/drug therapy ; Mutation ; Mice ; Gene Editing ; Reactive Oxygen Species/metabolism ; Drug Delivery Systems ; },
abstract = {This study investigates a novel approach to overcome Vemurafenib resistance in BRAF-mutant Anaplastic thyroid carcinoma (ATC) using CRISPR/Cas9 gene editing and TMTP1-modified extracellular vesicles (TMTP1-sgBRAF-EVs). By knocking out the BRAF gene, the study elucidates Vemurafenib-induced ferroptosis mechanisms involving lipid peroxidation and reactive oxygen species (ROS) generation in ATC cells. The developed TMTP1-sgBRAF-EVs system demonstrates superior tumour-targeting and drug delivery capabilities, significantly enhancing Vemurafenib efficacy in both in vitro and in vivo models. This innovative combination of gene editing technology with a nanoparticle delivery system shows promising potential as a therapeutic strategy for treating aggressive BRAF-mutant ATC.},
}
@article {pmid41000010,
year = {2025},
author = {Wang, Z and Wang, Q and Zhang, J and Li, B and Li, Y and Chen, Z and Guo, D and Feng, S},
title = {CRISPR-driven diagnostics: Molecular mechanisms, clinical efficacy and translational challenges.},
journal = {Clinical and translational medicine},
volume = {15},
number = {10},
pages = {e70482},
doi = {10.1002/ctm2.70482},
pmid = {41000010},
issn = {2001-1326},
support = {23ZX005//the Basic Research Project of the Key Research Program of Colleges and Universities in Henan Province/ ; 2023M731023//China Postdoctoral Science Foundation/ ; //Joint Funds of Science and Technology Research and Development Plan of Henan Province/ ; 232300421164//Natural Science Foundation of Henan Province/ ; //Basic Research Project of the Key Research Program of Colleges and Universities in Henan Province/ ; 82402600//National Natural Science Foundation of China/ ; },
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; Gene Editing/methods ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Translational Research, Biomedical ; },
abstract = {BACKGROUND: In the realm of public health, among the primary perils menacing human well-being, the issue of pathogen infection persists as a significant concern. Precise and timely diagnosis of diseases constitutes the bedrock for effective therapeutic interventions and epidemiological monitoring. Hence, it is crucial to develop quick, sensitive, and highly effective methods for identifying pathogen and their variants.<br><br>MATERIAL AND METHODS: This article reviews the recent research progress in the CRISPR/Cas system for detecting nucleic acids, with an emphasis on CRISPR/Cas9, CRISPR/Cas12, and CRISPR/Cas13. Initially, we provided a concise overview of the nucleic acid detection mechanism utilizing the CRISPR/Cas system. Subsequently, we dissect the molecular mechanisms of CRISPR tools, compare their clinical efficacy against traditional methods, and explore frontier innovations such as amplification-free detection and AI integration.<br><br>CONCLUSION: Ultimately, we argue that CRISPR diagnostics must evolve beyond technical optimization to embrace ecological adaptability, ensuring that precision medicine serves as a bridge-rather than a barrier-to global health equity.<br><br>KEY POINTS: Core Mechanism: Explains the molecular basis of CRISPR-Cas (Cas9, Cas12, Cas13) for nucleic acid detection, leveraging crRNA-guided targeting and trans-cleavage activity for ultra-sensitive (aM level) and specific pathogen identification. Superior Performance: Outperforms traditional methods in speed, sensitivity, and cost, making it ideal for point-of-care use in resource-limited settings. Cutting-Edge Innovations: Covers key advances like amplification-free detection, portable device integration, and multiplex platforms. Translation Challenges: Discusses hurdles in clinical adoption, including inhibitor interference in complex samples, scalability limitations, the need for multi-center clinical data, and varying regional regulations. Future Outlook: Highlights emerging directions such as integrated "sample-to-result" systems and AI integration, while also addressing associated biosafety and ethical concerns, calling for robust regulatory frameworks.},
}
@article {pmid40998781,
year = {2025},
author = {Porter, DF and Meyers, RM and Miao, W and Reynolds, DL and Hong, AW and Yang, X and Srinivasan, S and Mondal, S and Siprashvili, Z and Fabo, T and Zhou, R and Nguyen, T and Ducoli, L and Meyers, JM and Nguyen, DT and Ko, LA and Kellman, LN and Elfaki, I and Guo, M and Winge, MC and Jackrazi, LV and Lopez-Pajares, V and Liu, BB and Qu, Y and Porter, IE and Kim, SH and Kim, G and Tao, S and Engreitz, JM and Khavari, PA},
title = {Disease-linked regulatory DNA variants and homeostatic transcription factors in epidermis.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {8387},
pmid = {40998781},
issn = {2041-1723},
support = {AR076965, AR045192//U.S. Department of Health &amp; Human Services | NIH | National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS)/ ; HG010856//U.S. Department of Health &amp; Human Services | NIH | National Human Genome Research Institute (NHGRI)/ ; },
mesh = {Humans ; *Transcription Factors/metabolism/genetics ; *Epidermis/metabolism/pathology ; *Polymorphism, Single Nucleotide ; Homeostasis/genetics ; *Skin Diseases/genetics ; Genetic Predisposition to Disease ; *DNA/metabolism/genetics ; Multifactorial Inheritance/genetics ; Promoter Regions, Genetic ; Alleles ; CRISPR-Cas Systems ; },
abstract = {Identifying noncoding single nucleotide variants (SNVs) in regulatory DNA linked to polygenic disease risk, the transcription factors (TFs) they bind, and the genes they dysregulate is a goal in polygenic disease research. Here, we use massively parallel reporter analysis of 3451 SNVs linked to risk for polygenic skin diseases with disrupted epidermal homeostasis to identify 355 differentially active SNVs (daSNVs). daSNV target gene analysis, combined with daSNV editing, underscored dysregulated epidermal differentiation as a shared pathomechanism. CRISPR knockout screens of 1772 human TFs revealed 123 TFs essential for epidermal homeostasis, highlighting ZNF217 and CXXC1. Population sampling CUT&amp;RUN of 27 homeostatic TFs identified allele-specific DNA binding (ASB) differences at daSNVs enriched near epidermal homeostasis and monogenic skin disease genes, with notable representation of SP/KLF and AP-1/2 TFs. High TF-occupancy promoters were "buffered" against ASB. This resource implicates dysregulated binding of specific homeostatic TF families in risk for diverse polygenic skin diseases.},
}
@article {pmid40997767,
year = {2025},
author = {Shoeva, OY and Zedgenizova, VD and Egorova, AA and Gerasimova, SV and Kukoeva, TV and Vasiliev, GV and Kovaleva, ON and Zakhrabekova, S and Hansson, M and Hertig, CW and Hoffie, I and Kumlehn, J and Khlestkina, E},
title = {Analysis of Anthocyanin-Less 2 Diversity in Barley Reveals a Specific Allele to Cause Purple-Colored Grains.},
journal = {Journal of agricultural and food chemistry},
volume = {},
number = {},
pages = {},
doi = {10.1021/acs.jafc.5c05032},
pmid = {40997767},
issn = {1520-5118},
abstract = {The purple color of the barley (Hordeum vulgare L.) grain is attributed to anthocyanins which are beneficial for human health. Synthesis of these pigments in both grain and vegetative tissues is controlled by ANT1 and ANT2 that belong to the families R2R3-MYB and bHLH of transcription factors, respectively. Here, we investigated the role of the Ant2 gene in pigmentation of grains compared to vegetative tissue. After screening of 504 barley accessions, six relevant alleles of Ant2 were identified. These involve distinct insertions in the promoter and/or in intron 6. Allele Ant2.l was found in all purple-grained barley accessions and was more strongly expressed compared to the alleles present in the other lines. The crucial role of Ant2.l in grain pigmentation was validated by targeted mutagenesis using RNA-guided endonuclease Cas9. It is further shown that the other Ant2 alleles do not affect grain pigmentation while being essential for pigmentation of vegetative tissues.},
}
@article {pmid40996619,
year = {2025},
author = {Parkinson, JE and Baldwin, GE and Papotto, PH and Humphreys, NE and Day, AJ and Adamson, AD and Allen, JE and Sutherland, TE},
title = {Generation of a Ym1 deficient mouse utilising CRISPR-Cas9 in CB6 embryos.},
journal = {Transgenic research},
volume = {34},
number = {1},
pages = {44},
pmid = {40996619},
issn = {1573-9368},
support = {MR/K01207X/2/MRC_/Medical Research Council/United Kingdom ; MR/K01207X/2/MRC_/Medical Research Council/United Kingdom ; MR/K01207X/2/MRC_/Medical Research Council/United Kingdom ; MR/K01207X/2/MRC_/Medical Research Council/United Kingdom ; MRY0036831/MRC_/Medical Research Council/United Kingdom ; 097820/Z/11/B/WT_/Wellcome Trust/United Kingdom ; 203128/Z/16/Z/WT_/Wellcome Trust/United Kingdom ; 203128/Z/16/Z/WT_/Wellcome Trust/United Kingdom ; MRFAUK-2015-302//Asthma and Lung UK/ ; },
mesh = {Animals ; *CRISPR-Cas Systems/genetics ; Mice ; Mice, Inbred C57BL ; *Chitinase-3-Like Protein 1/genetics/deficiency ; Mice, Inbred BALB C ; Mice, Transgenic ; Female ; Embryo, Mammalian/metabolism ; },
abstract = {Chitinase-like proteins (CLPs) are of wide interest due to their significant roles during both homeostatic and pathological processes. Human CLPs such as YKL-40 have been proposed as biomarkers of disease severity in many conditions. Murine CLPs Brp39, Ym1, and Ym2 are similarly upregulated in multiple mouse models of pathology. Investigation of Ym1 and Ym2 is hampered by recent gene duplication events on the C57BL/6, but not BALB/c, background leading to complexity in the genomic locus. Here, we have generated a Ym1 deficient mouse using a novel CRISPR-Cas9 targeting approach involving CB6 (C57BL/6 X BALB/c) mixed background embryos. Validation using flow cytometry, ELISA, and immunofluorescence confirmed no expression of mature Ym1 protein. Additionally, expression of related genes including Chia, Chil1, and Chil4 were not altered in Ym1-deficent animals. This new transgenic mouse line will be key for future investigations of CLP functions and the utilised approach to genetic manipulation may provide a useful strategy for other genes which show differences in copy number between inbred mouse strains.},
}
@article {pmid40996539,
year = {2025},
author = {Nie, H and Wang, Z and Lin, Z and Gao, Y and Zhang, Y and Zheng, J and Cheng, Y},
title = {Enhancing lipase activity in Aspergillus niger through CRISPR/Cas9-mediated protease gene knockout and fermentation optimization.},
journal = {Biotechnology letters},
volume = {47},
number = {5},
pages = {114},
pmid = {40996539},
issn = {1573-6776},
support = {cstc2021jscx-jbgsX0002//Application Development Special Key Project of Chongqing/ ; 2022R01015//Leader-type Innovation and Entrepreneurship Team of Zhejiang/ ; },
mesh = {*Aspergillus niger/genetics/enzymology/metabolism ; *Lipase/metabolism/genetics ; *CRISPR-Cas Systems ; Fermentation ; *Gene Knockout Techniques/methods ; Fungal Proteins/genetics/metabolism ; *Peptide Hydrolases/genetics ; Metabolic Engineering/methods ; },
abstract = {The engineered Aspergillus niger strain AnCALB005 was selected as the research strain, which is a high-yield strain of Candida antarctica B lipase constructed in our laboratory. CRISPR/Cas9-mediated gene knockout was employed to construct the multiple protease-deficient strains targeting five genes (pepA, pepB, pepC, pepE and pepF) in the A. niger AnCALB005. Among the engineered variants, a triple-knockout strain lacking pepA, pepB, and pepF demonstrated 56% enhanced hydrolytic lipase activity relative to the parental strain. Fermentation culture conditions were initially screened through single-factor experiments. Building on these results, critical parameters were statistically determined via Plackett-Burman (PB) design. This was followed by a steepest ascent method combined with Box-Behnken (BB) response surface methodology. Key factors influencing lipase production (identified as maltose concentration, corn steep concentration, and shaking speed) were optimized. The final optimized fermentation conditions comprised: maltose (52 g/L), corn steep (52 g/L), K2HPO4 (5 g/L), soybean cake flour (30 g/L), initial pH 6.5, inoculation amount 10% (v/v), and shaking speed 220 rpm. Under the optimized fermentation conditions, Shake-flask validation of the engineered A. niger yielded a lipase activity of 46.66 U/mL, representing an increase of 92.01%. Scale-up fermentation in a 5 L bioreactor applying these optimized conditions over 120 h of cultivation achieved a lipase activity of 79.31 U/mL.},
}
@article {pmid40996239,
year = {2025},
author = {Calvert, RW and Knott, GJ},
title = {And… cut! - how conformational regulation of CRISPR-Cas effectors directs nuclease activity.},
journal = {The Biochemical journal},
volume = {482},
number = {19},
pages = {},
doi = {10.1042/BCJ20240481},
pmid = {40996239},
issn = {1470-8728},
mesh = {*CRISPR-Cas Systems ; *CRISPR-Associated Proteins/metabolism/chemistry/genetics ; RNA, Guide, CRISPR-Cas Systems/metabolism/genetics/chemistry ; Protein Conformation ; *Bacterial Proteins/metabolism/chemistry/genetics ; Nucleic Acid Conformation ; },
abstract = {Controlling the conformation of dynamic protein, RNA and DNA molecules underpins many biological processes, from the activation of enzymes and induction of signalling cascades to cellular replication. Clustered regularly interspaced short palindromic repeats (CRISPR)-associated (Cas) effectors are enzymes tightly controlled by conformational steps that gate activation of nuclease domains core to their function in bacterial adaptive immunity. These precise conformational checkpoints combined with programmable activation specified by RNA guides have driven the success of CRISPR-Cas tools in biotechnology, medicine and beyond. To illustrate the importance of conformation in controlling CRISPR-Cas activity, we review the discrete conformational checkpoints at play in class 2 CRISPR-Cas systems. Using Cas9, Cas12a and Cas13a as examples, we describe how protein and nucleic acid conformations precisely control the loading of guide RNA, the selection of target nucleic acids and the activation of nuclease domains. Much like a director controls the timing of transitions between scenes in a movie, CRISPR effectors use conformational checkpoints to precisely direct their enzymatic activity.},
}
@article {pmid40957828,
year = {2025},
author = {Qiu, M and Zhang, X and Zhang, J and Li, Y and Jiang, Y and Zhao, Q and Man, C and Zhang, X},
title = {CRISPR/Cas12a-Responsive Hydrogel Biosensing Platform Based on an Aggregation-induced Emission Metal-Organic Framework Nanozyme for Sensitive and Reliable Detection of Foodborne Pathogens.},
journal = {ACS sensors},
volume = {10},
number = {9},
pages = {6775-6787},
doi = {10.1021/acssensors.5c01523},
pmid = {40957828},
issn = {2379-3694},
mesh = {*Metal-Organic Frameworks/chemistry ; *Biosensing Techniques/methods ; *Hydrogels/chemistry ; *CRISPR-Cas Systems ; Colorimetry ; *Salmonella enterica/isolation &amp; purification ; Limit of Detection ; Food Microbiology ; },
abstract = {Programmable CRISPR/Cas12a has emerged as a promising and powerful tool for the construction of biosensors, yet the challenges persist regarding the signal transduction efficiency, sensitivity, and multisignal outputs. In this work, a metal-organic framework (MOF) nanozyme with an aggregation-induced emission (AIE) property and peroxidase (POD)-like activity was synthesized by encapsulating hemin into a Zr-based MOF using an in situ encapsulation strategy, named AIE MOFzyme. The prepared AIE MOFzyme showed an enhanced fluorescent property via the restriction of the intramolecular motion effect for boosting the significant fluorescent response and a highly efficient POD-like nanozyme activity for activating visual colorimetric response. Benefiting from these merits, AIE MOFzyme, as a difunctional signal probe, was embedded into a DNA hydrogel for the construction of a CRISPR-responsive AIE MOFzyme hydrogel platform (abbreviated as Cas12a-H-AIE). In this platform, the activated CRISPR/Cas12a system led to DNA hydrogel phase transitions to release AIE MOFzymes, triggering highly efficient fluorescent and colorimetric signal amplification. Importantly, the CRISPR-responsive AIE MOFzyme hydrogel platform for the detection of Salmonella enterica (S. enterica) showed a high sensitivity, which was superior to that of numerous existing methods. This work provided new insight for the design of multifunctional and programmable nanozyme hydrogels, which may also offer guidance for the development of novel CRISPR/Cas12a-based biosensors.},
}
@article {pmid40936456,
year = {2025},
author = {Huang, M and Shang, K and Ying, L and Han, Y and Hong, N and Yao, YF},
title = {CRISPR/Cas12-Driven Exponential Amplification Combined with a Lateral Flow Biosensor Enabling Rapid and Highly Sensitive DNA Detection.},
journal = {ACS sensors},
volume = {10},
number = {9},
pages = {6553-6563},
doi = {10.1021/acssensors.5c00944},
pmid = {40936456},
issn = {2379-3694},
mesh = {*Biosensing Techniques/methods ; Animals ; *CRISPR-Cas Systems ; Rabbits ; *Nucleic Acid Amplification Techniques/methods ; *DNA, Viral/analysis/genetics ; Humans ; Herpesvirus 1, Human/genetics/isolation &amp; purification ; Limit of Detection ; Point-of-Care Systems ; Tears/virology/chemistry ; *DNA/analysis ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {Rapid and precise detection of specific DNA is valuable for biological research and clinical disease diagnosis. Clustered regularly interspaced short palindromic repeat (CRISPR) technology can enhance existing DNA testing, making DNA detection faster, more portable, and more accurate. This study presents a new Cas12-driven exponential amplification-based lateral flow biosensor (CADEX-LF) for rapid and highly sensitive DNA detection. CADEX-LF takes full advantage of the highly specific target-loading-dependent trans-cleavage activity of Cas12 and the extremely high efficiency of nicking endonuclease-mediated exponential amplification. The adoption of lateral flow readout enables CADEX-LF for point-of-care (POC) use without requiring complicated supplementary equipment. CADEX-LF was shown to achieve a detection sensitivity of 2 × 10[-15] M within 45 min of measurement time and displayed outstanding specificity with double-base resolution. Furthermore, CADEX-LF could identify herpes simplex virus 1 (HSV-1) DNA in tears of rabbits and clinical patients with HSV-1 keratitis, exhibiting its practical application potential in clinical diagnosis. The proposed CADEX-LF biosensor may have great promise for point-of-care disease diagnosis in resource-limited environments.},
}
@article {pmid40921247,
year = {2025},
author = {Huerta, M&#xc1; and Codony, X and Ruiz-Cantero, MC and Porras, M and Tejada, M&#xc1; and Rickert-Llàcer, A and Artacho-Cordón, A and Zamanillo, D and Cobos, EJ and Nieto, FR},
title = {Generation and phenotypic characterization of a sigma-1 receptor knockout rat.},
journal = {Life sciences},
volume = {380},
number = {},
pages = {123953},
doi = {10.1016/j.lfs.2025.123953},
pmid = {40921247},
issn = {1879-0631},
mesh = {Animals ; *Receptors, sigma/genetics/metabolism ; Sigma-1 Receptor ; Rats ; Male ; Phenotype ; Gene Knockout Techniques ; Behavior, Animal ; Rats, Sprague-Dawley ; Neuralgia/genetics ; CRISPR-Cas Systems ; },
abstract = {The sigma-1 receptor (σ1R) is a chaperone involved in multiple physiological and pathological processes, including pain modulation, neuroprotection, and neurodegenerative diseases. Despite its functional significance, its precise roles remain unclear due to the lack of suitable models for detailed mechanistic studies. In this work, we describe the generation and phenotypic characterization of a novel σ1R knockout (σ1R KO) rat model. Using CRISPR/Cas9 technology, we introduced a specific 218-base-pair deletion into the σ1R gene, resulting in a complete loss of receptor expression, as confirmed by Western blot, immunohistochemistry, and binding assays. Comprehensive phenotypic analyses revealed no major developmental or behavioral abnormalities in σ1R KO rats under baseline conditions, suggesting that σ1R is not essential for development or survival. Additionally, no genotype-related differences were observed in cellular or biochemical blood parameters. Motor function tests (rotarod, grip strength, and wheel running) showed no deficits; however, σ1R KO rats displayed reduced exploratory behavior in actimetry and markedly diminished burrowing behavior. By contrast, no anxiodepressive-like behaviors were observed in the open field, startle, or forced swim tests. Sensory testing of naive rats revealed no significant genotype-related differences in responses to mechanical, heat, or cold stimuli, or in the formalin test (chemical-induced pain). However, σ1R KO rats displayed attenuated neuropathic pain after traumatic nerve injury (spared nerve injury), highlighting the role of σ1R in pain sensitization pathways. This study establishes the σ1R KO rat as a valuable tool for investigating σ1R-mediated mechanisms and for developing therapeutic strategies targeting σ1R for chronic pain, neurodegeneration, and psychiatric disorders.},
}
@article {pmid40676485,
year = {2025},
author = {Ran, F and Huang, H and Shang, B and Peng, W and Wu, L and Ling, K and Xie, X},
title = {An ultrasensitive sensing strategy based on CRISPR/Cas13a and T7 RNA polymerase amplification for detection of extracellular vesicles.},
journal = {Analytical sciences : the international journal of the Japan Society for Analytical Chemistry},
volume = {41},
number = {10},
pages = {1627-1636},
pmid = {40676485},
issn = {1348-2246},
support = {82272960//the National Science Foundation of China/ ; HBCH2024005//the Open Project of Hubei Clinical Research Center of Hypertension/ ; 24Y142//and the Science and Technology Key Program of Shiyan/ ; 24Y154//and the Science and Technology Key Program of Shiyan/ ; 24Y161//and the Science and Technology Key Program of Shiyan/ ; Y202522//the Young Talent Project of Affiliated Dongfeng Hospital/ ; Y202512//the Young Talent Project of Affiliated Dongfeng Hospital/ ; Y202518//the Young Talent Project of Affiliated Dongfeng Hospital/ ; },
mesh = {*Extracellular Vesicles/chemistry/metabolism ; *DNA-Directed RNA Polymerases/metabolism/genetics ; *CRISPR-Cas Systems/genetics ; Humans ; *Viral Proteins/metabolism/genetics ; *Nucleic Acid Amplification Techniques ; *Biosensing Techniques/methods ; Limit of Detection ; Aptamers, Nucleotide/chemistry ; },
abstract = {Extracellular vesicles (EVs) are important biomarkers for an early diagnosis of lung cancer. Herein, we proposed an ultrasensitive fluorescent sensing platform for EVs detection, which involves aptamer and streptavidin-modified magnetic nanoparticles (SA-MB) magnetic separation technology as well as T7 RNA polymerase-assisted CRISPR/Cas13a system, which can achieve target recycling signal amplification. In this detection method, biotin-modified CD63 aptamer hybridizes first with the aptamer Blocker (T7 promoter) and then binds to SA-MB. When adding EVs, the CD63 aptamer in CD63 aptamer/Blocker/SA-MB complex captures EVs causing the release of Blocker single chain. Subsequently, large amounts of ssRNAs, which are generated with the assistance of Blocker-initiated T7 RNA polymerase, were recognized by CRISPR/Cas13a and trigger its trans-cleavage report probe (F-Q). Eventually, the report probe labeled with fluorescent dye (FAM) and quench group (BHQ) at both ends was cut to produce fluorescent signal. The designed sensor combined this with a signal amplification strategy based on T7 RNA polymerase and CRISPR/Cas13a to significantly enhance the sensitivity and specificity of EVs detection. The use of magnetic separation technology eliminates interference from complex matrices and improves EVs detection efficiency, while the introduction of T7 RNA polymerase and CRISPR/Cas13a enables multiple amplifications of the sensor signals, and enhancing the accuracy and sensitivity of the method. Ultimately, the combination of multiple amplification reactions resulted in a detection limit (LOD) for EVs as low as 60 particles/mL (approximately 1 zmol/L). In addition, this detection method can specifically distinguish EVs from other confounding substances and efficiently detect plasma EVs from lung cancer and healthy individuals in actual samples. Indicating this sensing platform is a valuable tool for early lung cancer detection.},
}
@article {pmid40557518,
year = {2025},
author = {Li, K and Liu, C and Wan, G and Zhang, L and Fan, R and Zhang, X and Su, Y and He, J and Liu, N and Guan, F and Dong, W and Gao, S and Kong, W and Qi, X and Zhang, X and Ma, Y},
title = {Comparative analysis of two newly established Cre rat lines, NeuN-Cre and Thy1-Cre, for neurological research.},
journal = {Animal models and experimental medicine},
volume = {8},
number = {8},
pages = {1468-1479},
doi = {10.1002/ame2.70051},
pmid = {40557518},
issn = {2576-2095},
support = {Program CBYI202102//Research Project of China Baoyuan Investment Co., Ltd/ ; HH24KYZX0007//Haihe Laboratory of Cell Ecosystem Innovation Fund/ ; 2021-I2M-1-024//CAMS Innovation Fund for Medical Sciences/ ; 2021-I2M-1-034//CAMS Innovation Fund for Medical Sciences/ ; 2023-I2M-2-001//CAMS Innovation Fund for Medical Sciences/ ; 3332022040//Fundamental Research Funds for the Central Universities/ ; 3332023164//Fundamental Research Funds for the Central Universities/ ; 202411/WT_/Wellcome Trust/United Kingdom ; 2060204//State Key Laboratory Special Fund/ ; 2023-PT180-01.//the Non-profit Central Research Institute Fund of the Chinese Academy of Medical Sciences/ ; 202411/WT_/Wellcome Trust/United Kingdom ; },
mesh = {Animals ; Rats ; *Integrases/genetics/metabolism ; *Thy-1 Antigens/genetics/metabolism ; *Neurons/metabolism ; CRISPR-Cas Systems ; Male ; *Nerve Tissue Proteins/genetics ; Rats, Sprague-Dawley ; Rats, Transgenic ; },
abstract = {BACKGROUND: The Cre/loxP system is most popular in mice, but its application in rats has largely lagged far behind. The rat is vital laboratory animal, especially in toxicological and neurological studies. Generating genetic tools to manipulate neurons in rats could benefit neurological research.<br><br>METHODS: Using the CRISPR/Cas9 system, we inserted a Cre cassette into endogenous Thy1 and NeuN loci. Thy1-Cre rats featured a downstream P2A-linked insertion, while NeuN-Cre was inserted at the transcriptional start site. The Cre activity was assessed by crossing with a Cre reporter (Rosa26[imCherry]) rat and through analyzing mCherry expression patterns. The specificity of cell type was further confirmed by immunofluorescence with NeuN antibody. Phenotypic consequences were assessed by crossing with ND1[LSL] rats to deplete ND1, followed by monitoring weight/survival and conducting motor function tests.<br><br>RESULTS: We generated two neuron-specific rats (Thy1-Cre and NeuN-Cre), which exhibited high neuron-specific Cre expression in brain and spinal cord with minor leakage in other tissues. Thy1-Cre showed minor leakage in spleen, lung and kidney while NeuN-Cre showed minor leakage in spleen and kidney. ND1[Thy1-Cre] and ND1[NeuN-Cre] rats both showed decreased body weights and survival times. The ND1[NeuN-Cre] rats died within two weeks, while ND1[Thy1-Cre] rats lived longer with impaired motor function.<br><br>CONCLUSIONS: We successfully generated two neuron-specific NeuN-Cre and Thy1-Cre rats, and systemically analyzed their expression pattern.},
}
@article {pmid40175512,
year = {2025},
author = {Duan, M and Gao, P and Zhang, YZ and Hu, YL and Zhou, L and Xu, ZC and Qiu, HY and Tong, XH and Ji, RJ and Lei, XL and Yin, H and Guo, CL and Zhang, Y},
title = {TOPO-seq reveals DNA topology-induced off-target activity by Cas9 and base editors.},
journal = {Nature chemical biology},
volume = {21},
number = {10},
pages = {1554-1564},
pmid = {40175512},
issn = {1552-4469},
support = {82450105//National Natural Science Foundation of China (National Science Foundation of China)/ ; },
mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; *DNA/chemistry/genetics ; Humans ; RNA, Guide, CRISPR-Cas Systems/genetics ; *CRISPR-Associated Protein 9/metabolism/genetics ; High-Throughput Nucleotide Sequencing ; },
abstract = {With the increasing use of CRISPR-Cas9, detecting off-target events is essential for safety. Current methods primarily focus on guide RNA (gRNA) sequence mismatches, often overlooking the impact of DNA topology in regulating off-target activity. Here we present TOPO-seq, a high-throughput and sensitive method that identifies genome-wide off-target effects of Cas9 and base editors while accounting for DNA topology. TOPO-seq revealed that topology-induced off-target sites frequently harbor higher mismatches than the relaxed DNA sequence, with over 50% of off-target sites containing six mismatches, which are usually overlooked using previous methods. Applying TOPO-seq to three therapeutic gRNAs in hematopoietic stem cells identified 47 bona fide off-target loci, six of which are specifically induced by DNA topology. These findings highlight DNA topology as a regulator of off-target editing rates, establish TOPO-seq as a robust method for capturing DNA topology-induced off-target events and underscore its importance in off-target detection for developing safe genome-editing therapies.},
}
@article {pmid40995763,
year = {2025},
author = {Pramanik, D and Wang, K and Lee, K},
title = {CRISPR/Cas9-Mediated Gene Knockout in Cereal Crops.},
journal = {Current protocols},
volume = {5},
number = {9},
pages = {e70210},
doi = {10.1002/cpz1.70210},
pmid = {40995763},
issn = {2691-1299},
mesh = {*CRISPR-Cas Systems/genetics ; *Edible Grain/genetics ; *Gene Editing/methods ; *Crops, Agricultural/genetics ; *Gene Knockout Techniques/methods ; Plants, Genetically Modified/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; Genome, Plant ; },
abstract = {High-precision genome editing tools, such as programmable nucleases, are poised to transform crop breeding and significantly impact fundamental plant research. Among these tools, the CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)/Cas9 (CRISPR-associated 9) system is a programmable, RNA-guided nuclease that introduces targeted, site-specific double-stranded breaks in the target DNA loci. When these breaks are repaired, it often results in a frame-shift mutation via short insertion/deletion (indel), leading to gene knockout. Since its first successful use in plants, CRISPR/Cas9 has been widely adopted for targeting genes of agronomic and scientific importance in multiple crops, including rice, maize, wheat, and sorghum. These cereal crops ensure global food security, provide essential nutrition, and support economic stability. Additionally, such crops support biofuel production, livestock feed, and sustainable farming practices through crop rotation. This article outlines the strategies for implementing CRISPR/Cas9 genome editing in plants, including a step-by-step process of guide RNA target selection, oligonucleotide design, construct development, assembly, and analysis of genome edits. © 2025 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: CRISPR/Cas9 guide RNA target selection Support Protocol 1: Genomic DNA extraction in-house protocol Basic Protocol 2: Construction of a binary plasmid vector Support Protocol 2: Agrobacterium transformation with a binary vector construct and stability check Support Protocol 3: Plant transformation Basic Protocol 3: Genotyping of edited events.},
}
@article {pmid40994338,
year = {2025},
author = {Zhang, T and Wang, Z and Song, Y and Wang, J and Guo, F and Zhang, Y and Lu, F and Li, M},
title = {[Establishment and optimization of a genetic manipulation system for Staphylococcus pasteuri].},
journal = {Sheng wu gong cheng xue bao = Chinese journal of biotechnology},
volume = {41},
number = {9},
pages = {3604-3616},
doi = {10.13345/j.cjb.250167},
pmid = {40994338},
issn = {1872-2075},
mesh = {*Staphylococcus/genetics/metabolism/drug effects ; *Gene Editing/methods ; Electroporation/methods ; Plasmids/genetics ; CRISPR-Cas Systems ; Genetic Engineering/methods ; },
abstract = {One of the technical bottlenecks limiting the high yield of 1,4-butanediamine is the insufficient tolerance of strains to 1,4-butanediamine. Enhancing the tolerance of strains to 1,4-butanediamine is therefore a primary challenge that needs to be addressed for the construction of strains with high yields of 1,4-butanediamine. Staphylococcus pasteuri 326180 exhibits exceptional tolerance to high-concentration 1,4-butanediamine, serving as both an ideal model for studying the mechanism underlying the 1,4-butanediamine tolerance and a novel host for constructing strains capable of efficiently producing 1,4-butanediamine. However, for both the research on the tolerance mechanism and the modification of chassis strains, gene editing of S. pasteuri needs to be carried out at the molecular level. The research objective of this paper is to establish a genetic manipulation system for S. pasteuri, laying foundation for subsequent studies on tolerance mechanism and the modification of chassis strains. This study systematically optimized the electroporation conditions, including key parameters such as the growth phase of cells, electric field strength, electroporation buffer, and recovery medium, successfully establishing an electroporation method for S. pasteuri. Additionally, we constructed the gene editing plasmid pCpfOA by replacing the resistance expression cassette, optimized the selection markers for gene editing, and finally established a CRISPR/Cpf1-based gene editing technology for S. pasteuri, achieving an editing efficiency of 90%. The genetic manipulation system of S. pasteuri established in this study provides technical support for research into the tolerance mechanism of this bacterium and the genetic modification of chassis strains.},
}
@article {pmid40994249,
year = {2025},
author = {Zaheer, U and Munir, F and Qiao, Q and Salum, YM and Abbas, AN and Tariq, M and Huang, S and Zheng, C and Yang, G and He, W},
title = {Functional Role of the PxGRHPR2 Gene in the Host Plant Adaptation of Diamondback Moth (Plutella xylostella).},
journal = {Archives of insect biochemistry and physiology},
volume = {120},
number = {1},
pages = {e70100},
doi = {10.1002/arch.70100},
pmid = {40994249},
issn = {1520-6327},
support = {//This study was supported by the National Natural Science Foundation of China (32472659 and 32172503), Natural Science Foundation of Fujian Province in China (2023J01069), Open Research Project of the Fujian Key Laboratory for Monitoring and Integrated Management of Crop Pests (MIMCP-202402), and Innovation Fund of FAFU (KFB23014A)./ ; },
mesh = {Animals ; *Moths/genetics/growth &amp; development/physiology/enzymology ; Larva/genetics/growth &amp; development/physiology ; *Insect Proteins/genetics/metabolism ; CRISPR-Cas Systems ; Raphanus ; Adaptation, Physiological/genetics ; },
abstract = {The diamondback moth (Plutella xylostella), a major lepidopteran pest with a wide host range, presents persistent challenges to sustainable agriculture due to its high adaptability to cruciferous host plants. Although glyoxylate/hydroxypyruvate reductases (GRHPRs) have been well-characterized in plants and humans, their functional role in insects, particularly in host plant adaptation, remains largely unexplored. In this study, we characterized PxGRHPR2, a member of the GRHPR gene family, using a bioinformatics analysis, expression profiling, and CRISPR/Cas9-mediated gene knockout. RT-qPCR analysis showed that PxGRHPR2 was predominantly expressed in larval stage, with the highest transcript levels observed in the second instar and larval midgut tissues. Three homozygous PxGRHPR2 knockout strains were successfully generated using CRISPR/Cas9 system. Mutation of PxGRHPR2 led to significant reductions in larval weight, survival, and eclosion rates when larvae were fed on radish seedlings, whereas no such effects were observed under artificial diet conditions. These findings suggest that PxGRHPR2 plays a critical role in detoxification and metabolic regulation, thereby facilitating host plant adaptability in P. xylostella. Overall, this study provides new insights into insect-plant interactions and identifies PxGRHPR2 as a potential molecular target for developing sustainable pest management strategies.},
}
@article {pmid40994005,
year = {2025},
author = {Wang, S and Hu, Z},
title = {The marine diatom Phaeodactylum tricornutum as a versatile bioproduction chassis: Current progress, challenges and perspectives.},
journal = {Plant communications},
volume = {},
number = {},
pages = {101519},
doi = {10.1016/j.xplc.2025.101519},
pmid = {40994005},
issn = {2590-3462},
abstract = {Beyond its importance in diatom studies, the marine model diatom Phaeodactylum tricornutum has emerged as a versatile photosynthetic chassis for sustainable bioproduction, leveraging both native bioactive metabolites and engineered heterologous compounds through synthetic biology. Over the past three decades, transformative advances in genetic tool development, including transgenic element optimization, CRISPR/Cas genome editing and high-efficiency transformation systems, have driven strain engineering for elevated fucoxanthin, fatty acid, triacylglycerol yields and successful synthesis of diverse heterologous products, from terpenoids and therapeutic peptides to sustainable materials. Concurrently, advances in molecular toolkits have refined chassis optimization by elucidating fundamental biological mechanisms underlying nutrient uptake, environmental stress adaptation, stimuli sensing and cell development. Despite the progress, critical challenges persist, particularly suboptimal product yield, biomass limitations as well as a prohibitive production cost which hinder industrial translation. This review examines emerging strategies, such as chloroplastic gene expression, DNA site-specific integration and trophic alteration, promising for species improvement, while addressing other scale-up considerations including cultivation strategies, techno-economic analysis and regulatory policies. The integrative efforts could accelerate the transition of P. tricornutum from a model diatom to a scalable, eco-friendly biomanufacturing platform.},
}
@article {pmid40992924,
year = {2025},
author = {Warshauer, EM and Maier, PA and Runfeldt, G and Fuentes, I and Escamez, MJ and Valinotto, L and Natale, M and Manzur, G and Illera, N and Garcia, M and Del Rio, M and Mencia, A and Holguin, A and Larcher, F and Hellenthal, G and Brown, AR and Consuegra, L and Rivera, C and Nogueiro, I and Tang, J and Oro, A and Marinkovich, P and Palisson, F and Titeux, M and Hovnanian, AA and Sprecher, E and Skorecki, K and Norris, D and Bruckner, A and Kogut, I and Bilousova, G and Roop, D},
title = {Sephardic origins revealed for rare skin disorder, recessive dystrophic epidermolysis bullosa, in individuals carrying the unique c.6527insC mutation.},
journal = {Journal of medical genetics},
volume = {},
number = {},
pages = {},
doi = {10.1136/jmg-2025-110967},
pmid = {40992924},
issn = {1468-6244},
abstract = {BACKGROUND: Recessive dystrophic epidermolysis bullosa (RDEB) is a rare and severe blistering skin disorder caused by loss-of-function mutations in the type VII collagen gene (COL7A1). The COL7A1 c.6527insC mutation is curiously prevalent among individuals with RDEB and is found worldwide in Europe and the Americas. Previous research has suggested the possibility of a Sephardic Jewish origin of the mutation; however, individuals with RDEB are not known to have predominant Jewish ancestry.<br><br>METHODS: In this study, a global cohort of individuals with RDEB with the c.6527insC founder mutation from Spain, France, Argentina, Chile, Colombia and the USA were investigated by autosomal genotyping, pairwise identical-by-descent matching and a local ancestry analysis. Age estimation analysis was performed to determine when Jewish founders introduced the c.6527insC mutation into Iberian and Native American populations (~900&#x2009;CE and 1492 CE, respectively).<br><br>RESULTS: Sephardic ancestry was identified at the haplotype spanning the c.6527insC mutation in 85% of the individuals, despite mixed ancestry elsewhere in the genome and no known recent Sephardic ancestry. Identical-by-descent matching between this RDEB subpopulation and a known crypto-Jewish community in Belmonte, Portugal was also ascertained, providing support for crypto-Jewish ancestry in this RDEB subpopulation.<br><br>CONCLUSION: The identification of this unique RDEB subpopulation unified by the single most prevalent c.6527insC mutation holds great potential to facilitate promising new RDEB therapies using CRISPR Cas 9 gene and base editing. The identification of a single guide RNA allowing efficient and safe editing of this variant would represent a unique drug to treat a large cohort of patients with the same founder mutation.},
}
@article {pmid40991439,
year = {2025},
author = {Yang, W and Wang, S and Ji, S and Wang, J and Lian, S and Li, Z and Jansen, RA and Wu, W and Niu, K and Sun, Z and Jia, Q and Zheng, J and Zhu, H and Deng, X and Wang, L and Fan, Z and Shi, Y and Lieftink, C and Guan, M and Beijersbergen, RL and Qin, W and Gao, Q and Bernards, R and Jin, H},
title = {CRISPR screens identify the ATPase VCP as a druggable therapeutic vulnerability in cholangiocarcinoma.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {122},
number = {39},
pages = {e2519568122},
doi = {10.1073/pnas.2519568122},
pmid = {40991439},
issn = {1091-6490},
support = {W2411079//MOST | National Natural Science Foundation of China (NSFC)/ ; 82222047//MOST | National Natural Science Foundation of China (NSFC)/ ; 82403226//MOST | National Natural Science Foundation of China (NSFC)/ ; 82303081//MOST | National Natural Science Foundation of China (NSFC)/ ; 22XD1423100//Science and Technology Commission of Shanghai Municipality (STCSM)/ ; 23YF1443500//Science and Technology Commission of Shanghai Municipality (STCSM)/ ; 2022XD057//Shanghai Municipal Health Commission ()/ ; 12539//KWF Kankerbestrijding (DCS)/ ; },
mesh = {Humans ; *Valosin Containing Protein/genetics/antagonists &amp; inhibitors/metabolism ; *Cholangiocarcinoma/drug therapy/genetics/pathology/metabolism ; Animals ; CRISPR-Cas Systems ; Mice ; *Bile Duct Neoplasms/drug therapy/genetics/pathology/metabolism ; Cellular Senescence/drug effects ; Cell Line, Tumor ; Cell Proliferation/drug effects ; Xenograft Model Antitumor Assays ; Antineoplastic Agents/pharmacology ; },
abstract = {Cholangiocarcinoma (CCA) remains a lethal malignancy with limited therapeutic options. Through genome-wide CRISPR-Cas9 screening, we identified the adenosine triphosphatase (ATPase) valosin-containing protein (VCP) as a critical dependency in CCA. Compound screens revealed that the VCP inhibitor CB-5339 potently suppresses CCA proliferation in a panel of patient-derived organoids by inducing cellular senescence. It is known that senescent cells persist, and this can contribute to therapy resistance. To address this, we combined CB-5339 with senolytic agents (ABT-263 and conatumumab), which selectively eliminate senescent CCA cells, resulting in enhanced tumor suppression both in vitro and in vivo. Clinical analysis showed that VCP overexpression in CCA patients correlates with poor prognosis. Our study unveils a "one-two punch" strategy, targeting VCP-mediated senescence followed by senolytic clearance, offering a promising therapeutic approach for CCA.},
}
@article {pmid40991036,
year = {2025},
author = {Nikam, T and Rana, A and Saraf, SA and Awasthi, S},
title = {Micro- and nanoscale biosensing technologies for early diagnosis of Parkinson's disease.},
journal = {Mikrochimica acta},
volume = {192},
number = {10},
pages = {691},
pmid = {40991036},
issn = {1436-5073},
support = {BT/RLF/Re-Entry/40/2021//Department of Biotechnology, Ministry of Science and Technology, India/ ; },
mesh = {*Parkinson Disease/diagnosis ; Humans ; *Biosensing Techniques/methods ; Early Diagnosis ; Biomarkers/analysis ; Electrochemical Techniques ; },
abstract = {Parkinson's disease (PD), the second most prevalent neurodegenerative disorder, remains challenging to diagnose at its earliest stages due to the absence of definitive biomarkers and overlapping clinical features with other synucleinopathies, thereby delaying therapeutic intervention and effective disease management. This review provides an integrative evaluation of established and emerging approaches for detecting PD-specific biomarkers in biofluids and tissues with high sensitivity and specificity. Conventional assays such as seed amplification techniques, proximity ligation and extension methods, bead-based microarrays, and immunoassays including ELISA, electrochemiluminescence, and SIMOA are examined alongside their performance metrics and inherent limitations. We then highlight next-generation micro- and nanoscale biosensing platforms, including nanopore-based resistive pulse sensing, surface plasmon resonance (SPR), surface-enhanced Raman spectroscopy (SERS), field-effect transistors (FETs), electrochemical sensors, and lateral flow assays (LFAs), which are capable of ultrasensitive detection at nano- to attomolar concentrations. Particular emphasis is given to nucleic acid-based technologies such as aptasensors, genosensors, and CRISPR/Cas systems for their exceptional molecular recognition, programmable signal outputs, and portability. The potential of artificial intelligence and machine learning tools (e.g., SVM, RF, DNN) to improve biomarker interpretation, enable multiplexed analysis, and facilitate real-time monitoring is also discussed. Finally, we outline key translational challenges, including assay standardization, clinical validation, scalability, integration into wearable and point-of-care devices, and regulatory hurdles towards the development of robust, clinically deployable diagnostic platforms for early PD detection and monitoring.},
}
@article {pmid40988386,
year = {2025},
author = {Zuo, N and Zuo, F and Liu, Y and Xiang, B},
title = {Genome Editing Using the Endogenous Type I-E CRISPR-Cas System in Lactobacillus paracasei ATCC334.},
journal = {Biotechnology and applied biochemistry},
volume = {},
number = {},
pages = {},
doi = {10.1002/bab.70056},
pmid = {40988386},
issn = {1470-8744},
abstract = {Lactobacillus paracasei ATCC334 is a well-known beneficial strain that plays a crucial role in food industry and promotion of human health. However, despite its significance, our understanding of its gene functions remains limited due to obstacles in gene editing techniques. This gap hinders the full utilization and development of this beneficial bacterium. In this study, we targeted L. paracasei ATCC334 as editing chassis. Initially, bioinformatics tools were used to explore a type I-E endogenous clustered regularly interspaced short palindromic repeats (CRISPR)-Cas system within L. paracasei ATCC334. We further analyzed its repeat sequences, spacer sequences, and leader sequence predicted the protospacer adjacent motif (PAM) recognized by this system. To validate our findings, we assessed the accuracy of potential PAM, evaluated the cutting activity of the endogenous CRISPR-Cas system, and studied the impact of the artificial mini-CRISPR array through plasmid interference and genome interference experiments. These results helped us to achieve successful gene knockout and gene integration. Finally, we engineered a strain capable of nicotine degradation. Our study provides valuable insights for the broader development and application of lactobacilli.},
}
@article {pmid40988083,
year = {2025},
author = {Zhu, R and Ren, C and Bao, Z},
title = {Fueling chromosomal gene diversification and artificial evolution with CRISPR.},
journal = {Genome biology},
volume = {26},
number = {1},
pages = {297},
pmid = {40988083},
issn = {1474-760X},
support = {2023YFF1204500//National Key Research and Development Program of China/ ; 22308316//National Natural Science Foundation of China/ ; 226-2022-00214//Fundamental Research Funds for the Central Universities/ ; },
mesh = {*CRISPR-Cas Systems ; *Directed Molecular Evolution/methods ; *Gene Editing/methods ; *Chromosomes/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Humans ; *Genetic Variation ; Animals ; Evolution, Molecular ; },
abstract = {Gene diversification is an effective approach to massively dissecting variant functions and evolving sequences when paired with an appropriate assay. In vitro mutagenesis and ectopic gene expression, however, fail to simulate the endogenous regulatory environment of the variants. The development of clustered, regularly interspaced short palindromic repeats (CRISPR) systems has greatly boosted the efficiency of targeted gene diversification in various species. Here, we review recent CRISPR-assisted methods for chromosomal gene diversification and artificial evolution, focusing on the advantages and limitations of each approach, and propose possible strategies to overcome current limitations and directions in future technology development.},
}
@article {pmid40986369,
year = {2025},
author = {Ratna, TA and Sharon, BM and Barros Velin, CA and Palmer, K},
title = {Factors affecting CRISPR-Cas defense against antibiotic resistance plasmids harboured by Enterococcus faecalis laboratory model strains and clinical isolates.},
journal = {Microbiology (Reading, England)},
volume = {171},
number = {9},
pages = {},
doi = {10.1099/mic.0.001601},
pmid = {40986369},
issn = {1465-2080},
mesh = {*Enterococcus faecalis/genetics/drug effects/isolation &amp; purification ; *Plasmids/genetics ; *CRISPR-Cas Systems/genetics ; Gene Transfer, Horizontal ; Conjugation, Genetic ; Humans ; Anti-Bacterial Agents/pharmacology ; *Drug Resistance, Multiple, Bacterial/genetics ; Gram-Positive Bacterial Infections/microbiology ; },
abstract = {Enterococcus faecalis is a Gram-positive bacterium and opportunistic pathogen that acquires resistance to a wide range of antibiotics by horizontal gene transfer (HGT). The rapid increase of multidrug-resistant (MDR) bacteria including MDR E. faecalis necessitates the development of alternative therapies and a deeper understanding of the factors that impact HGT. CRISPR-Cas systems provide sequence-specific defense against HGT. From previous studies, we know that E. faecalis CRISPR-Cas provides sequence-specific anti-plasmid defense during agar plate biofilm mating and in the murine intestine. Those studies were mainly conducted using laboratory model strains with a single, CRISPR-targeted plasmid in the donor. MDR E. faecalis typically possess multiple plasmids that are diverse in sequence and may interact with each other to impact plasmid transfer and CRISPR-Cas efficacy. Here, we altered multiple parameters of our standard in vitro conjugation assays to assess CRISPR-Cas efficacy, including the number and genotype of plasmids in the donor, and laboratory model strains as donor versus recent human isolates as donor during conjugation. We found that the plasmids pTEF2 and pCF10, which are not targeted by CRISPR-Cas in our recipient, enhance the conjugative transfer of the CRISPR-targeted plasmid pTEF1 into both WT and CRISPR-Cas-deficient (via deletion of cas9) recipient cells. However, the effect of pTEF2 on pTEF1 transfer is much more pronounced, with a striking 6-log increase in pTEF1 conjugation frequency when pTEF2 is also present in the donor and recipients are deficient for CRISPR-Cas (compared with 4-log for pCF10). Overall, this study provides insight about the interplay between plasmids and CRISPR-Cas defence, opening avenues for developing novel therapeutic strategies to curb HGT among bacterial pathogens and highlighting pTEF2 as a plasmid for additional mechanistic study.},
}
@article {pmid40986360,
year = {2025},
author = {Ren, J and Duan, Y and Li, R and Zhang, X and Shi, Y and Zhou, S and Xie, K and Wu, X and Irish, VF and Deng, X and Zhang, F and Guo, W},
title = {Transcriptional regulation of thorn tip sclerification in plants.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {122},
number = {39},
pages = {e2510775122},
doi = {10.1073/pnas.2510775122},
pmid = {40986360},
issn = {1091-6490},
support = {2022YFF1003100//MOST | National Key Research and Development Program of China (NKPs)/ ; 2024YFD1200501//MOST | National Key Research and Development Program of China (NKPs)/ ; 32425048//MOST | NSFC | National Science Fund for Distinguished Young Scholars (National Science Foundation for Distinguished Young Scholars)/ ; HBZY2023B00501//Department of Agriculture &amp; Rural Affairs of Hubei Province/ ; CARS-26//MOA | Earmarked Fund for China Agriculture Research System/ ; 2306142//National Science Foundation (NSF)/ ; },
mesh = {*Gene Expression Regulation, Plant ; *Plant Proteins/genetics/metabolism ; *Citrus/genetics/growth &amp; development/metabolism ; CRISPR-Cas Systems ; Gene Editing ; Transcription Factors/genetics/metabolism ; Cell Wall/metabolism/genetics ; Plant Stems/genetics/growth &amp; development ; },
abstract = {A common characteristic of thorns, prickles, and spines is a hardened and sharp tip. This feature complicates cultivation and postharvest processing for many crops, yet the molecular mechanisms governing this specific sclerification process remain unclear. By genome editing screening, we identified a MYB family gene named SHORT and SOFT THORN 1 (SST1) that specifically promotes the development of sclerenchyma cells at Citrus thorn tips. CRISPR-Cas9 editing of SST1 results in the formation of short and soft thorns with significantly reduced secondary wall thickening at the tips, while leaving stem growth unaffected. SST1 directly activates NST1 and SND1, driving a hierarchical transcriptional regulatory network for secondary wall biosynthesis. Moreover, SST1 homologs in various species are specifically expressed in thorns, suggesting a potential conserved role. Our results uncover the molecular basis for hard plant protective structures, and modulation of this pathway could be an effective strategy for optimizing cultivation practices.},
}
@article {pmid40986203,
year = {2025},
author = {Sood, A and Sharma, P and Sharma, A and Chaudhary, DR and Manisha, },
title = {Genome editing in vegetable crops: a new era of sustainable agriculture.},
journal = {Molecular biology reports},
volume = {52},
number = {1},
pages = {944},
pmid = {40986203},
issn = {1573-4978},
}
@article {pmid40985903,
year = {2025},
author = {Palma-Cobo, M and Toribio, V and Morales, J and López-Martín, S and Enrich, C and Lu, A and Yáñez-Mó, M},
title = {Genome-Wide CRISPR/Cas9 Screening Identifies the COMMANDER Recycling Complex as a Key Player in EV Uptake.},
journal = {Journal of extracellular vesicles},
volume = {14},
number = {9},
pages = {e70166},
pmid = {40985903},
issn = {2001-3078},
support = {//Ministerio Espa&#xf1;ol de Ciencia e Innovaci&#xf3;n/ ; //Universidad Aut&#xf3;noma de Madrid/ ; PIF2023/SAL-GL-29726//FPI-UAM/ ; //GEIVEX/ ; //Comunidad de Madrid/ ; //PIPF-2023/SAL-GL- 29726/ ; },
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; K562 Cells ; *Extracellular Vesicles/metabolism/genetics ; HeLa Cells ; Cell Line, Tumor ; Genome, Human ; Melanoma/metabolism ; },
abstract = {Extracellular vesicles (EVs) hold immense potential in therapeutic delivery, warranting a comprehensive investigation of the mechanisms that regulate their uptake by target cells. To identify key molecular regulators of EV internalization, we conducted a genome-wide CRISPR (GWC) screen aimed to pinpoint candidate genes that influence EV uptake. We employed a GWC library spanning the entire human genome in K562 cells. 3.6 × 10[12] EVs isolated from the SKMEL147 human melanoma cell line were labelled with Alexa633-C5-Maleimide and incubated for 2 h with 500 × 10[6] K562 cells, providing a 2000× coverage of the library. The top 5% of high and low fluorescence populations were sorted. Next-generation sequencing (NGS) was performed to quantify sgRNA enrichment in the sorted populations compared to the unsorted control. Remarkably, among other genes, several members of the COMMANDER complex emerged as significant hits in our screen. We validated the hits in knockout (KO) cell lines of both K562 and HeLa cells using EVs derived either from melanoma or breast cancer cell lines. Kinetic follow-up of EV cargo, including surface or luminal proteins, suggests that the COMMANDER complex plays a pivotal role in the early stages of EV uptake but also in the final fate of EV components in the target cell.},
}
@article {pmid40938694,
year = {2025},
author = {Wei, Z and Zhang, L and Wang, Y and Xu, X and Cao, L and Lin, H and Sui, J and Wang, K and Wang, X},
title = {Development of a Label-Free Colorimetric and Fluorescent Diagnostic Platform for Foodborne Salmonella Based on RPA-CRISPR/Cas12 Assay in a Single Tube.},
journal = {Journal of agricultural and food chemistry},
volume = {73},
number = {38},
pages = {24447-24456},
doi = {10.1021/acs.jafc.5c06045},
pmid = {40938694},
issn = {1520-5118},
mesh = {*Salmonella/genetics/isolation &amp; purification ; *Colorimetry/methods/instrumentation ; CRISPR-Cas Systems ; Food Contamination/analysis ; Milk/microbiology ; Animals ; *Nucleic Acid Amplification Techniques/methods/instrumentation ; Fishes/microbiology ; Bacterial Proteins/genetics/metabolism ; Food Microbiology ; *Endodeoxyribonucleases/genetics/metabolism ; Recombinases/genetics/metabolism/chemistry ; Fluorescence ; CRISPR-Associated Proteins ; },
abstract = {Foodborne pathogen Salmonella poses a significant threat to public health, and therefore, it is important to establish accurate, convenient, and rapid detection methods. Herein, a label-free colorimetric and fluorescent diagnostic platform for foodborne Salmonella was developed, integrating recombinase polymerase amplification (RPA), CRISPR/Cas12, and water-soluble cationic conjugated polythiophene (PMNT) in a single-tube system. Upon recognition of Salmonella-specific gene invA targets, RPA products can stimulate the cis- and trans-cleavage activity of Cas12a in the presence of crRNA. This enzymatic activity degrades single-stranded DNA (ssDNA), leading to the release of PMNT from PMNT-ssDNA complexes, which, in turn, produces a detectable fluorescence increase along with a visible color transition from pink to yellow. The one-tube strategy enables sensitive detection of 1.9 × 10[1] copies/μL invA target and could detect as low as 10[3] CFU/mL of Salmonella in artificially spiked milk and fish samples without enrichment, while the detection limit improved to 10[0] CFU/mL after 8 h enrichment. Importantly, the assay demonstrated high specificity with no cross-reactivity with other bacteria with and without complex food matrices. This one-tube, dual-signal assay provides a rapid, reliable, and equipment-minimal solution for on-site detection of Salmonella, with a reduced risk of aerosol contamination.},
}
@article {pmid40985777,
year = {2025},
author = {Nguyen, GT and Raju, A and Schelling, MA and Sashital, DG},
title = {Rapid CRISPR-Cas9 target-strand nicking can provide phage resistance by reducing DNA abundance.},
journal = {Nucleic acids research},
volume = {53},
number = {18},
pages = {},
doi = {10.1093/nar/gkaf900},
pmid = {40985777},
issn = {1362-4962},
support = {1652661//National Science Foundation/ ; GM140876/NH/NIH HHS/United States ; },
mesh = {*CRISPR-Cas Systems ; Magnesium/metabolism ; *Bacteriophages/genetics ; *CRISPR-Associated Protein 9/metabolism/genetics/chemistry ; *DNA, Viral/metabolism/genetics ; DNA Breaks, Double-Stranded ; },
abstract = {Cas9 is an RNA-guided immune endonuclease that provides bacterial defense against bacteriophages. Cas9 relies on divalent metal ions for cleavage catalysis by two domains, HNH and RuvC, and to facilitate conformational changes that are required for cleavage activation. While Cas9 typically produces double-strand breaks (DSBs) in DNA targets, we observed that reduced, physiologically relevant Mg2+ concentrations can result in a slow rate of non-target strand cleavage by RuvC. This raised the question of whether rapid target-strand nicking by the Cas9 HNH domain is sufficient to provide protection against phage. To address this, we tested phage protection by Cas9 nickases, in which only the HNH or RuvC domain is catalytically active. We find that nicking by HNH, but not RuvC, can be sufficient to provide immunity. Target-strand nicking prevents phage DNA accumulation and can reduce the susceptibility of Cas9 to viral escape. Cleavage by RuvC is strongly impaired in the presence of other biomolecules that can compete for binding of free Mg2+, preventing formation of a DSB. Overall, our results suggest that HNH cleavage may occur more rapidly than RuvC cleavage under physiological conditions, resulting in an initial target-strand nick that may be sufficient to provide CRISPR-mediated immunity.},
}
@article {pmid40985776,
year = {2025},
author = {Park, SG and Park, JU and Dodero-Rojas, E and Bryant, JA and Sankaranarayanan, G and Kellogg, EH},
title = {Comprehensive profiling of activity and specificity of RNA-guided transposons reveals opportunities to engineer improved variants.},
journal = {Nucleic acids research},
volume = {53},
number = {18},
pages = {},
doi = {10.1093/nar/gkaf917},
pmid = {40985776},
issn = {1362-4962},
support = {//NIH/ ; 5R01GM144566-02/GM/NIGMS NIH HHS/United States ; //Cystic Fibrosis Foundation/ ; //Jane Coffin Childs Memorial Fund/ ; //Korea Health Industry Development Institute/ ; HI19C1095//Ministry of Health and Welfare/ ; //Hartwell Center for Bioinformatics &amp; Biotechnology/ ; //ALSAC/ ; P30 CA021765/CA/NCI NIH HHS/United States ; },
mesh = {*DNA Transposable Elements/genetics ; CRISPR-Cas Systems ; *RNA, Guide, CRISPR-Cas Systems/genetics ; *Genetic Engineering/methods ; Mutation ; Gene Editing/methods ; },
abstract = {Recently discovered CRISPR-associated transposons (CASTs) are natural RNA-guided DNA transposition systems capable of single-step genomic integration of large DNA cargo. Wild-type CASTs exhibit low integration activity in heterologous systems; therefore, engineering efforts are required to develop therapeutically relevant tools. Here we developed a high-throughput dual genetic screen capable of accurately quantifying the relative activity and specificity of a large pool of CAST variants. Under the conditions of our screen, we discovered that the wild-type V-K CAST system can consistently achieve between 88% and 95% on-site targeting specificity. We used site-saturation mutagenesis of the conserved core transposition machinery (TnsB, TnsC, and TniQ) to reveal novel mechanistic insights into the function of these transposon proteins. Furthermore, we found that different components have varying trade-offs between activity and specificity, a critical aspect overlooked in conventional screening pipelines. These findings provide clear engineering principles for further optimization of CASTs. Finally, we identified several mutations that, together, enhance CAST activity up to four-fold while minimally impacting targeting specificity. These methods are a powerful tool to characterize the sequence-function landscape across multiple functional parameters while also providing a robust platform for developing enhanced genome-editing tools.},
}
@article {pmid40985758,
year = {2025},
author = {Kippnich, J and Benz, F and Uecker, H and Baumdicker, F},
title = {Effectiveness of CRISPR-Cas in Sensitizing Bacterial Populations with Plasmid-Encoded Antimicrobial Resistance.},
journal = {Genetics},
volume = {},
number = {},
pages = {},
doi = {10.1093/genetics/iyaf192},
pmid = {40985758},
issn = {1943-2631},
abstract = {The spread of bacteria resistant to antibiotics poses a serious threat to human health. Genes that encode antibiotic resistance are often harbored on plasmids, extra-chromosomal DNA molecules found in bacteria. The emergence of multiresistance plasmids is particularly problematic and demands the development of new antibiotics and alternative strategies. CRISPR-Cas derived tools with their sequence specificity offer a promising new approach to combating antibiotic resistance. By introducing CRISPR-Cas encoding plasmids that %specifically target antibiotic resistance genes on plasmids, the susceptibility of bacteria to conventional antibiotics can be restored. However, genetic variation within bacterial populations can hinder the effectiveness of such CRISPR-Cas tools by allowing some mutant plasmids to evade CRISPR-mediated cleaving or gene silencing. In this study, we develop a model to test the effectiveness of CRISPR-Cas in sensitizing bacterial populations carrying resistance on non-transmissible plasmids and assess the success probability of a subsequent treatment with conventional antibiotics. We evaluate this probability according to the target interference mechanism, the copy number of the resistance-encoding plasmid, and its compatibility with the CRISPR-Cas encoding plasmid. Our results identify promising approaches to revert antibiotic resistance with CRISPR-Cas encoding plasmids: A DNA-cleaving CRISPR-Cas system on a plasmid incompatible with the targeted plasmid is most effective for low copy numbers, while for resistance plasmids with higher copy numbers gene silencing by CRISPR-Cas systems encoded on compatible plasmids is the superior solution.},
}
@article {pmid40983443,
year = {2025},
author = {Belizário, JE and Occhiucci, JM and Garay-Malpartida, M and Cunha da Silva, JR},
title = {Phageptosis: A bacterial cell death program induced by crispr-cas systems.},
journal = {Progress in molecular biology and translational science},
volume = {217},
number = {},
pages = {211-231},
doi = {10.1016/bs.pmbts.2025.07.003},
pmid = {40983443},
issn = {1878-0814},
mesh = {*CRISPR-Cas Systems/genetics ; *Bacteriophages/physiology ; *Bacteria/virology/cytology/genetics ; Cell Death ; Apoptosis ; Humans ; },
abstract = {Programmed cell death (PCD) is a fundamental mechanism that has evolved across both unicellular and multicellular organisms for species preservation and self-protection. In certain contexts, genetically regulated cell death can enable surviving cells to thrive, safeguarding the genotype from extinction. Recent research on bacteria and archaea has revealed an ancient defense mechanism involving CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) and Cas (CRISPR-associated) proteins. These systems identify and eliminate invading genetic elements, such as bacteriophages, transposons, and plasmids, using sequence-specific RNA-guided targeting. A protease complex called Craspase, activated by non-self RNA, regulates Cas nuclease activity, facilitating this primitive form of immunity. Interestingly, this pathway shows structural and mechanistic similarities to apoptosis, the first recognized form of programmed mammalian cell death, characterized by chromatin condensation, nuclear fragmentation, and membrane blebbing. Other regulated cell death pathways, including necroptosis and pyroptosis, also share overlapping features. Comparative genomic studies reveal a conserved molecular framework underpinning these diverse death pathways across life forms. In this article, we explore the emerging parallels and distinctions between apoptosis and CRISPR-Cas-mediated cell death, a process we refer to as "phageptosis," highlighting evolutionary links and their implications for understanding cell death mechanisms.},
}
@article {pmid40983435,
year = {2025},
author = {Zhang, H and Bai, X and Wang, C and Pang, W and Zhang, J and Song, J and Zhou, H and Gao, Z and Zheng, B},
title = {Bimetallic-satellite enhanced SERS reporter integrated with CRISPR-Cas12a for ultrasensitive biological rhythm monitoring.},
journal = {Analytica chimica acta},
volume = {1374},
number = {},
pages = {344544},
doi = {10.1016/j.aca.2025.344544},
pmid = {40983435},
issn = {1873-4324},
mesh = {*CRISPR-Cas Systems ; *Spectrum Analysis, Raman/methods ; Gold/chemistry ; *Biosensing Techniques/methods ; *Metal Nanoparticles/chemistry ; Silver/chemistry ; Humans ; RNA, Messenger/analysis/genetics ; Limit of Detection ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {BACKGROUND: Biorhythmic played an important role in physical health. Biorhythmic disorders led to various health issue, including cardiovascular diseases and metabolic disorders. Early diagnosis of biorhythmic disorders was crucial in slowing the progression of disease. Therefore, it was important to develop technologies for detecting biorhythmic and their disorder markers. This study selected HMGB1(High mobility group box 1), BMAL1 (Brain and Muscle Aryl hydrocarbon receptor nuclear translocator-like 1), and MICU (mitochondrial calcium uptake 1) mRNA for detection.<br><br>RESULTS: CRISPR/Cas12a/SERS (surface-enhanced Raman spectroscopy) ultrasensitive biosensing platform was developed for the detection of biorhythmic markers. The biosensor was partly an ultrabright Raman signal reporter (on) of a novel satellite structure formed by gold/silver bimetallic nanoparticles. This novel satellite structure utilizes the potential difference between gold and silver nanoparticles to promote the transfer of electrons from the core structure to the AuNPs and MBA (4-mercaptobenzoic acid) molecules, while enhancing the electromagnetic field distribution. Compared with the Raman signal of the satellite structure composed of Au/Au bimetallic, the Raman signal of the satellite structure in this study is increased by about 10 times. CRISPR/Cas12a recognized the target molecule and activated trans-cleavage activity, cleaving the DNA/RNA hairpin structure and releasing RNA2&#x2217;. RNA2&#x2217; caused the satellite structure to disperse, significantly attenuating the ultra-strong Raman signal (off), thereby enabling quantitative detection of the target nucleic acid. The LOD for three targets were of less than 1&#xa0;fM.<br><br>SIGNIFICANCE: In summary, the CRISPR/Cas12a/SERS biosensing platform enables mRNA detection without preamplification. This biosensing platform can detect target molecules at the fM level, achieving ultra-sensitive detection. In addition, the satellite structure exhibits long-term stability, providing a theoretical basis for the practical application of this biosensing platform.},
}
@article {pmid40981773,
year = {2025},
author = {Eloiflin, R and Pérez-Antón, E and Camara, A and Dujeancourt-Henry, A and Boiro, S and Djetchi, MN and Traoré, MB and Koffi, M and Kaba, D and Le Pennec, Y and Doukouré, B and Camara, AD and Kagbadouno, M and Campagne, P and Camara, M and Jamonneau, V and Thévenon, S and Bart, JM and Glover, L and Rotureau, B},
title = {A SHERLOCK toolbox for eco-epidemiological surveillance of African trypanosomes in domestic pigs from Western Africa.},
journal = {eLife},
volume = {14},
number = {},
pages = {},
pmid = {40981773},
issn = {2050-084X},
support = {ANR-21-CE17-0022-01 SherPa//Agence Nationale de la Recherche/ ; ANR-10-LABX-62-IBEID//Agence Nationale de la Recherche/ ; ANR-11-LABX-0024-PARAFRAP//Agence Nationale de la Recherche/ ; Bourse Calmette-Yersin//Institut Pasteur/ ; },
mesh = {Animals ; *Trypanosomiasis, African/epidemiology/veterinary/parasitology/diagnosis ; *Trypanosoma/isolation &amp; purification/genetics/classification ; Swine ; *Swine Diseases/epidemiology/parasitology/diagnosis ; *Epidemiological Monitoring/veterinary ; Africa, Western/epidemiology ; CRISPR-Cas Systems ; Cote d'Ivoire/epidemiology ; },
abstract = {Animal African trypanosomosis (AAT), caused by protist parasites of the genus Trypanosoma, puts upward of a million head of livestock at risk across 37 countries in Africa. The economic impact of AAT and the presence of human-infectious trypanosomes in animals place a clear importance on improving diagnostics for animal trypanosomes to map the distribution of the veterinary parasites and identify reservoirs of human-infectious trypanosomes. We have adapted the CRISPR-based detection toolkit SHERLOCK (Specific High-sensitivity Enzymatic Reporter unLOCKing) for trypanosomatid parasites responsible for AAT (SHERLOCK4AAT) including Pan-trypanosomatid, Trypanozoon, T. vivax, T. congolense, T. theileri, T. simiae, and T. suis assays. To test the applicability of this technique in the field, we analysed dried blood spots collected from 200 farm and 224 free-ranging pigs in endemic and historical human African trypanosomiasis foci in Guinea and Côte d'Ivoire, respectively. The results revealed that SHERLOCK4AAT can detect and discriminate between trypanosome species involved in multiple infections with a high sensitivity. 62.7% [58.1, 67.3] of pigs were found infected with at least one trypanosome species. T. brucei gambiense, a human-infectious trypanosome, was found in one animal at both sites, highlighting the risk that these animals may act as persistent reservoirs. These data suggest that, due to their proximity to humans and their attractiveness to tsetse flies, pigs could act as sentinels to monitor T. b. gambiense circulation using the SHERLOCK4AAT toolbox.},
}
@article {pmid40980906,
year = {2025},
author = {Croteau, FR and Tran, J and Hynes, AP},
title = {CRISPR adaptation in Streptococcus thermophilus benefits from phage environmental DNA.},
journal = {mSphere},
volume = {},
number = {},
pages = {e0045325},
doi = {10.1128/msphere.00453-25},
pmid = {40980906},
issn = {2379-5042},
abstract = {The CRISPR-Cas system is a bacterial adaptive immune system that protects against infection by phages: viruses that infect bacteria. To develop immunity, bacteria integrate spacers-fragments of the invading nucleic acids-into their CRISPR array to serve as the basis for sequence-targeted DNA cleavage. However, upon infection, a phage quickly takes over the metabolism of the bacterium, leaving little time for the bacterium to acquire new spacers, transcribe them, and use them to cut the invading DNA. To develop CRISPR immunity, bacteria must be safely exposed to phage DNA. Phage infection releases environmental DNA (eDNA) which could be involved in the development of CRISPR immunity. Using Streptococcus thermophilus and phages 2972 and 858 as a model for CRISPR immunity, we show that eDNA is involved in CRISPR immunity, as generation of phage-immune bacterial colonies decreases with eDNA digestion. Furthermore, it is phage eDNA specifically that impacts CRISPR immunity since only its addition increases the generation of phage-immune colonies. We also show that the effect of eDNA is phage-specific, sequence-specific, and can even be traced to a region of the genome covering the early-expressed genes, which differ between phages 2972 and 858. However, we also show that eDNA is not used as a source of genetic information for spacer acquisition. These results link eDNA to the CRISPR-Cas system, providing a better understanding of the context of the emergence of CRISPR immunity and could inform our understanding of the mechanisms through which bacteria detect phage infection.IMPORTANCEHow can a bacterial adaptive immune system (the CRISPR-Cas system) exist at all, when exposure to a virulent phage is so consistently lethal? We proposed that bacteria might actively sample their genetic environment for phage DNA through natural competence. In testing this hypothesis, we revealed that free phage DNA is important to CRISPR immunity-but not as the source of the immunological memory.},
}
@article {pmid40977958,
year = {2025},
author = {Alsallameh, SMS and Salman, HA and Al-Khafaji, K and Kuzukiran, O},
title = {Comprehensive Genomic Characterization of a Drug-Resistant Klebsiella pneumoniae Clinical Isolate in Iraq Using Whole Genome Sequencing.},
journal = {Iranian journal of medical sciences},
volume = {50},
number = {9},
pages = {638-648},
pmid = {40977958},
issn = {1735-3688},
mesh = {*Klebsiella pneumoniae/genetics/pathogenicity/isolation &amp; purification/drug effects ; Whole Genome Sequencing/methods ; Humans ; Iraq/epidemiology ; *Klebsiella Infections/microbiology/drug therapy/epidemiology ; Anti-Bacterial Agents/pharmacology/therapeutic use ; Drug Resistance, Multiple, Bacterial/genetics ; Genome, Bacterial ; Genomics/methods ; Microbial Sensitivity Tests ; },
abstract = {BACKGROUND: Klebsiella pneumoniae is a Gram-negative encapsulated opportunistic pathogen, which presents a major threat to public health due to its ability for multi-antibiotic drug resistance. It is responsible for 30% of Gram-negative bacterial infections, including nosocomial infections, pneumonia, septicemia, and urinary tract infections. The study aimed to analyze the key phenotypic and genetic features of clinical K. pneumoniae isolates.<br><br>METHODS: Between 2022 and 2023, a total of 91 strains of Klebsiella pneumoniae were collected from Al-Imamian Al-Kadhimiyain Medical City (IKMC) and characterized using the VITEK-2 technique. Whole-genome sequencing (WGS) was employed to characterize the extreme drug-resistant strain. The whole genome was extracted and sequenced using the Next Generation Sequencing (NGS) technique. The genome of our bacterial isolate was analyzed using different bioinformatics tools such as Galaxy workflow, SPAdes, PROKKA, and Staramr.<br><br>RESULTS: The analysis identified Klebsiella pneumoniae serotype K36:O2a and sequencing type ST-437, containing 15 different plasmids carrying 54 resistance genes and more than 100 virulence genes with one region of CRISPR and no Cas. The sample obtained four intact bacteriophages and two questionable ones. Seven insertion sequences were revealed in the analysis as part of Other Mobile Genetic Elements (OMG). Additionally, the 16SrRNA phylogenetic tree identified a higher relationship of the bacteria to the strains from the USA and India than from Iraq.<br><br>CONCLUSION: It is the first study in Iraq to utilize WGS to comprehensively characterize an opportunistic pathogen. The study emphasizes the need for WGS to track the development of resistance and virulence patterns in clinical strains of K. pneumoniae.},
}
@article {pmid40977706,
year = {2025},
author = {Wang, JW and Feng, Q and Liu, JH and Xun, JJ},
title = {Opportunities, challenges, and future perspectives of oncolytic virus therapy for malignant melanoma.},
journal = {Frontiers in immunology},
volume = {16},
number = {},
pages = {1653683},
pmid = {40977706},
issn = {1664-3224},
mesh = {Humans ; *Oncolytic Virotherapy/methods/adverse effects ; *Melanoma/therapy/immunology ; *Oncolytic Viruses/immunology ; Animals ; Tumor Microenvironment/immunology ; Immunotherapy/methods ; Dendritic Cells/immunology ; *Skin Neoplasms/therapy/immunology ; },
abstract = {Malignant melanoma is characterized by high heterogeneity, aggressive metastatic potential, and a profoundly immunosuppressive "cold" tumor microenvironment, contributing to broad therapeutic resistance and suboptimal responses to immunotherapy. Conventional PD-1 inhibitors yield an ORR of only 38%. As an emerging class of immunotherapeutic agents, oncolytic viruses (OV) induce ICD, promoting the release of DAMPs and activating innate immune pathways such as cGAS-STING, thereby transforming "cold" tumors into "hot" phenotypes and eliciting robust anti-tumor responses. Mechanistically, OV therapy increases the proportion of CD103[+] dendritic cells (DCs) in lymph nodes from 5% to 25% and enhances DC-tumor synapse formation by 300%, facilitating efficient cross-presentation of tumor antigens and T-cell priming. Clinically, T-VEC combined with pembrolizumab achieves a 48.6% ORR with grade ≥3 AEs occurring in <20% of patients-superior to either monotherapy or conventional chemoradiotherapy. Nonetheless, OV therapy faces challenges including tumor heterogeneity, core mechanistic limitations, viral shedding risks, and regulatory hurdles. Over the next 5-10 years, single-cell RNA sequencing is expected to unravel molecular heterogeneity in melanoma, while CRISPR/Cas systems may enable the design of tailored OV to overcome resistance. Additional strategies such as serotype switching, JAK/STAT inhibition, and arming OV with hyaluronidase or STING agonists are under investigation to overcome immune and stromal barriers. Integration of artificial intelligence with biomarkers-such as neutralizing antibody titers, ISG expression, and STING methylation-may further enable personalized OV-based therapies. This review discusses OV therapy's mechanisms, clinical impact, and future prospects in melanoma treatment.},
}
@article {pmid40947068,
year = {2025},
author = {Das, LJ and Venkatesan, G and Krishnaswamy, N and Shekhawat, I and Vijayapillai, U and Priyanka, M and Dechamma, HJ},
title = {Effect of CRISPR-Cas9 mediated knockout of IRF3 gene in BHK-21 cells on immune gene expression and foot-and-mouth disease virus replication.},
journal = {Microbial pathogenesis},
volume = {208},
number = {},
pages = {108022},
doi = {10.1016/j.micpath.2025.108022},
pmid = {40947068},
issn = {1096-1208},
mesh = {Animals ; *Foot-and-Mouth Disease Virus/physiology/immunology ; *Interferon Regulatory Factor-3/genetics ; *CRISPR-Cas Systems ; *Virus Replication/genetics ; Cell Line ; *Gene Knockout Techniques ; Foot-and-Mouth Disease/immunology/virology ; Cricetinae ; },
abstract = {Foot-and-mouth disease (FMD) is an acute highly contagious viral disease of cloven-hoofed animals. Currently, FMD vaccine production and research mainly depend on the BHK-21 cell line. BHK-21 is highly sensitive to FMD virus, however, still a lot of room for improvement that can result in higher antigen yield in vaccine production facilities. IRF3 (Interferon regulatory factor 3) is a key transcription regulatory factor involved in the interferon (IFN) pathway, the immediate antiviral response of the cells. In this study, IRF3 knock-out (KO) BHK-21 cells were established using the CRISPR-Cas9 method. The KO cell line was stable in growth and morphological characteristics, unveiled in growth curve analysis. After infection with FMDV, the viral copy number, virus titer, and plaque forming units (PFU) were significantly increased in KO cells than those in parental BHK-21 (NC) cells. The relative gene expression of type I IFNs and ISGs such as IRF3, IRF7, viperin, Mx1 and ISG15 upon FMDV infection was severely reduced in KO cells. Results preliminarily reveal the role of IRF3 in cellular antiviral immune response, and the IRF3-KO cell line could also serve as a useful tool for FMDV research and vaccine production.},
}
@article {pmid40944915,
year = {2025},
author = {Daliri, K and Clement, K},
title = {Protocol for permanent gene repression by CRISPR-adenine base editing of promoter CCAAT motifs.},
journal = {STAR protocols},
volume = {6},
number = {3},
pages = {104075},
doi = {10.1016/j.xpro.2025.104075},
pmid = {40944915},
issn = {2666-1667},
mesh = {*Gene Editing/methods ; *Promoter Regions, Genetic/genetics ; *CRISPR-Cas Systems/genetics ; Animals ; RNA, Guide, CRISPR-Cas Systems/genetics ; Mice ; Humans ; NIH 3T3 Cells ; Adenine ; Transfection ; },
abstract = {Here, we present a protocol to achieve permanent downregulation of gene expression by editing the CCAAT box in promoter regions using CRISPR-adenine base editors (ABEs). We outline steps for guide RNA (gRNA) design, transfection, genomic DNA extraction, Sanger sequencing, and gene expression quantification. The protocol is optimized for mammalian cell lines (e.g., NIH3T3). It allows for precise disruption of transcription factor binding site without double-strand breaks and offers a novel alternative to RNAi or CRISPR interference (CRISPRi). For complete details on the use and execution of this protocol, please refer to Daliri et al.[1].},
}
@article {pmid40938663,
year = {2025},
author = {Liu, WJ and Liu, J and Ma, F and Zhang, CY},
title = {CRISPR/Cas13a trans-Cleavage-Driven Programmable Autonomous Synthesis of a Functional G-Quadruplex for Ultrasensitive Detection of Circular RNAs with an Improved Signal-to-Background Ratio.},
journal = {Analytical chemistry},
volume = {97},
number = {37},
pages = {20323-20332},
doi = {10.1021/acs.analchem.5c03464},
pmid = {40938663},
issn = {1520-6882},
mesh = {*G-Quadruplexes ; *RNA, Circular/analysis/genetics ; Humans ; *CRISPR-Cas Systems ; Limit of Detection ; },
abstract = {Circular RNAs (circRNAs) are a prevalent class of endogenous RNAs that regulate gene expression in various biological pathways, and their dysregulation is closely linked to tumorigenesis. Herein, we demonstrate the CRISPR/Cas13a trans-cleavage-driven programmable autonomous synthesis of a functional G-quadruplex for ultrasensitive detection of circRNAs in living cells and clinical tissues. Upon specific binding to target circRNA, the activated Cas13a/crRNA enables collateral cleavage of a uracil ribonucleotide (rU)-containing substrate probe, releasing a trigger sequence with a 2',3'-cyclic phosphate at its 3'-end. Subsequently, the resultant trigger sequence initiates primer exchange reaction (PER) cascades after its 3'-end is repaired by T4 polynucleotide kinase (T4 PNK), generating numerous long G-quadruplex sequences with different lengths. The long G-quadruplex sequences can be lighted up by thioflavin T (ThT) to obtain a dramatically amplified fluorescence signal. Owing to the exceptional specificity of high-fidelity CRISPR/Cas13a, high amplification efficiency of PER cascades, and high signal-to-background ratio of the G-quadruplex/ThT complex, this method can achieve ultrasensitive detection of circRNA down to 0.83 aM and discriminate target circRNA from its mismatched variants with single-base resolution. Moreover, it can profile endogenous circRNA in single cancer cell and differentiate circRNA expression in breast cancer tissues and their healthy counterparts. Notably, this assay can be accomplished in a one-step and single-tube manner at physiological temperature with the characteristics of easy design, simplified operation, rapid turnaround, and cost-effectiveness, suitable for in-field molecular diagnostics with limited resources.},
}
@article {pmid40924991,
year = {2025},
author = {Zhao, J and Sui, Z and Wang, Y and Dong, H and Chen, B and Xu, J},
title = {Programmable Dual-Phase Electrochemical Biosensor Combines Homogeneous CRISPR/Cas12a Activation with Interfacial Poly-G Signaling for miRNA-21 Detection.},
journal = {Analytical chemistry},
volume = {97},
number = {37},
pages = {20515-20526},
doi = {10.1021/acs.analchem.5c04010},
pmid = {40924991},
issn = {1520-6882},
mesh = {*MicroRNAs/analysis/genetics/blood ; *Biosensing Techniques/methods ; *Electrochemical Techniques/methods ; Humans ; *CRISPR-Cas Systems ; DNA/chemistry ; Gold/chemistry ; *CRISPR-Associated Proteins/metabolism ; *Endodeoxyribonucleases/metabolism ; Limit of Detection ; Electrodes ; Bacterial Proteins ; },
abstract = {Despite the promise of electrochemical biosensors in amplified nucleic acid diagnostics, existing high-sensitivity platforms often rely on a multilayer surface assembly and cascade amplification confined to the electrode interface. These stepwise strategies suffer from inefficient enzyme activity, poor mass transport, and inconsistent probe orientation, which compromise the amplification efficiency, reproducibility, and practical applicability. To address these limitations, we report a programmable dual-phase electrochemical biosensing system that decouples amplification from signal transduction. In the homogeneous phase, a palindromic allosteric hairpin probe undergoes target-triggered polymerization and bidirectional strand extension, generating double-stranded DNA (dsDNA) amplicons. These dsDNAs activate CRISPR/Cas12a complexes, which, in turn, cleave thiolated reporter DNA immobilized on a gold electrode. The exposed 3'-hydroxyl termini then initiate terminal deoxynucleotidyl transferase (TdT)-mediated polyguanine (poly-G) extension in the presence of dGTP. Methylene blue, selectively binding to G-rich sequences, produces a strong voltammetric signal proportional to the original miRNA-21 concentration. By integration of homogeneous amplification with localized electrochemical signal generation, this dual-phase design circumvents the drawbacks of interface-bound cascades while leveraging their sensitivity advantages. The system achieves a detection limit of 25 attomolar for miRNA-21, excellent sequence specificity, and reliable performance in human blood samples. This approach provides a robust and generalizable platform for nucleic acid diagnostics with high sensitivity, modularity, and operational simplicity.},
}
@article {pmid40924964,
year = {2025},
author = {Guo, XM and Fu, SY and Wang, YQ and Chen, Y and Li, H and Zhang, YJ and Lin, JS and Li, JF},
title = {Background-Free Rolling Circle Amplification for SERS Bioassay Using a Chimeric Hairpin-Integrated CRISPR/Cas12a System.},
journal = {Analytical chemistry},
volume = {97},
number = {37},
pages = {20557-20566},
doi = {10.1021/acs.analchem.5c04250},
pmid = {40924964},
issn = {1520-6882},
mesh = {*CRISPR-Cas Systems ; *African Swine Fever Virus/isolation &amp; purification/genetics ; *Nucleic Acid Amplification Techniques/methods ; *Spectrum Analysis, Raman/methods ; Animals ; Swine ; DNA, Single-Stranded ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {Rolling circle amplification (RCA) has revolutionized nucleic acid detection owing to its isothermal simplicity. However, over two decades of clinical application have been hampered by off-target amplification and incompatibility with double-stranded DNA (dsDNA). Herein, a strategy, specifically cleavage of rationally designed DNA/RNA chimeric hairpin preprimer by dsDNA-targeted CRISPR/Cas12a to release ssRNA for initiating RCA (SCOPE-RCA), is proposed for nucleic acid identification of African swine fever virus (ASFV). Leveraging the transcleavage capability of CRISPR/Cas12a, the SCOPE-RCA system achieves 99% single-stranded DNA (ssDNA) cleavage within 20 min, thus largely suppressing nonspecific amplification. Simultaneously, dsDNA-to-ssRNA conversion is realized using a DNA/RNA chimeric hairpin primer, where RNA functions as an RCA activator, enabling dsDNA-compatible RCA. Eventually, cascaded with catalytic hairpin assembly (CHA) coupled with surface-enhanced Raman scattering (SERS) signal amplification, the SCOPE-RCA system can detect ASFV in mock-infected samples at levels as low as 59 copies/mL. With its ease of use and ultrasensitive detection capability, the SCOPE-RCA system demonstrates significant potential for nucleic acid research and clinical applications.},
}
@article {pmid40899816,
year = {2025},
author = {Fujii, C and Wang, D},
title = {An SRR1 domain-containing protein is required for efficient Orsay virus replication in Caenorhabditis elegans.},
journal = {Journal of virology},
volume = {99},
number = {9},
pages = {e0052125},
doi = {10.1128/jvi.00521-25},
pmid = {40899816},
issn = {1098-5514},
mesh = {Animals ; *Caenorhabditis elegans/virology/genetics/metabolism ; *Virus Replication ; *Caenorhabditis elegans Proteins/genetics/metabolism ; *Nodaviridae/physiology/genetics ; Protein Domains ; Host-Pathogen Interactions ; Mutation ; CRISPR-Cas Systems ; },
abstract = {Viruses depend on their hosts for completing their life cycle, and a better understanding of virus replication can inform therapeutic strategies. Using the Orsay virus-Caenorhabditis elegans experimental platform, we identified by a forward genetic screen the host gene Y55F3BL.4 (renamed viro-9) as a novel host factor critical for Orsay virus replication. Three distinct mutations of viro-9 each resulted in a >1,000-fold reduction in Orsay viral load, demonstrating a pro-viral function of viro-9. viro-9 had no previously described function in C. elegans, but in the absence of viral infection, deletion of the viro-9 locus by CRISPR/Cas9 led to a reduction in brood size and a shortened lifespan. VIRO-9 contains a sensitivity to red light reduced (SRR1) protein domain. While SRR1 domains are present in diverse organisms, including plants, yeast, and mammals, little is known about their function. The Caenorhabditis briggsae ortholog of viro-9, CBG23913, can functionally complement the C. elegans viro-9 defect, demonstrating that the pro-viral function of the SRR1 domain is conserved over at least 80 million years of evolution. Furthermore, we identified three conserved amino acid residues within the SRR1 domain that are required for Orsay virus infection. This study provides the first insights into amino acids necessary for functionality of the SRR1 domain and demonstrates the essential role of viro-9 in virus infection.IMPORTANCEHost factors required for viral replication could serve as therapeutic targets for various viral species. The Caenorhabditis elegans-Orsay virus experimental system offers a platform for identifying genes important for virus infection in nematodes that may also be important for human-infecting viruses. We determined that viro-9, a previously uncharacterized gene in C. elegans containing the SRR1 domain, is required for Orsay virus replication. The related gene in Caenorhabditis briggsae, a relative of C. elegans that diverged about 80 million years ago, can substitute for viro-9, demonstrating that this protein's ability to promote virus replication is functionally conserved. Because SRR1 domain-containing proteins exist in nematodes, fungi, Drosophila, plants, and mammals, including humans, these proteins could be important for facilitating virus infection in other organisms as well.},
}
@article {pmid40879383,
year = {2025},
author = {Wang, Y and Peng, D and Li, M and Yao, M and Li, T and Li, S and Qiu, H-J and Li, L-F},
title = {Organoids: physiologically relevant ex vivo models for viral disease research.},
journal = {Journal of virology},
volume = {99},
number = {9},
pages = {e0113225},
doi = {10.1128/jvi.01132-25},
pmid = {40879383},
issn = {1098-5514},
support = {32372983//National Natural Science Foundation of China/ ; Y2025YC117//Central Public-interest Scientific Institution Basal Research Fund/ ; },
mesh = {*Organoids/virology ; Humans ; *Virus Diseases/virology/drug therapy ; Gene Editing ; Animals ; Antiviral Agents/pharmacology ; CRISPR-Cas Systems ; SARS-CoV-2 ; Models, Biological ; Viruses ; },
abstract = {Viral diseases pose serious threats to human health, resulting in substantial economic losses. However, traditional disease models often fail to capture the full complexity of viral pathogenesis. Pluripotent and tissue stem cell-derived organoids help bridge this gap by closely mimicking the structure and function of native organs, thereby enabling new breakthroughs in studying viral pathogenesis. This review discusses the diverse applications of organoid models in virology, including infection modeling, host-virus interaction studies, CRISPR/Cas9-based gene editing, antiviral drug screening, and vaccine development. Here, we focus on human organoid models used to investigate viral infections, covering systemic viral infections (exemplified by viruses such as SARS-CoV-2, Zika virus, influenza virus, and monkeypox virus) as well as localized viral infections (exemplified by viruses including respiratory syncytial virus, herpes simplex virus 1, rotavirus, norovirus, hepatobiliary viruses, and cytomegalovirus). By advancing mechanistic insights and accelerating therapeutic discovery, organoid technology shows significant potential as a complementary tool for combating viral diseases.},
}
@article {pmid40829261,
year = {2025},
author = {Rauff, R and Dong, C and Ayyar, S and Liang, FS},
title = {Transcript and temporal-specific RNA nucleotide editing technologies.},
journal = {Bioorganic &amp; medicinal chemistry},
volume = {130},
number = {},
pages = {118346},
doi = {10.1016/j.bmc.2025.118346},
pmid = {40829261},
issn = {1464-3391},
mesh = {*RNA Editing ; Humans ; *RNA/genetics/metabolism ; Oligonucleotides, Antisense/genetics/chemistry/metabolism ; Animals ; CRISPR-Cas Systems ; *Nucleotides/genetics/metabolism ; },
abstract = {Modified RNA nucleotides have emerged as a rapidly growing field due to their critical roles in post-transcriptional gene regulation. Early approaches investigating epitranscriptomic regulation primarily involved altering the expression of RNA nucleotide editing enzymes. However, the global perturbation of RNA nucleotide modifications complicates the investigation of site-specific effects of individual modifications on transcripts. To address this limitation, researchers have developed novel RNA modification editing tools that use CRISPR, antisense oligonucleotides (ASOs), chemical ligands, and light to selectively target RNA editing enzymes to chosen RNA sites with precise temporal control. In this review, we provide a comprehensive overview of current spatiotemporal controlled RNA nucleotide editing technologies.},
}
@article {pmid40773349,
year = {2025},
author = {Sharma, D and Sinha, R and Lesch, BJ and Cromer, MK},
title = {Protocol for efficient CRISPR/AAV-mediated genome editing and erythroid differentiation of human hematopoietic stem and progenitor cells.},
journal = {STAR protocols},
volume = {6},
number = {3},
pages = {104018},
pmid = {40773349},
issn = {2666-1667},
mesh = {Humans ; *Gene Editing/methods ; *Hematopoietic Stem Cells/cytology/metabolism ; *CRISPR-Cas Systems/genetics ; *Dependovirus/genetics ; Cell Differentiation/genetics ; *Erythroid Cells/cytology/metabolism ; },
abstract = {Here, we present a protocol for genome editing in human hematopoietic stem and progenitor cells (HSPCs) using CRISPR-Cas9 ribonucleoproteins and adeno-associated virus (AAV)-mediated homology-directed repair. We describe steps for AAV production, purification, and titration; HSPC thawing and culture; genome editing; and quantification of editing frequencies. We then detail procedures for erythroid differentiation assays. This protocol ensures high editing efficiency while maintaining cell viability and engraftment potential. For complete details on the use and execution of this protocol, please refer to Chu et al.[1].},
}
@article {pmid40768339,
year = {2025},
author = {Adarska, P and Fox, E and Heyza, J and Barnaba, C and Schmidt, J and Bottanelli, F},
title = {Protocol for fast antibiotic resistance-based gene editing of mammalian cells with CRISPR-Cas9.},
journal = {STAR protocols},
volume = {6},
number = {3},
pages = {103949},
pmid = {40768339},
issn = {2666-1667},
support = {DP2 GM142307/GM/NIGMS NIH HHS/United States ; },
mesh = {Humans ; *Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; HeLa Cells ; RNA, Guide, CRISPR-Cas Systems/genetics ; *Drug Resistance, Microbial/genetics ; Plasmids/genetics ; Transfection ; Recombinational DNA Repair ; },
abstract = {Protein tagging with CRISPR-Cas9 enables the investigation of protein function in its native environment but is limited by low homology-directed repair (HDR) efficiency. Here, we present a protocol for fast antibiotic resistance-based gene editing with CRISPR-Cas9 (FAB-CRISPR), which streamlines N/C-terminal tagging using an antibiotic resistance cassette for rapid selection and enrichment of gene-edited cells. We describe in detail guide RNA and HDR donor plasmid cloning, transfection of editing reagents into HeLa cells, and subsequent enrichment and verification of gene-edited cells. For complete details on the use and execution of this protocol, please refer to Wong-Dilworth et al.,[1] Stockhammer et al.,[2] Stockhammer et al.,[3] Heyza et al.,[4] and Broadbent et al.[5].},
}
@article {pmid40762955,
year = {2025},
author = {Arnson, B and Ilich, E and von Beck, T and Li, S and Brooks, ED and Gheorghiu, D and He, G and Weinrub, M and Chan, SY and Kang, HR and Courtney, D and Everitt, JI and Cullen, BR and Koeberl, DD},
title = {Efficacious genome editing in infant mice with glycogen storage disease type Ia.},
journal = {JCI insight},
volume = {10},
number = {18},
pages = {},
doi = {10.1172/jci.insight.181760},
pmid = {40762955},
issn = {2379-3708},
mesh = {Animals ; *Gene Editing/methods ; *Glycogen Storage Disease Type I/therapy/genetics ; Mice ; *Genetic Therapy/methods ; Genetic Vectors ; Dependovirus/genetics ; *Glucose-6-Phosphatase/genetics ; Disease Models, Animal ; Humans ; Liver/pathology/metabolism ; CRISPR-Cas Systems ; Hypoglycemia/prevention &amp; control ; Animals, Newborn ; Male ; },
abstract = {Glycogen storage disease type Ia (GSD Ia) is caused by a deficiency of glucose-6-phosphatase (G6Pase) in the liver leading to lethal hypoglycemia. Gene therapy with adeno-associated virus (AAV) vectors encoding G6Pase fails to stably treat GSD Ia early in life. We evaluated genome editing in 12-day-old infant mice with GSD Ia using 2 AAV vectors, one containing Cas9 from Streptococcus pyogenes and a second Donor vector that expresses a guide RNA and a G6PC transgene. Gene therapy with the Donor vector only was compared with genome editing using both Donor and CRISPR vectors. Treatment with genome editing (total vector dose 0.2 × 1013 to 2 × 1013 vector genomes/kg) and bezafibrate (to stimulate autophagy) was efficacious, as assessed by hypoglycemia prevention and the frequency of transgene integration, which correlated with improved survival. This therapy achieved 5.9% chromosomal transgene integration through homology-directed repair, which surpassed a threshold to prevent long-term hepatic complications. No integration was detected in the absence of the CRISPR vector. Importantly for safety, CRISPR vector genomes were depleted, and no intact, integrated CRISPR genomes were detected by long-read sequencing. Thus, genome editing warrants further development as a potentially stable treatment for human infants with GSD Ia.},
}
@article {pmid40700012,
year = {2025},
author = {Parra-Rivas, LA and Sharma, R and Rust, TE and Bazick, HO and Carlson-Stevermer, J and Zylka, MJ and Ogawa, Y and Roy, S},
title = {Protocol for CRISPR-based manipulation and visualization of endogenous α-synuclein in cultured mouse hippocampal neurons.},
journal = {STAR protocols},
volume = {6},
number = {3},
pages = {103945},
pmid = {40700012},
issn = {2666-1667},
mesh = {Animals ; *alpha-Synuclein/genetics/metabolism ; *Hippocampus/cytology/metabolism ; Mice ; *Neurons/metabolism/cytology ; *CRISPR-Cas Systems/genetics ; Cells, Cultured ; Gene Editing/methods ; },
abstract = {CRISPR-Cas9 technology enables acute gene knockdown and endogenous tagging to study single-synapse function. Here, we present a protocol for depleting alpha-synuclein (α-syn) or visualizing native α-syn with an endogenously inserted fluorescent tag in cultured mouse hippocampal neurons. We describe detailed steps, including CRISPR design, virus packaging/transduction (delivery), and validation of on-/off-target editing. This protocol should be useful for assigning precise function to contentious synaptic proteins and for visualizing protein trafficking without overexpression in cultured hippocampal neurons-an established model system for synaptic biology. For complete details on the use and execution of this protocol, please refer to Parra-Rivas et al.[1].},
}
@article {pmid40700010,
year = {2025},
author = {Shelake, RM and Kim, JY},
title = {Protocol for assessment of CRISPR base editors and their components in Escherichia coli.},
journal = {STAR protocols},
volume = {6},
number = {3},
pages = {103973},
pmid = {40700010},
issn = {2666-1667},
mesh = {*Escherichia coli/genetics ; *Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; },
abstract = {Base editing (BE) is a CRISPR technique that allows precise nucleobase conversions. However, high expression of BE components is often toxic in Escherichia coli. Here, we present a protocol for analyzing BE at single or multiple target sites using promoter-terminators for single guide RNA (sgRNA) and BE component expression. We describe steps for designing and cloning sgRNA, synthetic target, and BE biomodules. We then detail procedures for BE module assemblage, E. coli transformation, and testing base editors and components. For complete details on the use and execution of this protocol, please refer to Shelake and Kim,[1] Shelake et al.,[2] and Shelake et al.[3].},
}
@article {pmid40674218,
year = {2025},
author = {Wilbie, D and Mastrobattista, E and de Jong, OG},
title = {Protocol to rapidly screen CRISPR-Cas9 gene editing outcomes in a cell population by mutating eGFP to a blue or non-fluorescent phenotype.},
journal = {STAR protocols},
volume = {6},
number = {3},
pages = {103950},
pmid = {40674218},
issn = {2666-1667},
mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; *Green Fluorescent Proteins/genetics ; Humans ; Mutation/genetics ; Phenotype ; },
abstract = {When designing genome editing therapy, it is crucial to measure outcomes of DNA damage repair. Here, we present a protocol to distinguish the outcome of targeted DNA damage repair from the bottom up, through a previously established readout of enhanced green fluorescent protein (eGFP) to blue fluorescent protein (BFP) mutations. We describe steps for producing eGFP-positive cells and differentiating between non-homologous end joining-induced gene knockout and homology-directed repair-induced-directed mutation in these cells. This protocol has potential for high-throughput and scalable assessment of gene editing techniques. For complete details on the use and execution of this protocol, please refer to Walther et al.[1] and Wilbie et al.[2].},
}
@article {pmid40618370,
year = {2025},
author = {Zhang, Y and Yang, H and Yang, Y and Lu, Z and Cheng, L and Tan, H and Zhang, JZ},
title = {Protocol for high-efficiency generation of iPSCs stably expressing Cas9-EGFP using the selection by essential gene exon knockin method.},
journal = {STAR protocols},
volume = {6},
number = {3},
pages = {103928},
pmid = {40618370},
issn = {2666-1667},
mesh = {*Induced Pluripotent Stem Cells/metabolism/cytology ; *CRISPR-Cas Systems/genetics ; *Exons/genetics ; *Gene Knock-In Techniques/methods ; *Green Fluorescent Proteins/genetics/metabolism ; *Gene Editing/methods ; Humans ; *Genes, Essential/genetics ; *CRISPR-Associated Protein 9/genetics/metabolism ; Animals ; },
abstract = {CRISPR-Cas9 is widely used for genome editing. However, Cas9 silencing occurs during the directed differentiation of induced pluripotent stem cells (iPSCs), even when it is inserted into the safe harbor locus. Here, we generate iPSC-Cas9-EGFP using selection by essential gene exon knockin technology. We describe steps for inserting the Cas9-EGFP into exon 9 of GAPDH, bypassing Cas9 silencing. We then detail procedures for Cas9 function validation. For complete details on the use and execution of this protocol, please refer to Zhang et al.[1].},
}
@article {pmid40569759,
year = {2025},
author = {Doorley, LA and Meza-Perez, V and Jones, SJ and Rybak, JM},
title = {A Candidozyma (Candida) auris-Optimized Episomal Plasmid-Induced Cas9-Editing System Reveals the Direct Impact of the S639F-Encoding FKS1 Mutation.},
journal = {The Journal of infectious diseases},
volume = {232},
number = {3},
pages = {e529-e536},
doi = {10.1093/infdis/jiaf285},
pmid = {40569759},
issn = {1537-6613},
support = {//St Jude Children's Research Hospital Children's Infection Defense Center/ ; //Society of Infectious Diseases Pharmacists Young Investigator Research/ ; //Hartwell Center for Bioinformatics and Biotechnology/ ; //St Jude Center for Advanced Genome Engineering/ ; //American Lebanese Syrian Associated Charities/ ; P30 CA021765/CA/NCI NIH HHS/United States ; P30 CA021765/CA/NCI NIH HHS/United States ; },
mesh = {*Glucosyltransferases/genetics ; Drug Resistance, Fungal/genetics ; *Plasmids/genetics ; Antifungal Agents/pharmacology ; *Gene Editing/methods ; Echinocandins/pharmacology ; Mutation ; *Fungal Proteins/genetics ; *Candida auris/genetics/drug effects ; Microbial Sensitivity Tests ; CRISPR-Cas Systems ; Humans ; beta-Glucans ; },
abstract = {BACKGROUND: Mutations in the Candidozyma (Candida) auris β-glucan synthase gene (FKS1) altering S639 are frequently associated with clinical echinocandin resistance. We have developed a novel C auris-optimized episomal plasmid-induced Cas9 (EPIC) gene-editing system capable of recyclable precision editing and demonstrate the contribution of FKS1S639F mutation to echinocandin resistance.<br><br>METHODS: The EPIC gene-editing system was generated for optimized use in C auris, and ADE2 modification was evaluated in 5 C auris clades. Mutations leading to Fks1S639F and Fks1WT were placed into echinocandin-susceptible and echinocandin-resistant isolates from clades III and I, respectively. Echinocandin susceptibility was determined by Clinical and Laboratory Standards Institute methods. Cell wall abundance of chitin and &#x3b2;-glucan was assessed by staining with calcofluor white and aniline blue.<br><br>RESULTS: The EPIC system was capable of targeted ADE2 editing in all tested C auris isolates and precise editing confirmed by sequencing. A single-nucleotide polymorphism (SNP) in FKS1 resulting in either the S639F substitution or a synonymous mutation was introduced in an echinocandin-susceptible clade III isolate. Precision FKS1 editing by the EPIC system was confirmed by whole genome sequencing. Introduction of the Fks1S639F allele increased echinocandin resistance, while correction of the Fks1S639F to the Fks1WT sequence in an echinocandin-resistant clade I isolate restored echinocandin sensitivity. Evaluation of cell wall composition showed strains harboring Fks1S639F to contain significantly elevated &#x3b2;-glucan and chitin content.<br><br>CONCLUSIONS: These data demonstrate the potential of our EPIC system in its ability to introduce SNPs in multiple C auris clade backgrounds while revealing the direct impact of the S639F-encoding FKS1 mutation on echinocandin resistance.},
}
@article {pmid40517388,
year = {2025},
author = {Jo, MJ and Nam, HJ},
title = {Protocol to screen chemicals to enhance homology-directed repair in CRISPR-Cas9 gene editing.},
journal = {STAR protocols},
volume = {6},
number = {3},
pages = {103896},
doi = {10.1016/j.xpro.2025.103896},
pmid = {40517388},
issn = {2666-1667},
mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; *Recombinational DNA Repair/drug effects/genetics ; *High-Throughput Screening Assays/methods ; Humans ; },
abstract = {CRISPR-Cas9-based gene editing via homology-directed repair (HDR) enables precise modifications, though its efficiency is limited by the prevalence of non-homologous end joining (NHEJ). Here, we present a protocol for enhancing HDR efficiency by identifying chemicals using high-throughput screening (HTS). We describe steps for designing 96-well plates, executing HTS, and performing data analysis. We then detail procedures for identifying small molecules that improve HDR-associated gene editing. This protocol has potential application in HTS analysis focused on discovering reliable HDR enhancers. For complete details on the use and execution of this protocol, please refer to Jang et al.[1].},
}
@article {pmid40974457,
year = {2025},
author = {Ma, D and Cai, F and Zhang, T},
title = {Advances in the detection of Drug-Resistant bacteria: current trends and innovations.},
journal = {European journal of clinical microbiology &amp; infectious diseases : official publication of the European Society of Clinical Microbiology},
volume = {},
number = {},
pages = {},
pmid = {40974457},
issn = {1435-4373},
abstract = {Facing challenges in the fields of microbial detection and antimicrobial resistance (AMR) monitoring, the scientific community is opening new research avenues with the help of cutting-edge technologies such as molecular biology, genomics, proteomics, nanotechnology, and bioinformatics. In this review, we comprehensively collate and elaborate on revolutionary detection methods and AMR surveillance strategies that go beyond traditional microbial culture techniques. These innovative methods have not only improved the sensitivity and speed of detection but also broadened our understanding of the microbial world, providing new weapons in the fight against drug-resistant microorganisms. Through the integration and innovation of interdisciplinary approaches, we are gradually constructing a more precise, efficient, and comprehensive new paradigm for microbial detection and AMR testing.},
}
@article {pmid40973454,
year = {2025},
author = {Cortolezzis, Y and Othman, Z and Agostini, F and Ibrahim, I and Picco, R and Salgado, GF and Di Giorgio, E and Xodo, LE},
title = {Post-transcriptional control of KRAS: functional roles of 5'UTR RNA G-quadruplexes, long noncoding RNA, and hnRNPA1.},
journal = {Nucleic acids research},
volume = {53},
number = {17},
pages = {},
pmid = {40973454},
issn = {1362-4962},
support = {IG 2017//Associazione Italiana per la Ricerca sul Cancro/ ; 19898//Associazione Italiana per la Ricerca sul Cancro/ ; //Italian Ministry of Education/ ; //SD-Cancer Contributions of physics, chemistry and engineering sciences to oncology/ ; //Minist&#xe8;re de l'Europe et des Affaires &#xc9;trang&#xe8;res/ ; },
mesh = {Humans ; *Proto-Oncogene Proteins p21(ras)/genetics/metabolism ; *5' Untranslated Regions/genetics ; *G-Quadruplexes ; *RNA, Long Noncoding/genetics/metabolism ; *Heterogeneous Nuclear Ribonucleoprotein A1/metabolism/genetics ; RNA, Messenger/genetics/metabolism ; RNA Stability ; Gene Expression Regulation, Neoplastic ; CRISPR-Cas Systems ; *RNA Processing, Post-Transcriptional ; Cell Line, Tumor ; },
abstract = {Previous studies have shown that human KRAS expression is regulated at the transcriptional level by G-quadruplex DNA structures within its promoter. Here we show an additional level of regulation involving a post-transcriptional mechanism centred on the 5'-untranslated region (5'UTR) of the messenger RNA (mRNA) characterized by G4 structures (rG4s). Long noncoding RNAs (lncRNAs) and the protein hnRNPA1 are also involved in this mechanism. RIP-seq confirmed the presence of rG4s in the 5'UTR. Deletion of the rG4 region using CRISPR/Cas9 resulted in a significant increase in KRAS mRNA levels, indicating the role of the 5'UTR in controlling mRNA levels. RIP shows that hnRNPA1 is recruited to the 5'UTR, where it unfolds the rG4 structures and potentially affects mRNA stability. In addition, lncRNAs transcribed from the LINC01750 locus can hybridize to the rG4 region of 5'UTR and form RNA duplexes leading to RNase III-assisted degradation of the targeted mRNA. Activation of the LINC01750 locus with dCas9-VP64 resulted in downregulation of KRAS mRNA, whereas its suppression with dCas9-KRAB led to upregulation of both KRAS mRNA and protein. Since lncRNA-mediated regulation of mRNA appears to be a crucial aspect of cellular homeostasis and its disruption contributes to various diseases, understanding these mechanisms may reveal promising new therapeutic targets.},
}
@article {pmid40973278,
year = {2025},
author = {Safarzadeh, S and Naghib, SM and Takdehghan, G and Forooshani, RS and Roozbahani, MH and Sharifianjazi, F and Tavamaishvili, K},
title = {Multimodal chitosan-based materials for combination immunotherapy in cancers: Structural engineering, immune regulatory mechanisms and synergistic therapeutic applications.},
journal = {Carbohydrate polymers},
volume = {369},
number = {},
pages = {124245},
doi = {10.1016/j.carbpol.2025.124245},
pmid = {40973278},
issn = {1879-1344},
mesh = {Humans ; *Immunotherapy/methods ; *Neoplasms/therapy/immunology ; *Chitosan/chemistry/therapeutic use ; Animals ; Combined Modality Therapy ; },
abstract = {Chitosan (CS)-based materials have gained considerable attention due to their intrinsic biodegradability, biocompatibility, and non-immunogenicity, establishing them as versatile candidates for state-of-the-art cancer immunotherapy. By harnessing the unique physicochemical and immunomodulatory properties of this polysaccharide, CS-based systems enable precise targeting, controlled release, and enhanced immune activation, thereby improving therapeutic efficacy while minimizing off-target effects. This review begins by outlining the cancer-immunity cycle and elucidating the structural and functional attributes of CS including cationic charge, chemical functionalization, biodistribution, targeting efficiency, and immune regulation that underpin its performance. The strategic roles of advanced CS derivatives, such as trimethyl CS (TMC), carboxymethyl CS (CMC), fluorinated CS (FCS), glycated CS (GC), hydroxypropyl CS (HPC), and thiolated CS (TC), are examined in optimizing these parameters. Cutting edge CS-based platforms, encompassing vaccines, immune checkpoint inhibitors, and nanostructures, are critically evaluated alongside their integration with synergistic modalities such as chemotherapy, radiotherapy (RT), phototherapy, sonodynamic therapy (SDT), gene therapy, and CRISPR-Cas mediated genome editing. The clinical and translational landscape is also addressed, highlighting emerging trials, manufacturing scalability, and regulatory considerations. Collectively, recent advances consolidate CS-based immunotherapy as a potent, adaptable, and clinically relevant approach capable of driving durable and tumor specific immune responses, with substantial promise for future cancer treatment.},
}
@article {pmid40973142,
year = {2025},
author = {Gong, M and Ding, Y and Jin, Y and Zhao, S and Jiang, X and Shao, S and Chen, S},
title = {CRISPR-based gene knockout in the model haloarchaeon Haloferax mediterranei.},
journal = {FEMS microbiology letters},
volume = {},
number = {},
pages = {},
doi = {10.1093/femsle/fnaf096},
pmid = {40973142},
issn = {1574-6968},
abstract = {Halophilic archaea, a specialized group of extremophiles that inhabit hypersaline environments, exhibit distinctive physiological and metabolic features. Traditional genetic manipulation of these organisms, predominantly reliant on homologous recombination techniques, suffers from limitations such as complex procedures and extended timelines, which hinder functional genomics research and the development of practical applications. This study established a CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)-mediated gene knockout system in the model halophilic archaeon Haloferax mediterranei. A polyethylene glycol (PEG)-mediated transformation method was used to deliver a plasmid carrying a mini-CRISPR array into H. mediterranei. The crtB gene, involved in pigment synthesis, was successfully knocked out, demonstrating the feasibility of CRISPR-based editing in H. mediterranei. To further validate the reliability and targeting accuracy of the system, the hlyR4 gene, encoding an extracellular serine protease, was also disrupted. The CRISPR-mediated gene knockout efficiency for hlyR4 reached 27%, significantly higher than the approximately 3% efficiency achieved with conventional homologous recombination. The establishment of this CRISPR-based gene knockout system provides a more efficient genetic tool for H. mediterranei and lays a new experimental foundation for exploiting microbial resources from extreme environments.},
}
@article {pmid40904316,
year = {2025},
author = {Li, Z and Song, Y and Huang, H and Chen, R and Liu, M and Yang, X and Luo, Z and Liu, BM and Wang, J},
title = {Highly stable Cas9 promotes HBV genome destruction by antagonizing HSC70-mediated degradation.},
journal = {Emerging microbes &amp; infections},
volume = {14},
number = {1},
pages = {2556728},
doi = {10.1080/22221751.2025.2556728},
pmid = {40904316},
issn = {2222-1751},
mesh = {Humans ; *HSC70 Heat-Shock Proteins/metabolism/genetics ; *Hepatitis B virus/genetics/physiology ; *CRISPR-Cas Systems ; *Genome, Viral ; Autophagy ; *CRISPR-Associated Protein 9/metabolism/genetics ; Proteolysis ; *Hepatitis B/virology ; Lysosomes/metabolism ; },
abstract = {As a naturally existing adaptive immune system of prokaryotes against phages and foreign genetic materials, the CRISPR/Cas9 system has been widely used to combat various viral infections. However, its ability to destroy the constantly replicating viral genome and subsequently clear viral infections still needs further improvement. This study found that Cas9 protein was mainly degraded through the chaperone-mediated autophagy (CMA)-lysosome pathway in human cells, which was mediated by the binding between heat shock cognate protein 70 (HSC70) and Cas9 protein. HRS could stabilize Cas9 protein by competing with HSC70 to bind to Cas9 and subsequently inhibiting its degradation via the CMA-lysosome pathway. The stability of Cas9 protein with mutant KFERQ-like motifs located at aa 670-674 and aa 894-898 was significantly increased by antagonizing the HSC70-mediated CMA degradation, thus this Cas9 mutant was referred to as a highly stable Cas9 (HSCas9). The enhanced ability of HSCas9 to destroy the constantly replicating hepatitis B virus (HBV) genome promoted the CRISPR/Cas9 system to clear HBV infection without exhibiting cytotoxicity or increasing off-target effects. In summary, this study uncovers the degradation mechanism of Cas9 protein in human cells and provides a strategy to enhance the ability of the CRISPR/Cas9 system to clear HBV infection.Abbreviations: ALP: autophagy-lysosome pathways; AR7: 7-Chloro-3-(4-methylphenyl)-2H-1,4-benzoxazine; cccDNA: covalently closed circular DNA; CMA: chaperone-mediated autophagy; CRISPR/Cas9: clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated nuclease 9 (Cas9); gRNA: single-guide RNA; HBV: hepatitis B virus; HRS: hepatocyte growth factor-regulated tyrosine kinase substrate; HSC70: heat shock cognate protein 70; HSCas9: highly stable Cas9; rcDNA: relaxed circular DNA; SNP: single nucleotide polymorphism; UPS: ubiquitin-proteasome system.},
}
@article {pmid40972923,
year = {2025},
author = {Sun, H and Bu, S and Wang, J and Zhang, Z and Li, C and Wan, Y and Zhao, Y and Zhou, H and Hao, Z and Feng, X and Li, C and Wan, J},
title = {PAM-free CRISPR/Cas12a biosensor for PNAs-assisted isothermal amplification detection of S. typhimurium.},
journal = {International journal of biological macromolecules},
volume = {328},
number = {Pt 2},
pages = {147721},
doi = {10.1016/j.ijbiomac.2025.147721},
pmid = {40972923},
issn = {1879-0003},
abstract = {CRISPR/Cas12a (Cpf1) have been generally used for detecting pathogenic microorganisms. However, rules for a protospacer adjacent motif (PAM) on target double-stranded DNA (dsDNA) hindered their application. To overcome this shortcoming, we developed a novel isothermal amplification scheme, peptide nucleic acids (PNAs)-assisted self-folding isothermal amplification for a PAM-free CRISPR/Cas12a biosensor (PSCas) to detect salmonella typhimurium (S. typhimurium). The PSCas biosensor uses PNAs to precisely invade the target gene dsDNA to form single-stranded DNA (ssDNA), which in turn binds to a self-folding primer (SP). When the self-folding strand of SP is complementary to the target DNA, the hairpin structure is formed, which exposes the start-up site of the new SP continuous hybridization. Consequently, this amplification used only one primer to produce a large number of ssDNA amplifiers, and exhibited a remarkable diagnostic sensitivity of 3 CFU•mL[-1] S. typhimurium. This platform provided a novel approach for developing innovative PAM-free CRISPR/Cas biosensors for PNAs-assisted isothermal amplification.},
}
@article {pmid40769810,
year = {2025},
author = {Liu, W and Zhu, X and Xue, Y and Wang, X and Chang, L and Wang, S and Li, L and Huang, J and Zhang, Y and Jiang, C and Chi, H and Zhang, Y and Li, CZ and Liang, T},
title = {RNA target-independent non-canonical activation (RINCA) of Cas13 trans-nuclease activity.},
journal = {Science bulletin},
volume = {70},
number = {18},
pages = {3005-3018},
doi = {10.1016/j.scib.2025.07.015},
pmid = {40769810},
issn = {2095-9281},
mesh = {*CRISPR-Cas Systems/genetics ; Humans ; *CRISPR-Associated Proteins/genetics/metabolism/chemistry ; *RNA/metabolism/genetics ; Animals ; Aptamers, Nucleotide/genetics ; Ribonucleases/metabolism/genetics ; Mutation ; Mice ; },
abstract = {A thorough understanding of unintended Cas13 activity is critical for ensuring the safe in vivo application of CRISPR-Cas13. In this study, we uncover the RNA target-independent, non-canonical activation (RINCA) of Cas13 by crRNA alone and elucidate its structural basis. RINCA poses both challenges and opportunities for Cas13-based technologies. On one hand, it constrains Cas13's utility due to its uncontrolled RNase activity. To address this, we screened over 40 rationally designed Cas13a mutants to identify those with diminished RINCA potential while preserving target-dependent responses. These optimized Cas13a variants improve biosensing sensitivity and significantly reduce cytotoxicity in vivo. On the other hand, RINCA can be harnessed for novel applications. By integrating RNA aptamers into spacer regions, the RINCA-capable LwCas13a/ap-crRNA system was engineered to sense intracellular metabolite levels, drive systemic RNA degradation, and demonstrate anti-cancer efficacy across multiple models. This study not only discovers the RINCA model of Cas13 activity but also provides effective mitigation strategies and establishes novel RINCA-based applications.},
}
@article {pmid40972653,
year = {2025},
author = {Serpa, G and Gong, Q and De, M and Rana, PSJB and Montgomery, CP and Wozniak, DJ and Long, ME and Hemann, EA},
title = {Detection of pre-existing immunity to bacterial Cas9 proteins in people with cystic fibrosis.},
journal = {ImmunoHorizons},
volume = {9},
number = {10},
pages = {},
doi = {10.1093/immhor/vlaf041},
pmid = {40972653},
issn = {2573-7732},
support = {LONG19F5-CI//Cystic Fibrosis Foundation/ ; LONG21R3//Cystic Fibrosis Foundation/ ; },
mesh = {Humans ; *Cystic Fibrosis/immunology/genetics ; *CRISPR-Associated Protein 9/immunology/genetics ; CRISPR-Cas Systems/immunology ; Gene Editing ; Male ; Adult ; Female ; Genetic Therapy ; Staphylococcus aureus/immunology ; Streptococcus pyogenes/immunology ; Cystic Fibrosis Transmembrane Conductance Regulator/genetics ; Immunoglobulin G/blood/immunology ; T-Lymphocytes/immunology ; Young Adult ; Adolescent ; Interferon-gamma/metabolism ; },
abstract = {Cystic fibrosis (CF) is caused by homozygous mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, resulting in multi-organ dysfunction and decreased lifespan and quality of life. A durable cure for CF will likely require a gene therapy approach to correct CFTR. Rapid advancements in genome editing technologies, including CRISPR/Cas9, have already resulted in Food and Drug Administration (FDA) approval for cell-based gene editing therapies, providing new therapeutic avenues for many rare diseases. However, immune responses to gene therapy delivery vectors and editing tools remain a challenge, especially for strategies targeting complex in vivo tissues such as the lung. Previous findings in non-CF healthy individuals reported pre-existing antibody and T cell responses to recombinant Cas9 proteins, suggesting potential additional obstacles for incorporation of clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technologies in gene therapies. To determine whether pre-existing immunity to Cas9 from S. aureus or S. pyogenes was present or augmented in people with CF, anti-Cas9 IgG levels and Cas9-specific T cell responses were determined from peripheral blood samples of people with CF and non-CF healthy controls. Overall, non-CF control and CF samples displayed evidence of pre-existing antibody and T cell responses to both S. aureus and S. pyogenes Cas9, although there were no significant differences between these populations. However, we observed global changes in CF activation of Th1 and CD8 T cell responses as measured by interferon γ (IFN-γ) and tumor necrosis factor (TNF) that warrant further investigation and mechanistic understanding as this finding has implications not only for CRISPR/Cas9 gene therapy for people with CF but also for protection against infectious disease.},
}
@article {pmid40972525,
year = {2025},
author = {Tatarakis, A and Saini, H and Yu, J and Feng, W and Pinzon-Arteaga, CA and Moazed, D},
title = {Requirements for establishment and epigenetic stability of mammalian heterochromatin.},
journal = {Molecular cell},
volume = {85},
number = {18},
pages = {3388-3406.e12},
doi = {10.1016/j.molcel.2025.08.025},
pmid = {40972525},
issn = {1097-4164},
mesh = {*Heterochromatin/genetics/metabolism ; Animals ; Mice ; *Histones/metabolism/genetics ; DNA Methylation ; *Epigenesis, Genetic ; *Mouse Embryonic Stem Cells/metabolism ; Chromatin Assembly and Disassembly ; Cell Differentiation ; Histone-Lysine N-Methyltransferase/metabolism/genetics ; Methylation ; CRISPR-Cas Systems ; Histone Deacetylases/metabolism/genetics ; },
abstract = {Heterochromatic domains of DNA account for a large fraction of mammalian genomes and play critical roles in silencing transposons and genes, but the mechanisms that establish and maintain these domains are not fully understood. Here, we use a CRISPR-based genetic screen to investigate the requirements for establishment and maintenance of histone H3 lysine 9 trimethylation (H3K9me3) heterochromatin. In mouse embryonic stem cells (mESCs), we show that transiently induced H3K9me3 heterochromatin is inherited for a limited number of cell divisions, independently of sequence-dependent recruitment, but becomes stable upon differentiation, concomitant with downregulation of enzymes erasing H3K9me and DNA methylation. In addition, ordered and non-redundant activities of multiple H3K9 and DNA methyltransferases, together with histone deacetylases, chromatin remodeling complexes, and RNA processing factors, are required for heterochromatin maintenance. Our findings suggest that a newly acquired H3K9me3 domain can be maintained like an imprint but requires reinforcement by DNA methylation and other pathways.},
}
@article {pmid40970972,
year = {2025},
author = {Yang, C and Li, B and Yu, H and Wang, Y and An, Z and Chen, M and He, C},
title = {GmCDC7 is involved in coordinating seed size and quality in soybean.},
journal = {TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik},
volume = {138},
number = {10},
pages = {253},
pmid = {40970972},
issn = {1432-2242},
support = {2023ZD0406904//Biological Breeding-National Science and Technology Major Project/ ; 2023ZD0406802//Biological Breeding-National Science and Technology Major Project/ ; 31930007//National Natural Science Foundation of China/ ; 31525003//National Natural Science Foundation of China/ ; XDA08010105//Chinese Academy of Sciences/ ; },
mesh = {*Glycine max/genetics/growth &amp; development ; *Seeds/genetics/growth &amp; development ; Gene Expression Regulation, Plant ; *Plant Proteins/genetics/metabolism ; *Cell Cycle Proteins/genetics/metabolism ; Phenotype ; CRISPR-Cas Systems ; Plants, Genetically Modified ; },
abstract = {We revealed essential roles of GmCDC7 in modulating seed size/weight and seed protein/oil content in soybean, presenting potential new targets for improving yield and quality of soybean and other crops. Seed size/weight is a critical factor determining crop yield; however, a limited number of genes regulating this trait have been characterized in soybean. In this study, we identified a Glycine max CELL DIVISION CYCLE 7 (GmCDC7) and revealed its essential roles in seed development. The putative GmCDC7 was highly conserved in both sequences and structure across various species. GmCDC7 transcripts were detectable in multiple tissues, with peak expression occurring during early seed development, while the GmCDC7 proteins were predominantly localized within the nucleus. CRISPR/Cas9-mediated knockout of GmCDC7 led to a significant increase in seed size and 100-seed weight, while overexpression of this gene resulted in a reduction in both seed size and weight. Further cytological analysis demonstrated that GmCDC7 promoted cell expansion and inhibited cell proliferation in seeds. Notably, the gene-edited gmcdc7 mutants showed a substantial increase in protein content alongside a reduction in oil content in seeds. Correspondingly, transcriptomic analyses revealed that GmCDC7 may significantly influence multifaceted regulatory pathways related to cell cycle-related activities, storage protein accumulation, and lipid transport and metabolism during seed development. These findings suggest that GmCDC7 plays pivotal roles in modulating seed size/weight and quality, offering new gene resources and insights into biotechnological strategies for soybean breeding.},
}
@article {pmid40970094,
year = {2025},
author = {Shu, J and Xie, X and Wang, S and Du, Z and Huang, P and Chen, Y and He, Z},
title = {CRISPR/Cas-edited iPSCs and mesenchymal stem cells: a concise review of their potential in thalassemia therapy.},
journal = {Frontiers in cell and developmental biology},
volume = {13},
number = {},
pages = {1595897},
pmid = {40970094},
issn = {2296-634X},
abstract = {Thalassemia, a prevalent single-gene inherited disorder, relies on hematopoietic stem cell or bone marrow transplantation as its definitive treatment. However, the scarcity of suitable donors and the severe complications from anemia and iron overload pose significant challenges. An immediate need exists for a therapeutic method that addresses both the illness and its associated complications. Advancements in stem cell technology and gene-editing methods, such as clustered regularly interspaced short palindromic repeats along with its associated protein (CRISPR/Cas), offer encouraging prospects for a therapy that could liberate patients from the need for ongoing blood transfusions and iron chelation treatments. The potential of genetic reprogramming using induced pluripotent stem cells (iPSCs) to address thalassemia is highly promising. Furthermore, mesenchymal stem cells (MSCs), recognized for their capacity to self-renew and differentiate into multiple lineages that include bone, cartilage, adipose tissue, and liver, demonstrate potential in alleviating several complications faced by thalassemia patients, including osteoporosis, cirrhosis, heart conditions, respiratory issues, and immune-related disorders. In this review, we synthesize and summarize relevant studies to assess the therapeutic potential and predict the curative effects of these cellular approaches.},
}
@article {pmid40968372,
year = {2025},
author = {Harle, V and Offord, V and Gökbağ, B and Fotopoulos, L and Williams, T and Alexander, D and Mehta, I and Thompson, NA and Olvera-León, R and Peidli, S and Iyer, V and Gonçalves, E and Kebabci, N and De Kegel, B and van de Haar, J and Li, L and Ryan, CJ and Adams, DJ},
title = {A compendium of synthetic lethal gene pairs defined by extensive combinatorial pan-cancer CRISPR screening.},
journal = {Genome biology},
volume = {26},
number = {1},
pages = {284},
pmid = {40968372},
issn = {1474-760X},
support = {20/FFP-P/8641 and 18/CRT/6214//Health Research Charities Ireland/ ; },
mesh = {Animals ; Humans ; *CRISPR-Cas Systems ; Mice ; Cell Line, Tumor ; *Neoplasms/genetics ; *Synthetic Lethal Mutations ; Mice, Knockout ; *Genes, Lethal ; },
abstract = {BACKGROUND: Synthetic lethal interactions are attractive therapeutic candidates as they enable selective targeting of cancer cells in which somatic alterations have disrupted one member of a synthetic lethal gene pair while leaving normal tissues untouched, thus minimising off-target toxicity. Despite this potential, the number of well-established and validated synthetic lethal gene pairs is modest.<br><br>RESULTS: We generate a dual-guide CRISPR/Cas9 Library and analyse 472 predicted synthetic lethal pairs in 27 cancer cell Lines from melanoma, pancreatic and lung cancer Lineages. We report a robust collection of 117 genetic interactions within and across cancer types and explore their candidacy as therapeutic targets. We show that SLC25A28 is an attractive target since its synthetic lethal paralog partner SLC25A37 is homozygously deleted pan-cancer. We generate knockout mice for Slc25a28 revealing that, except for cataracts in some mice, these animals are normal; suggesting inhibition of SLC25A28 is unlikely to be associated with profound toxicity.<br><br>CONCLUSIONS: We provide and validate an extensive collection of synthetic lethal interactions across cancer types.},
}
@article {pmid40968144,
year = {2025},
author = {Wang, M and Zhang, Z and Wang, X and Zhang, L and Chen, X and Li, N and Sun, Q and Lu, Y and He, Z and Yang, H and Tan, F and Qi, J and Chai, R},
title = {Optimized in vivo base editing restores auditory function in a DFNA15 mouse model.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {8322},
pmid = {40968144},
issn = {2041-1723},
support = {82030029//National Science Foundation of China | National Natural Science Foundation of China-Yunnan Joint Fund (NSFC-Yunnan Joint Fund)/ ; 82330033//National Science Foundation of China | National Natural Science Foundation of China-Yunnan Joint Fund (NSFC-Yunnan Joint Fund)/ ; 92468302//National Science Foundation of China | National Natural Science Foundation of China-Yunnan Joint Fund (NSFC-Yunnan Joint Fund)/ ; 92149304//National Science Foundation of China | National Natural Science Foundation of China-Yunnan Joint Fund (NSFC-Yunnan Joint Fund)/ ; BK20232007//Natural Science Foundation of Jiangsu Province (Jiangsu Provincial Natural Science Foundation)/ ; BK20241692//Natural Science Foundation of Jiangsu Province (Jiangsu Provincial Natural Science Foundation)/ ; BG2024037//Natural Science Foundation of Jiangsu Province (Jiangsu Provincial Natural Science Foundation)/ ; JCYJ20240813161801003//Shenzhen Science and Technology Innovation Commission/ ; 2024M750455//China Postdoctoral Science Foundation/ ; 2025T180661//China Postdoctoral Science Foundation/ ; 82401369//National Natural Science Foundation of China (National Science Foundation of China)/ ; 82271183//National Natural Science Foundation of China (National Science Foundation of China)/ ; 82222017//National Natural Science Foundation of China (National Science Foundation of China)/ ; 82192864//National Natural Science Foundation of China (National Science Foundation of China)/ ; 82371161//National Natural Science Foundation of China (National Science Foundation of China)/ ; 82371162//National Natural Science Foundation of China (National Science Foundation of China)/ ; U23A200440//National Natural Science Foundation of China (National Science Foundation of China)/ ; RF1028623028//Southeast University (SEU)/ ; CZXM-GSP-RC04//Southeast University (SEU)/ ; tsqn202408320//Taishan Scholar Project of Shandong Province/ ; 7252089//Natural Science Foundation of Beijing Municipality (Beijing Natural Science Foundation)/ ; },
mesh = {Animals ; *Gene Editing/methods ; Disease Models, Animal ; Mice ; CRISPR-Cas Systems ; *Transcription Factor Brn-3C/genetics ; *Deafness/genetics/therapy ; Genetic Therapy/methods ; Mutation ; Dependovirus/genetics ; Humans ; RNA, Guide, CRISPR-Cas Systems/genetics ; Female ; Male ; },
abstract = {Genetic mutations cause hereditary deafness, in which mutations in the POU4 transcription factor 3 gene (POU4F3) lead to autosomal dominant non-syndromic deafness 15 (DFNA15), for which no effective clinical treatment currently exists. Gene editing holds promise for precisely repairing mutated nucleotides, thus offering a potential cure for hereditary hearing loss. Here, we establish a Pou4f3[WT/Q113*] mutant mouse model mimicking DFNA15. We develop and screen adenine base editors (ABEs) targeting the Pou4f3[Q113*] allele by fusing diverse adenine deaminases to Cas9 we discovered before. SchABE8e accomplishes highly precise and efficient editing (up to 48.5%) at sgRNA3 in vitro. Neonatal Pou4f3[WT/Q113*] mice are treated via synthetic AAV (Anc80L65)-delivered SchABE8e-sgRNA3, resulting in near-complete hearing recovery, with the effect persisting for at least four months. Biosafety analyses further support the feasibility of base editing, providing a therapeutic strategy for DFNA15.},
}
@article {pmid40966844,
year = {2025},
author = {Sihamok, W and Islam, SI and Khang, LTP and Dangsawat, O and Sangsawad, P and Tu, TA and Thao, CP and Dinh-Hung, N and Permpoonpattana, P and Linh, NV},
title = {Genomic insights into Bacillus sp. KNSH11 from Litopenaeus vannamei intestine: Probiotic potential, safety, and aquaculture applications.},
journal = {Comparative biochemistry and physiology. Part D, Genomics &amp; proteomics},
volume = {56},
number = {},
pages = {101633},
doi = {10.1016/j.cbd.2025.101633},
pmid = {40966844},
issn = {1878-0407},
abstract = {In the context of sustainable aquaculture, probiotics represent a promising alternative to antibiotics for promoting shrimp health and disease resistance. In this study, Bacillus sp. KNSH11, a Gram-positive, rod-shaped bacterium isolated from the intestine of whiteleg shrimp (Litopenaeus vannamei), was characterized to assess its probiotic potential. The strain exhibited excellent sporulation efficiency (> 99 %), supporting its resilience under harsh environmental conditions. Functional assays demonstrated that KNSH11 retained high viability under various stressors, including acidic pH (2-4), bile salts, elevated temperatures (up to 95 °C), and lysozyme exposure, indicating robust tolerance to gastrointestinal and processing challenges. Metabolic profiling revealed substantial lactic acid production with minimal levels of acetate and propionate, distinguishing it from conventional lactic acid bacteria. The strain also exhibited strong antioxidant activity and moderate antibiofilm effects against pathogenic bacteria. Antibiotic susceptibility testing showed sensitivity to amoxicillin, chloramphenicol, kanamycin, and tetracycline (all at 30 μg/disc), while resistance was observed against ampicillin and penicillin (10 μg/disc each). Whole genome sequencing confirmed the absence of virulence genes and identified the presence of mobile genetic elements, a CRISPR/Cas system, and gene clusters potentially responsible for bacteriocin production. Collectively, these results indicate that Bacillus sp. KNSH11 exhibits key probiotic characteristics and genomic features consistent with a safe profile, supporting its potential application in sustainable shrimp aquaculture, pending further in vitro and in vivo validation.},
}
@article {pmid40966510,
year = {2025},
author = {Rananaware, SR and Shoemaker, GM and Pizzano, BLM and Vesco, EK and Sandoval, LSW and Lewis, JG and Bodin, AP and Flannery, SJ and Lange, IH and Pedada, D and Fang, A and Antal, SG and Aguilar, D and Rakestraw, NR and Karalkar, VN and Meister, KS and Nguyen, LT and Jain, PK},
title = {AsCas12a tolerates insertions in target DNA.},
journal = {Nucleic acids research},
volume = {53},
number = {17},
pages = {},
doi = {10.1093/nar/gkaf887},
pmid = {40966510},
issn = {1362-4962},
support = {//University of Florida/ ; //UF Herbert Wertheim College of Engineering/ ; //Shah Foundation/ ; //Exxon Mobil Gator Alumni Faculty/ ; R21AI156321/GF/NIH HHS/United States ; R21AI168795/AI/NIAID NIH HHS/United States ; 5R61AI181016/AI/NIAID NIH HHS/United States ; R35GM147788/AI/NIAID NIH HHS/United States ; },
mesh = {*CRISPR-Associated Proteins/chemistry/genetics/metabolism ; *CRISPR-Cas Systems ; *DNA/chemistry/genetics/metabolism ; RNA, Guide, CRISPR-Cas Systems/genetics/metabolism/chemistry ; *Endodeoxyribonucleases/metabolism/genetics/chemistry ; *Acidaminococcus/enzymology/genetics ; *Bacterial Proteins/metabolism/chemistry/genetics ; Gene Editing ; DNA Cleavage ; Models, Molecular ; },
abstract = {CRISPR-Cas12a enzymes are RNA-guided nucleases widely used for programmable genome editing and diagnostics. Perfect complementarity between guide RNA and target DNA is essential for efficient binding and cleavage by Cas12a. However, we report that a particular ortholog of Cas12a, Acidaminococcus sp. Cas12a (AsCas12a), shows an unexpected tolerance to noncomplementary insertions at various positions in its DNA target. AsCas12a remains functional despite DNA bubbles or loops in the CRISPR-RNA (crRNA)-target DNA duplex, displaying both cis- and trans-cleavage activities even when the target harbors insertions of lengths 1-20 nucleotides in the crRNA-binding region. This activity is sequence-independent and works for ssDNA and is observed on dsDNA in vitro for specific insertion lengths/positions and DNA topologies but is strongly diminished in cells. Among 12 Cas12a orthologs tested, only AsCas12a exhibits this tolerance, making it a unique member of the Cas12a family. Structural analysis suggests a distinctive α-helix in AsCas12a's WED domain is required for this flexibility. Upon deleting this α-helix, AsCas12a loses its ability to tolerate insertions. This discovery can be utilized to detect single-nucleotide polymorphisms and enable protospacer adjacent motif (PAM)-flexible DNA cleavage with Cas12a. Our findings expand our fundamental understanding of CRISPR-Cas12a systems. In conclusion, we uncover and characterize a unique property of AsCas12a to tolerate insertions in its target.},
}
@article {pmid40966507,
year = {2025},
author = {Obi, I and Sengupta, P and Sabouri, N},
title = {CRISPR-Cas9 targeting of G-Quadruplex DNA in ADH1 promoter highlights its role in transcriptome and metabolome regulation.},
journal = {Nucleic acids research},
volume = {53},
number = {17},
pages = {},
doi = {10.1093/nar/gkaf853},
pmid = {40966507},
issn = {1362-4962},
support = {//Insamlingsstiftelsen at Ume&#xe5; University/ ; 22 2380 Pj 01 H//Swedish Cancer Society/ ; VR-MH 2021-02&#xa0;468//Swedish Research Council/ ; KAW 2021.0173//Knut and Alice Wallenberg Foundations/ ; UPD2020-0097//Wenner-Gren Foundations/ ; 24 0907 PT 01 H//Swedish Cancer Society/ ; },
mesh = {*G-Quadruplexes ; *Promoter Regions, Genetic ; *CRISPR-Cas Systems ; *Schizosaccharomyces/genetics/metabolism ; *Metabolome/genetics ; *Alcohol Dehydrogenase/genetics/metabolism ; *Transcriptome/genetics ; Gene Expression Regulation, Fungal ; Mutation ; *Schizosaccharomyces pombe Proteins/genetics/metabolism ; TATA Box ; NAD/metabolism ; },
abstract = {G-quadruplex (G4) structures are critical regulators of gene expression, yet the role of an individual G4 within its native chromatin remains underexplored, especially outside human systems. Here, we used CRISPR-Cas9 to introduce guanine-to-thymine mutations at a G4-forming motif within the adh1+ promoter in yeast Schizosaccharomyces pombe, creating two mutant strains: one with G4-only mutations and another with both G4 and TATA-box mutations. Chromatin immunoprecipitation using BG4 antibody confirmed reduced G4 enrichment in both mutants, validating G4 structure formation in the wild-type chromatin. Detailed characterizations demonstrated that the G4 mutations alter its dynamics without fully preventing its formation. These mutations significantly reduce adh1 transcript levels, with G4 TATA-box mutant causing the strongest transcriptional suppression. This indicates a positive regulatory role for the G4 structure in transcription. Furthermore, both mutants displayed altered transcriptomic profiles, particularly impacting the oxidoreductase pathway. Metabolomic analyses by mass spectrometry further highlighted substantial disruptions in NAD+/NADH metabolism, a key energy reservoir for metabolic regulation. These results highlight that tuning G4 dynamics, without abolishing the structure, can still profoundly affect gene expression and metabolism, unlike prior studies on the human MYC promoter that disrupted G4 formation. This represents the first such finding in yeast.},
}
@article {pmid40949944,
year = {2025},
author = {Guy, J and Hein, E and Alexander-Howden, B and von Bock Und Polach, T and Mathieson, T and Kleinstiver, BP and Zoghbi, HY and Bird, AP},
title = {Translational reading frame determines the pathogenicity of C-terminal frameshift deletions in MeCP2: an alternative therapeutic approach.},
journal = {bioRxiv : the preprint server for biology},
volume = {},
number = {},
pages = {},
pmid = {40949944},
issn = {2692-8205},
abstract = {Mutations in the MECP2 gene cause the severe neurological disorder Rett syndrome. A cluster of frameshift-causing C-terminal deletions (CTDs) lead to loss of ~100 amino acids at the C-terminus of the MeCP2 protein, and account for approximately 10% of RTT-causing mutations. The pathogenicity of C-terminal deletions (CTDs) is unexpected, as this C-terminal domain is non-essential in mice. Utilising databases of pathogenic and benign human MECP2 mutations, we find that some individuals with apparently typical CTDs do not exhibit Rett syndrome, confirming that C-terminal truncations are not intrinsically pathogenic. Using human DNA sequence data and mouse models, we demonstrate that pathogenicity results from a drastic reduction in MeCP2 levels and is determined by the presence of the short amino acid motif proline-proline-stop (-PPX) at the C-terminus, which results from a shift to the +2 reading frame. Individuals with CTDs that shift to the +1 frame avoid this motif and do not develop Rett syndrome. Mutating the stop codon of the PPX motif to tryptophan rescues MeCP2 expression and RTT-like phenotypes in a CTD mouse model. Finally, we demonstrate that an adenine base editor can efficiently introduce this tryptophan substitution in cultured cells. Overall, our findings uncover a simple and reliable prognostic distinction between benign and pathogenic CTDs and provide proof-of-concept for an editing strategy that potentially corrects all disease-causing CTD mutations.},
}
@article {pmid40928467,
year = {2025},
author = {Rijal, S and Standage-Beier, K and Zhang, R and Stone, A and Youssef, A and Wang, X and Tian, XJ},
title = {CRISPRi-Linked Multimodule Negative Feedback Loops to Address Winner-Take-All Resource Competition.},
journal = {ACS synthetic biology},
volume = {14},
number = {9},
pages = {3646-3654},
doi = {10.1021/acssynbio.5c00394},
pmid = {40928467},
issn = {2161-5063},
mesh = {*CRISPR-Cas Systems/genetics ; *Gene Regulatory Networks/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; *Feedback, Physiological ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Synthetic Biology/methods ; },
abstract = {Cellular resource limitations create unintended interactions among synthetic gene circuit modules, compromising circuit modularity. This challenge is particularly pronounced in circuits with positive feedback, where uneven resource allocation can lead to Winner-Takes-All (WTA) behavior, favoring one module at the expense of others. In this study, we experimentally implemented a Negatively Competitive Regulatory (NCR) controller using CRISPR interference (CRISPRi) and evaluated its effectiveness in mitigating WTA behavior in two gene circuits: dual self-activation and cascading bistable switch. We chromosomally integrated a tunable dCas9 gene and designed module-specific gRNAs, with each module encoding its own gRNA to self-repress via competition for limited dCas9. This configuration introduces strong negative feedback to the more active module while reallocating resources to the less active one, promoting balanced module activation. Compared to the control group lacking dCas9-mediated repression, the NCR controller significantly increased module coactivation and suppressed WTA behavior. Our quantitative results demonstrate that NCR provides an effective strategy for regulating resource competition and improving the modularity of synthetic gene circuits.},
}
@article {pmid40824237,
year = {2025},
author = {Holland, KL and Blancher, I and McKesey, M and Silas, M and Gandhi, S and Nickerson, A and Jackson, K and Blazeck, J},
title = {RNA Polymerase III Promoters Compatible with CRISPR Gene Regulation in Saccharomyces cerevisiae.},
journal = {ACS synthetic biology},
volume = {14},
number = {9},
pages = {3387-3400},
doi = {10.1021/acssynbio.5c00122},
pmid = {40824237},
issn = {2161-5063},
mesh = {*RNA Polymerase III/genetics/metabolism ; *Saccharomyces cerevisiae/genetics ; *Promoter Regions, Genetic/genetics ; *CRISPR-Cas Systems/genetics ; Gene Expression Regulation, Fungal ; RNA, Guide, CRISPR-Cas Systems/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Kluyveromyces/genetics ; },
abstract = {Saccharomyces cerevisiaeis a model organism commonly used to study gene regulation and function recently via CRISPR-(d)Cas9 technologies. Modulating the expression of multiple gene targets simultaneously is often important for synthetic biology and metabolic engineering applications and is crucial for genetic interaction studies. CRISPR-based systems can be used to target multiple genetic loci via expression of multiple single-guide RNAs (sgRNAs) in a single cell. However, there are currently a limited number of well-characterized RNA polymerase III (Pol III) promoters (e.g., pSNR52) for sgRNA expression in S. cerevisiae. Herein, we characterize 20 RNA Pol III promoters from different yeast species, from S. cerevisiae itself or from mammals, for their utility toward effectively mediating CRISPR activation and repression in S. cerevisiae. We show that the Pol III promoter cross-species functionality is impacted by promoter architecture and inclusion of core sequence motifs and that scaffold-mediated recruitment of multiple effectors can rescue poor promoter function in some contexts. Also, we highlight two Kluyveromyces lactis Pol III promoters that mediate CRISPR function as well as the gold standard S. cerevisiae pSNR52 and previously described tRNA promoters. Finally, we show that these non-native promoters enable effective simultaneous CRISPR-mediated activation and repression of endogenous S. cerevisiae genes to enhance resistance to hydrogen peroxide. The Pol III promoters described here highlight the cross-species compatibility of genetic units in simple eukaryotes and will be useful for synthetic biology and phenotype engineering applications in yeast.},
}
@article {pmid40810600,
year = {2025},
author = {Zhang, H and Khoury, LR and Xu, P},
title = {Multiplexed Genome Editing and Transcriptional Knockdown in Yarrowia lipolytica by CRISPR-Cpf1 and an Orthogonal T7 System.},
journal = {ACS synthetic biology},
volume = {14},
number = {9},
pages = {3377-3386},
doi = {10.1021/acssynbio.5c00104},
pmid = {40810600},
issn = {2161-5063},
mesh = {*Yarrowia/genetics/metabolism ; *Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; Promoter Regions, Genetic/genetics ; *Gene Knockdown Techniques/methods ; Transcription, Genetic ; Bacteriophage T7/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; },
abstract = {Yarrowia lipolytica, a nonconventional yeast, has become an industrial workhorse to synthesize valuable compounds, including lipids, oleochemicals, and nutraceuticals. While the synthetic biology toolkits to engineer the genome and endogenous metabolic pathways are not as developed as Baker's yeast, it has emerged as a promising microbial host for industrial applications. In this study, we examined the multiplexed editing capability of the CRISPR-AsCpf1 coupled with gRNAs generated from either a yeast native promoter or an orthogonal T7 promoter, which yielded 73.3% editing efficiency for up to four target genes and 100% editing efficiency for two genes. We also attempted two strategies to enhance homology-directed recombination (HDR) efficiency; only minor improvements were observed. We further demonstrated that CRISPR-dAsCpf1 with T7-driven gRNA achieved significant gene knockdown compared to the CRISPR-RfxCas13d system. Its knockdown efficiency was comparable to that of an antisense T7 promoter system. Taken together, this work provides a facile toolkit that enables efficient and multiplexed genome editing and transcriptional knockdown of critical genes by combining CRISPR-Cpf1 with an orthogonal T7 transcription system in Y. lipolytica.},
}
@article {pmid40492356,
year = {2025},
author = {Sha, R and Qin, H and Yuan, A and Deng, Z and Liao, W and Qu, G and Wen, B and Xie, W and Peng, H},
title = {Cellular Inflammation-Induced Cleavage of Phosphorothioate DNA Locker Activates CRISPR/Cas9 Regulator for Gene Editing.},
journal = {Chemistry, an Asian journal},
volume = {20},
number = {17},
pages = {e01696},
doi = {10.1002/asia.202401696},
pmid = {40492356},
issn = {1861-471X},
support = {2023YFA0915102//National Key Research and Development Program of China/ ; XDB0750100//Strategic Priority Research Program of the Chinese Academy of Sciences/ ; 22276199//National Natural Science Foundation of China/ ; 22206201//Youth Fund from National Natural Science Foundation of China/ ; },
mesh = {Humans ; *Gene Editing ; *CRISPR-Cas Systems/genetics ; HeLa Cells ; RNA, Guide, CRISPR-Cas Systems/genetics/metabolism ; *Inflammation/metabolism ; *Phosphorothioate Oligonucleotides/chemistry/metabolism ; *DNA/chemistry/metabolism/genetics ; Green Fluorescent Proteins/genetics ; },
abstract = {The CRISPR technology is a highly promising strategy for developing a versatile toolbox to engineer genetic circuits. However, achieving precise and specific control over the activity of the CRISPR/Cas9 system in response to intracellular processes remains a challenging endeavor. In this study, we present a cellular inflammation-induced activation of an engineered CRISPR/Cas9 regulator for gene regulation. A phosphorothioate (PS)-modified DNA sequence, referred as the "locker," is employed to deactivate single guide RNA (sgRNA), whose locker sequence complements the spacer region of sgRNA. In the presence of myeloperoxidase during cellular inflammation, a halogenation process is triggered, leading to the generation of HClO, specifically cleaving the PS site of locker and activating CRISPR/Cas9 for gene editing. The target GFP gene has been successfully edited, downregulating the GFP protein expression in HeLa cells. This study provides valuable insights into the CRISPR-based gene regulation through specific endogenous processes.},
}
@article {pmid40394347,
year = {2025},
author = {Kumar, N},
title = {Genome Editing in Gynecological Oncology: The Emerging Role of CRISPR/Cas9 in Precision Cancer Therapy.},
journal = {Therapeutic innovation &amp; regulatory science},
volume = {59},
number = {5},
pages = {937-948},
pmid = {40394347},
issn = {2168-4804},
mesh = {Humans ; *Gene Editing/methods ; *CRISPR-Cas Systems ; Female ; *Genital Neoplasms, Female/therapy/genetics ; *Precision Medicine/methods ; Genetic Therapy/methods ; },
abstract = {INTRODUCTION: Gynecological cancers, including cervical, ovarian, and endometrial cancers, represent a significant global health challenge due to their high prevalence and profound impact on mortality and quality of life. This narrative review explores the transformative capability of genome editing, specifically clustered regularly interspaced short palindromic repeats (CRISPR/Cas9) technology, in advancing the management of these cancers. Genome editing offers great opportunities to develop targeted therapies by enabling precise modifications of genes involved in cancer initiation, progression, and chemoresistance.<br><br>METHODOLOGY: A comprehensive literature search was conducted from October 2004 to October 2024. Only peer-reviewed relevant English articles with substantial insights into the impact of genome editing on cancer therapies were considered using keywords such as "CRISPR/Cas9," "genome editing," "gynecological cancers," and specific cancer types like "cervical cancer," "ovarian cancer," and "endometrial cancer."<br><br>CONCLUSION: Genome editing, particularly CRISPR/Cas9, holds substantial capacity for revolutionizing the treatment landscape of gynecological cancers by enabling highly specific, gene-targeted therapies that can overcome conventional treatment limitations such as chemoresistance and tumor recurrence. Emerging preclinical studies demonstrate the feasibility of correcting oncogenic mutations and enhancing the sensitivity of tumors to existing therapies. However, before clinical translation can be realized, critical challenges-including off-target effects, delivery system optimization, and immune responses-must be systematically addressed through rigorous research and clinical trials. Advancing these solutions will be essential for safely integrating CRISPR-based interventions into personalized medicine approaches for gynecological malignancies.},
}
@article {pmid40966498,
year = {2025},
author = {Xiao, Y and Chen, J and Hou, X and Wang, H and Zhang, K and Xu, S and Jiang, T and Huo, Y and Zhang, F and Gu, L},
title = {A NanoLock-enabled, Craspase-based strategy for highly sensitive RNA detection.},
journal = {Nucleic acids research},
volume = {53},
number = {17},
pages = {},
doi = {10.1093/nar/gkaf907},
pmid = {40966498},
issn = {1362-4962},
support = {2024YFC27007400//Key Technologies Research and Development Program/ ; 2020CXGC011305//Key Technology Research and Development Program of Shandong Province/ ; 32371276//National Natural Science Foundation of China/ ; 32400148//National Natural Science Foundation of China/ ; ts20220107//Instrument Improvement Funds of Shandong University Public Technology Platform/ ; XDB37010301//Strategic Priority Research Program of the Chinese Academy of Sciences/ ; },
mesh = {Humans ; *SARS-CoV-2/genetics/isolation &amp; purification ; *CRISPR-Cas Systems ; *RNA, Viral/genetics/analysis ; *COVID-19/diagnosis/virology ; RNA, Guide, CRISPR-Cas Systems/genetics ; Limit of Detection ; *Caspases/genetics/metabolism ; },
abstract = {Rapid and sensitive detection of RNA is important in fields such as biomedical research and clinical diagnostics. However, current methods typically involve an amplification process, require substantial time, and are susceptible to aerosol contamination. Herein, we introduce a NanoLock-powered, amplification-free assay based on the type III-E clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated system for rapid, highly sensitive, and specific RNA diagnostics. This innovative platform, designated CRISPR-guided caspase (Craspase)-NanoLock-Csx30 (CNC), harmoniously integrates the precise protease activity of Craspase with the remarkable luminescent sensitivity of NanoLock, creating a novel and streamlined approach for RNA detection. The CNC platform exhibited exceptional sensitivity in detecting severe acute respiratory syndrome coronavirus-2 N gene RNA through the integration of three guide RNAs, achieving a detection limit of 250 fM in just 10 min without amplification. Preliminary studies further revealed the platform's extended diagnostic potential for detecting influenza A virus and human immunodeficiency virus. These findings collectively establish the CNC platform as an appealing tool for infectious disease detection and significantly broaden the scope of CRISPR-based diagnostic applications.},
}
@article {pmid40966253,
year = {2025},
author = {Takahashi, M and Nashimoto, M},
title = {Cleavage of MALAT1 RNA by 14-nt sgRNA-guided tRNase ZL.},
journal = {PloS one},
volume = {20},
number = {9},
pages = {e0318968},
doi = {10.1371/journal.pone.0318968},
pmid = {40966253},
issn = {1932-6203},
mesh = {*RNA, Long Noncoding/genetics/metabolism ; Humans ; *Endoribonucleases/genetics/metabolism ; *RNA, Guide, CRISPR-Cas Systems/genetics ; Gene Silencing ; Cell Line, Tumor ; *RNA Cleavage ; },
abstract = {We have been developing a gene suppression technology, tRNase ZL-utilizing efficacious (TRUE) gene silencing, in which artificially designed small guide RNA (sgRNA) guides tRNase ZL to cleave cellular target RNA. In this study, we examined 14-nt linear-type sgRNAs, which are fully 2'-O-methylated and have full phosphorothioate linkages, for their ability to suppress a level of a nuclear-localized long non-coding RNA, Metastasis Associated Lung Adenocarcinoma Transcript 1 (MALAT1). The MALAT1 RNA is implied to be involved in stress responses and diseases including cancers. Specifically, we designed six 14-nt linear-type sgRNAs, sgRM1 - sgRM6 that target the human MALAT1 RNA. sgRM1, sgRM2 and sgRM6 suppressed the MALAT1 RNA level, while the other sgRNAs showed little effect. In order to demonstrate that the suppression effect of sgRM1, sgRM2 and sgRM6 on the MALAT1 RNA level is caused by TRUE gene silencing, we performed in vitro tRNase ZL cleavage assay, microscopic analysis for nuclear existence of sgRNA, and tRNase ZL knockdown experiment. For the in vitro tRNase ZL cleavage assay, three 30-nt MALAT1 RNA fragments, TM1, TM2 and TM6 were prepared, which were RNA targets for sgRM1, sgRM2 and sgRM6, respectively. All of the sgRNAs guided recombinant tRNase ZL in vitro to cleave their own targets, although the cleavage efficiency changed depending on target/sgRNA pairs. By fluorescence microscopy, a 14-nt 5'-Alexa568-labeled sgRNA released from liposome was observed to be distributed ubiquitously in A549 cells with higher density in the nucleus, where both the target MALAT1 RNA and tRNase ZL exist. Knockdown of tRNase ZL by siRNA attenuated the suppression effect of sgRM1, sgRM2 and sgRM6 on the MALAT1 RNA level. We also demonstrated that the effective sgRNAs sgRM1, sgRM2 and sgRM6 reduce A549 cell viability.},
}
@article {pmid40964963,
year = {2025},
author = {Shigeta, M and Inoue, KI and Shimada, N and Tobe, A and Abe, T and Kiyonari, H},
title = {Generation of Knock-In Syrian Hamsters via Zygote Microinjection Using CRISPR/Cas9 Genome Editing.},
journal = {Genesis (New York, N.Y. : 2000)},
volume = {63},
number = {5},
pages = {e70027},
doi = {10.1002/dvg.70027},
pmid = {40964963},
issn = {1526-968X},
support = {//RIKEN Intramural/ ; JP20H05767//JSPS KAKENHI/ ; JP23H04945//JSPS KAKENHI/ ; JP23H04939//JSPS KAKENHI/ ; },
mesh = {Animals ; *CRISPR-Cas Systems ; *Mesocricetus/genetics ; *Gene Editing/methods ; *Zygote/metabolism ; *Gene Knock-In Techniques/methods ; Microinjections/methods ; Cricetinae ; Female ; Male ; },
abstract = {Syrian hamsters (Mesocricetus auratus) have long served as valuable model organisms in diverse research fields such as oncology, immunology, and physiology owing to their unique biological and pathological characteristics. Although embryo manipulation techniques such as embryo collection, pronuclear microinjection, and embryo transfer have been established, gene knock-in (KI) hamsters have not yet been reported. Here, we report the successful generation of gene KI Syrian hamsters by microinjecting CRISPR/Cas9 components and plasmid DNA into pronuclear-stage zygotes. Targeted insertion of a DNA cassette up to 8 kb was achieved at the ROSA26 orthologous locus and other genomic sites. Importantly, we confirmed functional expression of a reporter cassette inserted at the ROSA26 site, providing evidence of transcriptional activity at this locus in Syrian hamsters. Furthermore, we demonstrated that frozen-thawed KI embryos could give rise to live offspring using a simplified freezing and thawing protocol originally developed for mice and rats. These results confirm the feasibility and applicability of advanced genome editing technologies in Syrian hamsters. These technological advancements enable the development of versatile KI models for applications such as gene expression monitoring and conditional mutagenesis, thereby expanding the utility of Syrian hamsters as model organisms, comparable to mice and rats.},
}
@article {pmid40963829,
year = {2025},
author = {Ton, LB and Qayyum, Z and Amas, J and Thomas, WJW and Edwards, D and Batley, J and Dolatabadian, A},
title = {Applications of CRISPR/Cas tools in improving stress tolerance in Brassica crops.},
journal = {Frontiers in plant science},
volume = {16},
number = {},
pages = {1616526},
pmid = {40963829},
issn = {1664-462X},
abstract = {Brassica species, which include economically important Brassica crops grown around the globe, are important as popular vegetables, forage, and oilseed crops, supplying food for humans and animals. Despite their importance, these crops face increasing challenges from biotic and abiotic stresses, exacerbated by climate change and the evolving threat of crop pathogens. Enhancing crop resilience against these stresses has become a key priority to ensure stable crop production. Recent advancements in genomic studies on Brassica crops and their pathogens have facilitated the deployment of CRISPR/Cas systems in breeding major Brassica crops. This review highlights recent progress in CRISPR/Cas-based gene editing technologies to improve resistance to pathogens and enhance tolerance to drought, salinity, and extreme temperatures. It also summarises the molecular mechanisms underlying crop responses to these stresses. Furthermore, the review discusses the workflow for employing the CRISPR/Cas system to boost stress tolerance and resistance, outlines the associated challenges, and explores prospects based on gene editing research in Brassica species.},
}
@article {pmid40963206,
year = {2025},
author = {Nie, YF and Yue, SJ and Huang, P and Hu, DK and Xu, Z and Aguilar-Vera, A and Carreri, JU and Zhang, XH and Hu, HB},
title = {Harnessing the Endogenous Type I-F CRISPR/Cas System for Efficient Genome Engineering and Gene Repression in Pseudomonas chlororaphis LX24.},
journal = {ACS synthetic biology},
volume = {},
number = {},
pages = {},
doi = {10.1021/acssynbio.5c00371},
pmid = {40963206},
issn = {2161-5063},
abstract = {Pseudomonas chlororaphis, a nonpathogenic plant growth-promoting rhizobacterium, holds immense potential for agricultural and industrial applications due to its ability to biosynthesize bioactive metabolites. However, the lack of efficient genetic tools has hindered its metabolic engineering. In this study, we first characterized an endogenous type I-F CRISPR/Cas system in P. chlororaphis LX24 and established a programmable genome editing toolkit based on this system. Concurrently, the plasmid transformation efficiency of P. chlororaphis LX24 was enhanced by identifying and deleting the restriction-modification systems. We further demonstrated the DNA interference capability with different PAM sequences of the type I-F CRISPR/Cas system, which also exhibited various editing efficiencies ranging from 22 to 87% in P. chlororaphis LX24. By introducing the λ-Red recombination system, the knockout efficiency of the phenazine cluster (8.3 kb) increased by over 9-fold. Next, introducing the sacB-based counterselection marker achieved a 100% plasmid curing success within 36 h. The optimized toolkit was further applied to single-step gene insertion and replacement with 100% success rates. Additionally, we established a CRISPR interference (CRISPRi) system for transcriptional repression in P. chlororaphis LX24 by knocking out nuclease Cas3. Through modulating the induction time and concentration of IPTG, the production of phenazines was reduced to 21-89% within 24 h in P. chlororaphis LX24. Overall, our work developed a convenient and precise genetic tool for the P. chlororaphis LX24, and the methods may also provide a reference for repurposing endogenous CRISPR systems in non-model prokaryotes.},
}
@article {pmid40962795,
year = {2025},
author = {Huang, Z and Song, Z and Zeng, J and Liu, X and Fang, M and Wu, Z and Zhao, Y and Chen, Y and Li, D and Huang, H and Fu, L and Xu, P and Ning, B and Chen, J and Guan, M and Sun, L and Lyon, CJ and Fan, XY and Lu, S and Hu, T},
title = {Sensitive pathogen DNA detection by a multi-guide RNA Cas12a assay favoring trans- versus cis-cleavage.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {8257},
pmid = {40962795},
issn = {2041-1723},
support = {R01HD090927, R01HD103511//U.S. Department of Health &amp; Human Services | NIH | Eunice Kennedy Shriver National Institute of Child Health and Human Development (NICHD)/ ; R01AI144168, R01AI175618, R01AI173021, R01AI174964, R01AI177986, R01AI179714, R21AI169582-01A1//U.S. Department of Health &amp; Human Services | NIH | National Institute of Allergy and Infectious Diseases (NIAID)/ ; W8IXWH1910026//U.S. Department of Defense (United States Department of Defense)/ ; Weatherhead Presidential Endowment fund//Tulane University/ ; 82302614//National Natural Science Foundation of China (National Science Foundation of China)/ ; },
mesh = {Humans ; *Mycobacterium tuberculosis/genetics/isolation &amp; purification ; *CRISPR-Cas Systems/genetics ; Sensitivity and Specificity ; *DNA, Bacterial/genetics ; *RNA, Guide, CRISPR-Cas Systems/genetics ; *CRISPR-Associated Proteins/genetics ; *Molecular Diagnostic Techniques/methods ; Adult ; Feces/microbiology ; Tuberculosis, Meningeal/diagnosis/microbiology/cerebrospinal fluid ; Child ; Bacterial Proteins ; Endodeoxyribonucleases ; },
abstract = {Most CRISPR assays lack clinical utility due to their complex workflows and limited validation. Here we present a streamlined "one-pot" asymmetric CRISPR tuberculosis assay that attenuates amplicon degradation to achieve 5 copies/μL sensitivity within 60 min and detect positive patient samples within 15 min. This assay exhibited 93%, 83%, and 93% sensitivity with adult respiratory, pediatric stool, and adult cerebral spinal fluid specimens, and detected 64% of clinically diagnosed tuberculous meningitis cases, in a cohort of 603 clinical samples. This assay achieves complete specificity and greater sensitivity (74% vs. 56%) than the most sensitive reference test with prospectively collected tongue swabs, and exhibits similar performance when adapted to a lateral flow assay format and employed to analyze self-collected tongue swabs. These results demonstrate the utility of this approach across diverse specimen types, including those suitable for use in remote and resource-limited settings, to improve access to molecular diagnostics.},
}
@article {pmid40962474,
year = {2025},
author = {Gao, YH and Deng, JJ and Xiao, X and Pan, LY and He, MD and Zhang, YB},
title = {Role of ppp6r3 in zebrafish gonadal differentiation and gametogenesis.},
journal = {Yi chuan = Hereditas},
volume = {47},
number = {9},
pages = {1023-1031},
doi = {10.16288/j.yczz.25-093},
pmid = {40962474},
issn = {0253-9772},
mesh = {Animals ; *Zebrafish/genetics/metabolism ; *Gametogenesis/genetics ; Male ; *Zebrafish Proteins/genetics/metabolism ; Female ; *Phosphoprotein Phosphatases/genetics/metabolism ; *Gonads/growth &amp; development/metabolism/cytology ; Cell Differentiation/genetics ; Mutation ; Spermatogenesis/genetics ; Testis/growth &amp; development/metabolism ; CRISPR-Cas Systems ; },
abstract = {The gonad differentiation and gametogenesis of fish is regulated by various factors. Protein phosphatase (PP) have the function of catalyzing the dephosphorylation of proteins in organisms. As a member of the protein phosphatase family, protein phosphatase type 6 (PP6) plays an important role in gonadal development and gametogenesis. The role of ppp6r3, which encodes the regulatory subunit 3 of protein phosphatase 6, in gonadal differentiation and gametogenesis is still unclear. In this study, two zebrafish ppp6r3 mutant lines were constructed using CRISPR/Cas9 technology. It was found that the absence of ppp6r3 leads to the development of only male zebrafish, and these mutants are incapable of fertilizing wild-type eggs. Further investigations revealed that in the testes of ppp6r3 mutants, the transition of spermatocytes to mature sperm was blocked, disrupting spermatogenesis. In summary, this study established a ppp6r3 mutant model, exhibiting defects in gonadal differentiation and gametogenesis, which provides a new model for further research on the mechanisms by which Ppp6r3 regulates germ cell proliferation, differentiation, and sex determination.},
}
@article {pmid40961787,
year = {2025},
author = {Zhang, H and Wu, Y and Liu, D and Feng, S and Xuan, X and Dong, G and Cheng, J and Qin, Y and Ngo, HH},
title = {Insights into microalgal biotechnology: Current applications, key challenges, and future prospects.},
journal = {Journal of environmental management},
volume = {394},
number = {},
pages = {127263},
doi = {10.1016/j.jenvman.2025.127263},
pmid = {40961787},
issn = {1095-8630},
abstract = {Microalgae have emerged as multifunctional biofactories capable of simultaneously supporting carbon capture, renewable energy production, environmental remediation, and the synthesis of high value bioproducts. Despite this promise, large-scale deployment remains limited by techno-economic barriers, particularly the high costs of biomass harvesting and dewatering. Recent advances including bioflocculation, magnetic separation, and solar-assisted drying are helping to reduce energy inputs and enhance feasibility. In parallel, breakthroughs in synthetic biology, such as CRISPR/Cas genome editing, are enabling the development of engineered strains with enhanced lipid, carbohydrate, and hydrogen productivity. Innovations in photobioreactor design have further improved light-use efficiency, reduced contamination risks, and supported high-density cultivation. Life cycle assessments indicate that integrating microalgal systems with flue gas utilization and wastewater treatment can substantially lower freshwater use and greenhouse gas emissions. To unlock the full potential of this technology, future efforts should prioritize modular biorefinery systems, intelligent process control, and supportive policy frameworks that incentivise negative-emission technologies. These integrated strategies can help position microalgae as a key enabler of a sustainable, circular bioeconomy.},
}
@article {pmid40959562,
year = {2025},
author = {Tang, S and Chen, X and Tong, X and Zhu, L},
title = {Overcoming the Delivery Challenges in CRISPR/Cas9 Gene Editing for Effective Cancer Treatment: A Review of Delivery Systems.},
journal = {International journal of medical sciences},
volume = {22},
number = {14},
pages = {3625-3649},
pmid = {40959562},
issn = {1449-1907},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; *Neoplasms/therapy/genetics ; *Genetic Therapy/methods ; Animals ; Genetic Vectors/genetics/administration &amp; dosage ; *Gene Transfer Techniques ; Extracellular Vesicles/genetics ; },
abstract = {Therapeutic strategies based on gene editing provide the ability to modify faulty genes contributing to the development of diseases such as cancer by directly altering the cellular machinery. The clustered regularly interspaced short palindromic repeats associated nuclease 9 (CRISPR/Cas9) system is currently the primary tool used for gene editing. Several effective Cas9 variants have already been established to address the complex genetic modifications that arise during diseases. Although gene-editing systems have made significant advancements, a primary obstacle that requires attention is the transportation of CRISPR/Cas to diverse target cells, both in vivo and in vitro, to render them suitable for clinical implementation. Various strategies can be utilized to facilitate the transportation of the CRISPR/Cas systems into mammalian cells. Herein, we reviewed contemporary research about delivery systems for gene-editing systems that interact effectively in biological systems. This review explores the benefits and drawbacks of using extracellular vesicles and viral vectors as vehicles for delivering the CRISPR/Cas system in the context of cancer treatment.},
}
@article {pmid40938937,
year = {2025},
author = {Hossain, MF and Popsuj, S and Vitrinel, B and Kaplan, NA and Stolfi, A and Christiaen, L and Ruggiu, M},
title = {Neuron-specific Agrin splicing by Nova RNA-binding proteins regulates conserved neuromuscular junction development in chordates.},
journal = {PLoS biology},
volume = {23},
number = {9},
pages = {e3003392},
doi = {10.1371/journal.pbio.3003392},
pmid = {40938937},
issn = {1545-7885},
support = {R01 HD096770/HD/NICHD NIH HHS/United States ; R01 GM096032/GM/NIGMS NIH HHS/United States ; R01 HD104825/HD/NICHD NIH HHS/United States ; R15 GM119099/GM/NIGMS NIH HHS/United States ; R01 HL108643/HL/NHLBI NIH HHS/United States ; R35 GM158421/GM/NIGMS NIH HHS/United States ; },
mesh = {Animals ; *Neuromuscular Junction/metabolism/growth &amp; development ; *Agrin/genetics/metabolism ; *RNA-Binding Proteins/metabolism/genetics ; Alternative Splicing ; Neuro-Oncological Ventral Antigen ; *Nerve Tissue Proteins/metabolism/genetics ; Motor Neurons/metabolism ; Receptors, Cholinergic/metabolism ; Neurons/metabolism ; Humans ; CRISPR-Cas Systems ; },
abstract = {In mammals, neuromuscular synapses rely on clustering of acetylcholine receptors (AChRs) in the muscle plasma membrane, ensuring optimal stimulation by motor neuron-released acetylcholine neurotransmitter. This clustering depends on a complex pathway based on alternative splicing of Agrin pre-mRNAs by the RNA-binding proteins Nova1/2. Neuron-specific expression of Nova1/2 ensures the inclusion of small "Z" exons in Agrin, resulting in a neural-specific form of this extracellular proteoglycan carrying a short peptide motif that is required for binding to Lrp4 receptors on the muscle side, which in turn stimulate AChR clustering. Here we show that this intricate pathway is remarkably conserved in Ciona robusta, a non-vertebrate chordate in the tunicate subphylum. We use in vivo tissue-specific CRISPR/Cas9-mediated mutagenesis and heterologous "minigene" alternative splicing assays in cultured mammalian cells to show that Ciona Nova is also necessary and sufficient for Agrin Z exon inclusion and downstream Lrp4-mediated AChR clustering. We present evidence that, although the overall pathway is well conserved, there are unexpected differences in Nova structure-function between Ciona and mammals. We further show that, in Ciona motor neurons, the transcription factor Ebf is a key activator of Nova expression, thus ultimately linking this RNA switch to a conserved, motor neuron-specific transcriptional regulatory network.},
}
@article {pmid40889206,
year = {2025},
author = {Li, T and Wang, Y and Zhang, X and Wu, Z and Zhang, L},
title = {CRISPR-SDA: an integrated isothermal amplification and CRISPR-Cas12a biosensing platform for sensitive detection of microRNA-21.},
journal = {Analytical methods : advancing methods and applications},
volume = {17},
number = {36},
pages = {7266-7271},
doi = {10.1039/d5ay01227a},
pmid = {40889206},
issn = {1759-9679},
mesh = {*MicroRNAs/genetics/analysis/blood ; *CRISPR-Cas Systems/genetics ; *Nucleic Acid Amplification Techniques/methods ; *Biosensing Techniques/methods ; Humans ; Limit of Detection ; Animals ; Cattle ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {MicroRNA-21 (miRNA-21), a critical oncogenic biomarker, poses detection challenges due to low abundance and limitations of conventional methods. Herein, we developed a novel CRISPR-SDA biosensing platform by integrating strand displacement amplification (SDA) with CRISPR-Cas12a, leveraging SDA's efficient isothermal amplification of miRNA-21 and Cas12a's precise target recognition and trans-cleavage activity for signal amplification. Optimized conditions achieved high sensitivity with a detection limit of 10.1 fM and segmented linear ranges of 0.05-25 pM and 25-500 pM. It showed excellent selectivity against other miRNAs and anti-interference in complex matrices (e.g., salmon sperm DNA). Spike recovery experiments in fetal bovine serum yielded recoveries of 92.0 ± 7.8% to 105.6 ± 3.3%, confirming its reliability in complex biological samples. This CRISPR-SDA platform overcomes the drawbacks of conventional methods, enabling rapid, sensitive, and equipment-friendly detection. It holds great potential for early cancer diagnosis and point-of-care testing and provides a versatile framework for detecting other disease-associated nucleic acids.},
}
@article {pmid40865701,
year = {2025},
author = {Gao, J and Li, W and Li, M and Ji, G and Liu, Z},
title = {Cd36 deficiency enhances the immune defence against Grass Carp Reovirus in zebrafish.},
journal = {Developmental and comparative immunology},
volume = {170},
number = {},
pages = {105446},
doi = {10.1016/j.dci.2025.105446},
pmid = {40865701},
issn = {1879-0089},
mesh = {Animals ; *Zebrafish/immunology/virology/genetics ; *Reoviridae/immunology/physiology ; *CD36 Antigens/genetics/metabolism ; *Reoviridae Infections/immunology ; *Fish Diseases/immunology/virology ; Immunity, Innate ; CRISPR-Cas Systems/genetics ; *Zebrafish Proteins/genetics/metabolism ; Carps/immunology ; Transcriptome ; Liver ; Gene Expression Profiling ; Viral Load ; },
abstract = {CD36, a single-chain transmembrane glycoprotein belonging to the class B scavenger receptor family, remains enigmatic in terms of its antiviral immune function, with some conflicting conclusions. This study aims to elucidate the role of Cd36 in antiviral immunity using zebrafish as a model organism. We generated cd36[-/-] zebrafish mutants using CRISPR/Cas9 technology and challenged them with Grass Carp Reovirus (GCRV), a dsRNA virus. Compared to wild-type (WT) zebrafish, cd36[-/-] zebrafish exhibited stronger antiviral immunity, reduced viral load, and lower mortality. Transcriptome sequencing analysis of liver tissue revealed that the differentially expressed genes (DEGs) between WT and cd36[-/-] zebrafish were primarily enriched in metabolic pathways, such as glycan biosynthesis and fatty acid metabolism. This suggests that Cd36 may modulate antiviral immune responses by influencing metabolic activities. Our findings provide a foundation for further investigations into the functional and molecular mechanisms of Cd36 in antiviral immunity in fish.},
}
@article {pmid40959550,
year = {2025},
author = {Mahto, RK and B S, C and Singh, RK and Kumar, A and Kumar, S and Yadav, R and Dey, D and Hamwieh, A and Kumar, R},
title = {Symbiotic nitrogen fixation for sustainable chickpea yield and prospects for genome editing in changing climatic situations.},
journal = {Frontiers in plant science},
volume = {16},
number = {},
pages = {1621191},
pmid = {40959550},
issn = {1664-462X},
abstract = {Chickpea (Cicer arietinum L.) is a vital/essential legume crop valued for its nutritional, agricultural, and economic importance, with a relatively large genome size of approximately 738 megabases. Chickpea roots establish symbiotic relationships with soil microorganisms, resulting in the formation of root nodules essential for biological nitrogen fixation. In this study, 20 chickpea genotypes were selected from a genome-wide association panel to assess nodulation traits under eight different treatment combinations involving biofertilizers (Rhizobium, vesicular-arbuscular mycorrhiza - VAM) and inorganic fertilizers (NPK) using a randomized block design with three replications. Pre-planting soil preparation included the application of fertilizers and biofertilizers. Comprehensive analyses including descriptive statistics, correlation, path analysis, principal component analysis, agglomerative hierarchical clustering, and gene expression studies were conducted. Among treatments, the NPK+Rhizobium combination significantly enhanced nodulation across genotypes, while the Rhizobium+VAM (T7) treatment identified ICC-9085 as a superior donor for the number of nodules, aiming for sustainable chickpea productivity. Gene expression profiling through qRT-PCR revealed that the RZ+VAM treatment notably upregulated several key genes, including CaNFP, GST, Leghemoglobin, Nodulin6, and CaLYK3, with CaNFP emerging as a pivotal regulator of nodulation. The marked upregulation of CaNFP underlines its potential as a target for enhancing symbiotic efficiency. The availability of the chickpea draft genome opens new avenues for employing genome editing tools such as CRISPR/Cas systems. Targeted editing of the CaNFP gene offers a promising strategy to improve nodule formation, nitrogen fixation, and overall plant vigor. Integrating CaNFP gene through genome editing with potential genotypes and use of microbial treatments can accelerate the development of elite chickpea cultivars, enhancing productivity while reducing reliance on chemical fertilizers and supporting sustainable agricultural practices.},
}
@article {pmid40959136,
year = {2025},
author = {Zhou, D and Yu, H and Shao, Y and Gao, C and Xia, C and Qi, Y},
title = {Rapid and accurate detection method for bluetongue virus based on CRISPR-Cas13a combined with RT-ERA.},
journal = {Frontiers in cellular and infection microbiology},
volume = {15},
number = {},
pages = {1621012},
pmid = {40959136},
issn = {2235-2988},
mesh = {*Bluetongue virus/isolation &amp; purification/genetics ; Animals ; *Bluetongue/diagnosis/virology ; Sensitivity and Specificity ; *CRISPR-Cas Systems ; *Nucleic Acid Amplification Techniques/methods ; *Molecular Diagnostic Techniques/methods ; Sheep ; RNA, Viral/genetics ; },
abstract = {INTRODUCTION: Bluetongue virus (BTV), a vector-borne pathogen of domestic and wild ruminants, poses substantial global threats to livestock health and trade. Conventional detection methods, such as RT-qPCR, remain constrained by reliance on specialized equipment and prolonged turnaround times, limiting their utility in field settings.<br><br>METHODS: To overcome these challenges, we developed an integrated isothermal amplification-CRISPR detection platform-Reverse Transcription-Enzymatic Recombinase Amplification coupled with CRISPR-Cas13a (RT-ERA/CRISPR-Cas13a)-enabling rapid, sensitive, specific and visual pan-serotype detection of BTV.<br><br>RESULTS: The assay demonstrated a sensitivity of 20 RNA copies/reaction within 55 min using three readout modalities: fluorescence values, visual fluorescence signals, and lateral flow test strips. Specificity evaluation revealed no cross-reactivity with 9 non-target pathogens, including epidemiologically significant viruses such as EHDV, AKAV, and CHUV. Clinical validation using 263 field samples demonstrated that RT-ERA/CRISPR-Cas13a achieved clinical sensitivities of 100%, 100%, and 96% with fluorescence values, fluorescence signals, and lateral flow strips, respectively, while maintaining 100% clinical specificity via all modalities. Field adaptation using Nucleic Acid Release Reagent (NARR) simplified crude sample processing, delivering 97% clinical sensitivity and 100% clinical specificity even in the presence of inhibitors from unpurified samples.<br><br>CONCLUSION: This work represents the first CRISPR-Cas13a-based platform for pan-serotype BTV detection, combining portability, cost-efficiency, and detective accuracy suitable for point-of-care deployments. By bridging the gap between high laboratory sensitivity and practical field applicability, this system enables real-time BTV surveillance and facilitates timely outbreak containment in resource-constrained agricultural and veterinary settings.},
}
@article {pmid40957902,
year = {2025},
author = {Ekins, TG and Rybicki-Kler, C and Deng, T and Brooks, IAW and Jedrasiak-Cape, I and Donoho, E and Ahmed, OJ},
title = {Psychedelic neuroplasticity of cortical neurons lacking 5-HT2A receptors.},
journal = {Molecular psychiatry},
volume = {},
number = {},
pages = {},
pmid = {40957902},
issn = {1476-5578},
support = {R01MH129282//U.S. Department of Health &amp; Human Services | NIH | National Institute of Mental Health (NIMH)/ ; R34NS127101//U.S. Department of Health &amp; Human Services | NIH | National Institute of Neurological Disorders and Stroke (NINDS)/ ; P50NS123067//U.S. Department of Health &amp; Human Services | NIH | National Institute of Neurological Disorders and Stroke (NINDS)/ ; AARG-NTF-21-846572//Alzheimer&apos;s Association/ ; T32DA007268//U.S. Department of Health &amp; Human Services | NIH | National Institute on Drug Abuse (NIDA)/ ; T32DC000011//U.S. Department of Health &amp; Human Services | NIH | National Institute on Deafness and Other Communication Disorders (NIDCD)/ ; T32DC000011//U.S. Department of Health &amp; Human Services | NIH | National Institute on Deafness and Other Communication Disorders (NIDCD)/ ; },
abstract = {Classical psychedelic drugs show promise as a treatment for major depressive disorder and related psychiatric disorders. This therapeutic efficacy stems from long-lasting psychedelic-induced neuroplasticity onto prefrontal cortical neurons and is thought to require the postsynaptic expression of serotonin 2A receptors (5-HT2AR). However, other cortical regions such as the granular retrosplenial cortex (RSG) - important for memory, spatial orientation, fear extinction, and imagining oneself in the future, but impaired in Alzheimer's disease - lack 5-HT2AR and are thus considered unlikely to benefit from psychedelic therapy. Here, we show that RSG pyramidal cells lacking postsynaptic 5-HT2A receptors still undergo long-lasting psychedelic-induced synaptic enhancement. A newly engineered CRISPR-Cas-based conditional knockout mouse line reveals that this form of psychedelic-induced retrosplenial plasticity requires presynaptic 5-HT2A receptors expressed on anterior thalamic axonal inputs to RSG. These results highlight a broader psychedelic therapeutic utility than currently appreciated, suggesting potential for augmenting RSG circuit function in Alzheimer's disease, post-traumatic stress disorder, and other neuropsychiatric conditions, despite the lack of postsynaptic 5-HT2A receptors.},
}
@article {pmid40956304,
year = {2025},
author = {Zhuang, Q and Xiao, C and Tang, S and Song, Y and Chen, F and Zhao, G},
title = {Single-Cas, Single-Reporter, and Time-Resolved CRISPR-Cas by Stoichiometry Coding of Multiplex crRNAs in a Single Tube for Brucella Species and ABO Genotype Identification.},
journal = {ACS sensors},
volume = {},
number = {},
pages = {},
doi = {10.1021/acssensors.5c01034},
pmid = {40956304},
issn = {2379-3694},
abstract = {Genotyping is significant in identifying pathogen gene types and disease-related allele genes. Multiplex nucleic acid detection is convenient and useful for genotyping. Though CRISPR-Cas has great advantages in nucleic acid detection, multiplex detection strategies remain to be developed. Here, we propose a novel CRISPR-Cas strategy characterized by using stoichiometry coding of multiplex crRNAs. Different targets can be recognized by the corresponding crRNA with a certain amount, leading to distinguishable fluorescence difference. We employed this strategy to achieve Brucella species identification and human cell ABO blood genotyping. The detection can be conveniently visualized in a time-resolved way using a single tube, single Cas, and single reporter. The strategy can be employed in a PCR-coupled, RAA-coupled, or amplification-free way. The multiplex crRNA-coding CRISPR-Cas has potential application in differentiating gene types in various fields.},
}
@article {pmid40956103,
year = {2025},
author = {Babajanyan, SG and Garushyants, SK and Wolf, YI and Koonin, EV},
title = {Evolution of antivirus defense in prokaryotes, depending on the environmental virus prevalence and virome dynamics.},
journal = {mBio},
volume = {},
number = {},
pages = {e0240925},
doi = {10.1128/mbio.02409-25},
pmid = {40956103},
issn = {2150-7511},
abstract = {UNLABELLED: Prokaryotes can acquire antivirus immunity via two fundamentally distinct types of processes: direct interaction with the virus, as in clustered regularly interspaced short palindromic repeats (CRISPR)-Cas adaptive immunity systems, and horizontal gene transfer (HGT), which is the main route of transmission of innate immunity systems. These routes of defense evolution are not mutually exclusive and can operate concomitantly, but observations suggest that at least in some bacterial and archaeal species, one or the other route dominates the defense landscape. We hypothesized that the observed dichotomy stems from different life-history trade-offs characteristic of these organisms. To test this hypothesis, we analyzed a mathematical model of a well-mixed prokaryote population under a stochastically changing viral prevalence. Optimization of the long-term population growth rate reveals two contrasting modes of defense evolution. In stable, predictable environments, direct interaction with the virus is the optimal route of immunity acquisition. In fluctuating, unpredictable environments with a moderate viral prevalence, horizontal transfer of defense genes is preferred. In the HGT-dominant mode, we observed a universal distribution of the fraction of microbes with different immune repertoires. Under very low virus prevalence, the cost of immunity exceeds the benefits such that the optimal state of a prokaryote is complete absence of defense systems. By contrast, under very high virus prevalence, horizontal spread of defense systems dominates regardless of the stability of the virome. These findings might explain consistent but enigmatic patterns in the spread of antivirus defense systems among prokaryotes, such as the ubiquity of adaptive immunity in hyperthermophiles contrasting their patchy distribution among mesophiles.<br><br>IMPORTANCE: The virus-host arms race is a major component of the evolutionary process in all organisms that drove the evolution of a broad variety of immune mechanisms. In the last few years, over 200 distinct antivirus defense systems have been discovered in prokaryotes. There are two major modes of immunity acquisition: innate immune systems spread through microbial populations via HGT, whereas adaptive-type immune systems acquire immunity via direct interaction with the virus. We developed a mathematical model to explore the short-term evolution of prokaryotic immunity and showed that in stable environments with predictable viral repertoires, adaptive-type immunity is the optimal defense strategy, whereas in fluctuating environments with unpredictable virus composition, HGT dominates the immune landscape.},
}
@article {pmid40954158,
year = {2025},
author = {Gottschling, DC and Döring, F},
title = {The novel HRD motif kinase SPE-60 is required for sperm development and motility in Caenorhabditis elegans.},
journal = {Scientific reports},
volume = {15},
number = {1},
pages = {32539},
pmid = {40954158},
issn = {2045-2322},
mesh = {*Caenorhabditis elegans/genetics/physiology/metabolism ; Male ; Animals ; *Sperm Motility/genetics ; *Caenorhabditis elegans Proteins/metabolism/genetics/chemistry ; *Spermatozoa/metabolism ; *Protein Serine-Threonine Kinases/metabolism/genetics ; *Spermatogenesis ; Amino Acid Motifs ; },
abstract = {Sexual reproduction requires sperm development, activation and motility. Many of these post-meiotic processes remain incompletely understood. We identified the gene spe-60, which encodes a tau-tubulin kinase with a conserved catalytic HRD motif. Localisation via immunostaining reveals that SPE-60 is present in sperm and dynamically colocalises with membranous organelles (MOs), that store factors essential for activation and fertility. spe-60 null mutants are sterile due to immotile sperm that undergo normal MO-plasma membrane fusion but fail to extend normal pseudopods essential for motility and fertilisation. Furthermore, mutant sperm exhibit arrested spermatocyte development. The functional loss observed in spe-60 null sperm is phenocopied by sperm carrying a CRISPR/Cas-mediated point mutation within the HRD motif. Genetic epistasis places SPE-60 downstream of the serine/threonine kinase SPE-6 in the canonical sperm activation pathway. Thus, SPE-60 is required for sperm development and motility, but not MO fusion. These findings suggest that the HRD-containing kinase SPE-60 fulfils a dual function as a key evolutionarily conserved factor.},
}
@article {pmid40938948,
year = {2025},
author = {He, W and Yang, Z and Wang, J and Yang, F and Li, N and Xu, R and Zeng, S and Xiao, L and Feng, Y and Guo, Y},
title = {Cryptosporidium parvum protease INS6 plays an important role in parasite proliferation and pathogenicity.},
journal = {PLoS neglected tropical diseases},
volume = {19},
number = {9},
pages = {e0013532},
doi = {10.1371/journal.pntd.0013532},
pmid = {40938948},
issn = {1935-2735},
mesh = {*Cryptosporidium parvum/pathogenicity/genetics/enzymology/growth &amp; development ; Animals ; *Cryptosporidiosis/parasitology/pathology ; Mice ; *Protozoan Proteins/genetics/metabolism ; Humans ; Mice, Knockout ; Male ; Virulence ; *Peptide Hydrolases/genetics/metabolism ; CRISPR-Cas Systems ; Gene Deletion ; },
abstract = {BACKGROUND: Cryptosporidium parvum is a common protozoan pathogen responsible for moderate to severe diarrhea in humans and animals. Parasite invasion and egress cause damage to intestinal epithelial cells, which is mediated by a variety of secretory proteins from several unique organelles, such as micronemes. Previous spatial proteomic analysis has identified insulinase-like protease 6 (INS6) as a putative microneme protein in C. parvum. However, the functional contribution of INS6 to Cryptosporidium pathogenicity remains poorly characterized. In this study, we used genetic manipulation techniques to investigate the expression and biological functions of INS6 in C. parvum.<br><br>The INS6 gene was tagged and deleted in C. parvum using CRISPR/Cas9 technology. The expression of INS6 was determined by immunofluorescence analysis, ultrastructure-expansion microscopy, and immunoelectron microscopy. Endogenous labelling showed low levels of INS6 expression, which is found in C. parvum micronemes and is absent during the male gamont stage. The effect of INS6 deletion on parasite growth and pathogenicity was assessed in vitro using HCT-8 cultures and in vivo by infection of interferon-&#x3b3; knockout mice. Deletion of the INS6 gene impaired C. parvum proliferation in vitro and significantly reduced the parasite burden in infected mice. In addition, mice infected with the &#x394;ins6 strain showed a significant reduction in the intestinal villus-to-crypt ratio, attenuated body weight loss and increased survival rates, compared to those infected with the INS6-3HA strain.<br><br>CONCLUSIONS/SIGNIFICANCE: These results indicate that INS6 protein is involved in C. parvum proliferation and plays a critical role in modulating the pathogenicity of this zoonotic parasite. Deletion of this gene affects the invasion efficiency and pathogenicity of the parasite.},
}
@article {pmid40907495,
year = {2025},
author = {Bayraktar, E and Rodriguez-Aguayo, C and Stur, E and Kumar, S and Mangala, LS and Jennings, NB and Bayram, NN and Corvigno, S and Asare, A and Ivan, C and Kim, MS and Vu, TC and Hanjra, P and Kim, S and Ahumada, AL and Wu, W and Lee, S and Szymanowska, A and Oztatlici, H and Estecio, MR and Lee, JS and Jain, AK and Sahni, N and Hagan, JP and Baylin, S and Liu, J and Lopez-Berestein, G and Pradeep, S and Sood, AK},
title = {Epigenetic modulation of BARD1 to enhance anti-VEGF therapy.},
journal = {Cell reports. Medicine},
volume = {6},
number = {9},
pages = {102329},
doi = {10.1016/j.xcrm.2025.102329},
pmid = {40907495},
issn = {2666-3791},
mesh = {Humans ; *Epigenesis, Genetic/drug effects ; *Ubiquitin-Protein Ligases/genetics/metabolism ; Animals ; *Vascular Endothelial Growth Factor A/antagonists &amp; inhibitors/metabolism ; Female ; *Tumor Suppressor Proteins/genetics/metabolism ; DNA Methylation/drug effects/genetics ; Cell Line, Tumor ; Mice ; *Ovarian Neoplasms/genetics/drug therapy/pathology ; Neovascularization, Pathologic/genetics/drug therapy ; Promoter Regions, Genetic ; CRISPR-Cas Systems/genetics ; Drug Resistance, Neoplasm/genetics/drug effects ; Mixed Function Oxygenases ; Proto-Oncogene Proteins ; },
abstract = {Despite the clinical use of anti-vascular endothelial growth factor (VEGF) antibodies (AVAs) in cancer therapy, resistance frequently develops, leading to disease progression. To address this, we identify a previously unknown role for breast cancer type 1 susceptibility protein (BRCA1)-associated RING domain 1 (BARD1) in modulating AVA sensitivity. Epigenetic modulation-via global and targeted DNA methylation-reveals BARD1 as a key regulator of angiogenesis. Sequential treatment with azacytidine overcomes AVA resistance in vivo. To enable precise epigenetic reactivation, we develop a liposomal CRISPR-deactivated Cas9 (dCas9)-TET1 system guided by BARD1-targeting single-guide RNAs (sgRNAs). This platform achieves CpG-specific demethylation of the BARD1 promoter, restores expression, and enhances AVA response. Additionally, BARD1 restoration, through either dCas9-TET1 or small interfering RNA (siRNA), significantly reduces tumor growth in combination with AVA in ovarian cancer models. These findings uncover a previously unrecognized function of BARD1 in tumor angiogenesis and demonstrate the potential of gene-specific epigenetic targeting to overcome AVA resistance.},
}
@article {pmid40905401,
year = {2025},
author = {Chen, G and Shan, Y and Wang, J and Zhang, Q and Yao, L and Chen, X},
title = {Multiple Lignocellulosic Inhibitor-Tolerant Saccharomyces cerevisiae Strains Developed by Evolutionary Engineering and CRISPR/Cas9 Gene Editing Technology.},
journal = {Journal of agricultural and food chemistry},
volume = {73},
number = {37},
pages = {23486-23497},
doi = {10.1021/acs.jafc.5c04039},
pmid = {40905401},
issn = {1520-5118},
mesh = {*Lignin/metabolism/antagonists &amp; inhibitors ; *Saccharomyces cerevisiae/genetics/metabolism/growth &amp; development/drug effects ; CRISPR-Cas Systems ; Gene Editing ; Saccharomyces cerevisiae Proteins/genetics/metabolism ; Furaldehyde/metabolism/pharmacology ; Acetic Acid/metabolism/pharmacology ; Benzaldehydes/pharmacology/metabolism ; Ethanol/metabolism ; Fermentation ; },
abstract = {Through evolutionary engineering strategies, scientists have successfully cultivated multiple strains of Saccharomyces cerevisiae with enhanced tolerance, demonstrating significant potential in improving S. cerevisiae resistance. In this study, S. cerevisiae CEN.PK113-7D was continuously cultured for 80 days in a medium containing lignocellulosic inhibitors (furfural, acetic acid, and vanillin). The evolved strain, S. cerevisiae CEN.PK113-AL80-4, exhibited 12 h reduction in lag phase under multiple stress conditions and 17% increase in the ethanol conversion rate. The double mutant strain RG was constructed by mutating genes such as Rad18 and Gcn1 using CRISPR/Cas9 gene editing technology. Under the stress of 2 g/L furfural, 3 g/L acetic acid, and 1.5 g/L vanillin, ethanol yield reached 5.88 ± 0.28 g/L (the conversion rate was 0.29 ± 0.01 g/g). However, the original strain cannot grow. Mechanism studies have shown that Rad18 and Gcn1 significantly enhance stress tolerance by increasing the activities of catalase (CAT) (75%) and superoxide dismutase (SOD) (27.6%), increasing intracellular glycerol content, and strengthening carbon metabolism and oxidative stress responses. This study lays a solid theoretical foundation for developing more robust strains and advancing efficient utilization of lignocellulosic biomass.},
}
@article {pmid40897678,
year = {2025},
author = {Liu, S and Lun, J and Hu, L and Pan, L},
title = {Rapid and Field-Deployable Detection of the S59G Mutation in Glutamine Synthetase Endowing Glufosinate Resistance in Eleusine indica.},
journal = {Journal of agricultural and food chemistry},
volume = {73},
number = {37},
pages = {23292-23299},
doi = {10.1021/acs.jafc.5c08583},
pmid = {40897678},
issn = {1520-5118},
mesh = {*Glutamate-Ammonia Ligase/genetics/metabolism ; *Aminobutyrates/pharmacology ; *Herbicide Resistance ; *Herbicides/pharmacology ; *Plant Proteins/genetics/metabolism ; *Eleusine/genetics/drug effects/enzymology ; Mutation ; CRISPR-Cas Systems ; },
abstract = {Glufosinate is a crucial nonselective herbicide used in both conventional and transgenic cropping systems. Its effectiveness is increasingly compromised by resistant weed species like Eleusine indica (L.) Gaertn. A single nucleotide mutation in the GS1-1 gene, specifically the S59G substitution in glutamine synthetase, is the only known target-site mutation conferring resistance to glufosinate in E. indica. This study presents a rapid and sensitive detection system for the GS1-1 S59G mutation, integrating recombinase-aided amplification (RAA) with CRISPR/Cas12a technology. We optimized key components to achieve robust performance, detecting target sequences at concentrations as low as approximately 10[4] aM. The system effectively differentiates between susceptible and S59G-mutated resistant E. indica using fluorescence readouts or lateral flow assays. This RAA-CRISPR/Cas12a detection system serves as an efficient molecular diagnostic tool for monitoring glufosinate resistance in E. indica, aiding in effective weed management strategies.},
}
@article {pmid40842431,
year = {2025},
author = {Erol, A and Celebi-Birand, D and Yilmaz, E and Polat, C and Kasap, OE and Alten, B and Duman, M},
title = {A novel CRISPR-Cas12a-based diagnostic for rapid and highly sensitive detection of West Nile virus.},
journal = {Journal of materials chemistry. B},
volume = {13},
number = {36},
pages = {11318-11328},
doi = {10.1039/d5tb01268f},
pmid = {40842431},
issn = {2050-7518},
mesh = {*West Nile virus/isolation &amp; purification/genetics ; *CRISPR-Cas Systems/genetics ; *West Nile Fever/diagnosis/virology ; Animals ; Humans ; *CRISPR-Associated Proteins/genetics/metabolism ; Nucleic Acid Amplification Techniques ; *Endodeoxyribonucleases/genetics/metabolism ; Bacterial Proteins ; },
abstract = {Climate change is increasing the global threat of vector-borne diseases, including West Nile Virus (WNV), a significant human and animal pathogen transmitted primarily by Culex mosquitoes. Current WNV diagnostic methods, while including sensitive techniques like RT-PCR, have limitations in early detection, practicality, and cost-effectiveness. There is an urgent need to develop novel and more efficient strategies to address these challenges and to facilitate the surveillance and management of WNV infections and their spread. This study presents a highly specific and sensitive CRISPR-Cas12a-based detection protocol for WNV detection. Through systematic analysis of key reaction parameters (time: 0-60 min; reporter concentration: 1-80 nM, Cas12a and crRNA concentration: 5.625-90 nM; and template amount: 10[-2]-10[5] pg) and integration with reverse transcriptase recombinase polymerase amplification to enhance sensitivity through an isothermal technique, this assay demonstrates a novel strategy for the rapid detection of WNV, achieving 10 femtomolar sensitivity within one hour. Moreover, the assay retained its efficacy at different temperatures (25 °C and 37 °C) and in biological matrices containing the host (fly or human) genetic material, which supports its applicability in resource-limited settings. Therefore, the method presented here has the potential for broad application in diverse point-of-care settings for rapid diagnosis of WNV.},
}
@article {pmid40757862,
year = {2025},
author = {Fernbach, J and Baggenstos, J and Svorjova, E-A and Riedo, J and McCallin, S and Loessner, MJ and Kilcher, S},
title = {CRISPR-Cas9 enables efficient genome engineering of the strictly lytic, broad-host-range staphylococcal bacteriophage K.},
journal = {Applied and environmental microbiology},
volume = {91},
number = {9},
pages = {e0201424},
doi = {10.1128/aem.02014-24},
pmid = {40757862},
issn = {1098-5336},
support = {PZ00P3_174108//Schweizerischer Nationalfonds zur F&#xf6;rderung der Wissenschaftlichen Forschung/ ; },
mesh = {*CRISPR-Cas Systems ; *Staphylococcus Phages/genetics/physiology ; *Staphylococcus aureus/virology ; Host Specificity ; *Genetic Engineering/methods ; Animals ; *Genome, Viral ; Staphylococcal Infections/microbiology/veterinary/diagnosis ; Cattle ; Humans ; },
abstract = {Staphylococcus aureus is a major opportunistic pathogen, increasingly difficult to treat due to rising resistance to methicillin, vancomycin, and other antimicrobials. Bacteriophages offer a promising alternative, particularly when conventional therapies fail and their efficacy can be enhanced through genetic engineering. Among S. aureus phages, the strictly lytic, broad-host-range members of the Twortvirinae subfamily are among the most promising therapeutic candidates. However, their large genome sizes make them notoriously difficult to engineer. In this study, we utilized Twortvirus K as a model to develop an efficient phage engineering platform, leveraging homologous recombination and CRISPR-Cas9-assisted counterselection. As proof of principle, this platform was utilized to construct a nanoluciferase (nluc)-encoding reporter phage (K::nluc) and tested as a bioluminescence-based approach for identifying viable Staphylococcus cells. Independent of their phage-resistance profile, 100% of tested clinical S. aureus isolates emitted bioluminescence upon K::nluc challenge. This diagnostic assay was further adapted to complex matrices such as human whole blood and bovine raw milk, simulating S. aureus detection scenarios in bacteremia and bovine mastitis. Beyond reporter phage-based diagnostics, our engineering technology opens avenues for the design and engineering of therapeutic Twortvirinae phages to combat drug-resistant S. aureus strains.IMPORTANCEPhage engineering, the process of modifying bacteriophages to enhance or customize their properties, offers significant potential for advancing precision antimicrobial therapies and diagnostics. While methods for engineering small Staphylococcus phage genomes are well-established, larger Staphylococcus phages have historically been challenging to modify. In this study, we present a novel method that enables the engineering of Twortvirinae, a subfamily of Staphylococcus phages known for their broad host range and strictly lytic lifestyle, making them highly relevant for diagnostic and therapeutic applications. Using this method, we successfully developed a phage-based diagnostic tool capable of rapid and sensitive detection of S. aureus cells across various matrices. This approach has the potential to extend beyond diagnostics, enabling applications such as phage-mediated delivery of antimicrobial effector proteins in the future.},
}
@article {pmid40752587,
year = {2025},
author = {Liang, G and Lu, B and Dai, S and Li, M and Yao, J and Liu, H and Liu, X and Liu, X and Wang, D},
title = {Creation of colorless transparent tilapia using CRISPR/Cas9 mediated multi-gene mutation.},
journal = {New biotechnology},
volume = {89},
number = {},
pages = {163-176},
doi = {10.1016/j.nbt.2025.07.009},
pmid = {40752587},
issn = {1876-4347},
mesh = {Animals ; *CRISPR-Cas Systems/genetics ; *Tilapia/genetics ; *Mutation/genetics ; *Gene Editing ; },
abstract = {Transparent mutant fish have been obtained from small-sized fish (medaka, zebrafish, and killifish), and are often used as experimental models in biological and medical research. However, transparent fish have never been created in medium- or large-sized fish through gene editing. In this study, mutants without xanthophores, erythrophores and pigmented melanophores were first obtained by crossing to form tyrb;csf1ra double mutant tilapia. Subsequently, single mutants lacking reflective platelets without visible iridophores were obtained by mutation of pnp4a and tfec using CRISPR/Cas9 gene editing, the recovery of iridophores were observed in pnp4a mutants but not in tfec mutants in the later stage. In addition, we also found that tfec mutation led to a decrease in number of melanophores and diameter of melanophores/erythrophores. Finally, transparent triple mutant homozygotes without visible pigment cells were obtained by triple mutation of tyrb;csf1ra;pnp4a (named amber) and tyrb;csf1ra;tfec (named ruby), through crossing of double and single mutants. The two mutant lines were no longer transparent at 60 dpf (days post fertilization) and 120 dpf, respectively, due to the recovery of iridophores in the former, and thickening of the body wall in the latter. In summary, this study created two transparent lines without visible pigment cells through aggregation of multiple gene mutation and crossing. The transparent fish is suitable for in vivo imaging, and is currently the only medium-sized transparent fish obtained through gene editing.},
}
@article {pmid40705967,
year = {2025},
author = {Uçar, SK and Yıldırım, C and Opladen, T},
title = {A CRISPR/Cas9 knockout model for AADC deficiency reveals structural loop3 instability as a key driver of catalytic failure.},
journal = {The FEBS journal},
volume = {292},
number = {18},
pages = {4804-4807},
doi = {10.1111/febs.70204},
pmid = {40705967},
issn = {1742-4658},
mesh = {*CRISPR-Cas Systems/genetics ; Humans ; *Aromatic-L-Amino-Acid Decarboxylases/genetics/deficiency/chemistry/metabolism ; *Amino Acid Metabolism, Inborn Errors/genetics/pathology/metabolism ; Gene Knockout Techniques ; },
abstract = {The CRISPR-Cas9 dopa decarboxylase (DDC) gene knockout SH-SY5Y model for aromatic L-amino acid decarboxylase (AADC) deficiency provides a valuable neuronal platform for functional and structural investigation of pathogenic variants. In their study, Bertoldi et al. successfully recapitulate the biochemical and metabolic hallmarks of AADC deficiency using the AADC catalytic variants R347Q and L353P. Their combined structural and cellular approach identifies loop3 dynamics as a critical determinant of enzymatic dysfunction. This model may pave the way for the development of precision therapies.},
}
@article {pmid40681150,
year = {2025},
author = {Xi, C and Chiu, S and Voje, WE and Carothers, JM and Moon, TS},
title = {Conditional guide RNA deactivation by mRNA and small molecule triggers in Saccharomyces cerevisiae.},
journal = {New biotechnology},
volume = {89},
number = {},
pages = {105-118},
pmid = {40681150},
issn = {1876-4347},
support = {R01 AT009741/AT/NCCIH NIH HHS/United States ; },
mesh = {*Saccharomyces cerevisiae/genetics/metabolism ; *RNA, Messenger/genetics/metabolism ; *RNA, Guide, CRISPR-Cas Systems/genetics/metabolism ; CRISPR-Cas Systems/genetics ; Gene Expression Regulation, Fungal ; },
abstract = {CRISPR interference (CRISPRi) technologies have revolutionized bioengineering by providing precise tools for gene expression modulation, enabling targeted gene perturbation and metabolic pathway optimization. Despite these advances, achieving dynamic control over gene expression by CRISPR-based regulation remains a challenge due to its inherently static nature. Utilizing toehold-mediated strand displacement and ligand-responsive ribozymes (aptazymes), this study introduces switchable guide RNAs (gRNAs) that facilitate tunable gene expression mediated by mRNA or small molecule signals. We demonstrate complete silencing of gRNA via strategically designed 5' or 3' extensions that impede the gRNA spacer or the dCas9 handle, with subsequent restoration of function through sequestration or cleavage of the obstructive sequence. The resulting toehold-embedded or aptazyme-embedded gRNAs can be deactivated by specific signals, including two full-length translatable mRNAs and two small molecule triggers, thereby lifting CRISPRi repression on targeted genes. This modular approach allows for gRNA-based biocomputing through multi-layer or multi-input genetic logic gates in Saccharomyces cerevisiae. Offering a versatile strategy for post-CRISPR regulation in response to environmental signals or cellular states, this methodology expands the toolkit in eukaryotic systems for reversible control of gene expression.},
}
@article {pmid40318155,
year = {2025},
author = {Carmona-Carmona, CA and Bisello, G and Franchini, R and Lunardi, G and Galavotti, R and Perduca, M and Ribeiro, RP and Belviso, BD and Giorgetti, A and Caliandro, R and Lievens, PM and Bertoldi, M},
title = {The CRISPR-Cas9 knockout DDC SH-SY5Y in vitro model for AADC deficiency provides insight into the pathogenicity of R347Q and L353P variants: a cross-sectional structural and functional analysis.},
journal = {The FEBS journal},
volume = {292},
number = {18},
pages = {4833-4853},
doi = {10.1111/febs.70120},
pmid = {40318155},
issn = {1742-4658},
support = {PRIN2022 code 2022BTMTP8//Ministero dell'Universit&#xe0; e della Ricerca, National Recovery and Resilience Plan (NRRP)/ ; IIS154//PTC Therapeutics/ ; PE0000006//National Recovery and Resilience Plan, #NEXTGENERATIONEU (NGEU)/ ; 155311.10.2022//A Multiscale integrated approach to the study of the nervous system in health and disease/ ; },
mesh = {Humans ; *Aromatic-L-Amino-Acid Decarboxylases/genetics/deficiency/chemistry/metabolism ; *CRISPR-Cas Systems/genetics ; *Amino Acid Metabolism, Inborn Errors/genetics/pathology/metabolism ; Cell Line, Tumor ; Dopamine/metabolism ; Gene Knockout Techniques ; Crystallography, X-Ray ; Molecular Dynamics Simulation ; },
abstract = {Aromatic amino acid decarboxylase (AADC) deficiency is a severe inherited recessive neurotransmitter disorder caused by an impairment in dopamine synthesis due to the lack/modification of AADC, the enzyme converting l-dopa to dopamine. Patients exhibit severe movement disorders and neurodevelopmental delay, with a high risk of premature mortality. Given the lack of a reliable model for the disease, we developed a dopa decarboxylase knockout model using CRISPR/Cas9 technology in the SH-SY5Y neuroblastoma cell line. This model showed a deficiency in AADC protein and activity, with an altered dopamine metabolites profile (low homovanillic acid and high 3-O-methyldopa) and a modified expression of key enzymes, such as dopamine beta-hydroxylase and monoamine oxidases, which are involved in the catecholamine pathway. We then transfected the DDC-KO cells with two AADC catalytic variants, R347Q and L353P, which resulted in loss-of-function and an altered profile of dopamine metabolites. By combining several structural approaches (X-ray crystallography, molecular dynamics, small angle X-ray scattering, dynamic light scattering, and spectroscopy), we determined that both variants alter the flexibility of the structural element to which they belong, whose integrity is essential for catalysis. This change causes a mispositioning of essential residues at the active site, leading, in turn, to an unproductive external aldimine, identifying the molecular basis for the loss-of-function. Overall, the DDC-KO model recapitulates some key features of AADC deficiency, is useful to study the molecular basis of the disease, and represents an ideal system for small molecule screening regarding specific enzyme defects, paving the way for a precision therapeutic approach.},
}
@article {pmid39484758,
year = {2025},
author = {Sharma, B and Chauhan, I and Kumar, G and Bhardwaj, K and Tiwari, RK},
title = {Nanoparticle Carriers: A New Era of Precise CRISPR/Cas9 Gene Editing.},
journal = {MicroRNA (Shariqah, United Arab Emirates)},
volume = {14},
number = {2},
pages = {101-111},
pmid = {39484758},
issn = {2211-5374},
mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; Humans ; *Nanoparticles/chemistry ; Animals ; Drug Delivery Systems/methods ; },
abstract = {The revolutionary CRISPR/Cas9 gene editing technology holds immense potential for treating genetic diseases and tackling conditions like cancer. However, efficient delivery remains a significant challenge. This is where nanoparticles come into play, emerging as powerful allies in the realm of drug delivery. Nanoparticles can accommodate larger insertion sizes, enabling the incorporation of larger Cas9 enzymes and complex guide RNAs, thus opening up the possibility of editing previously inaccessible genetic regions. Their relatively straightforward and scalable production processes make them cost-effective options for wider applications. Notably, nanoparticles excel in vivo, demonstrating efficient tissue penetration and targeted delivery, which are crucial for maximizing therapeutic impact while minimizing side effects. This review aims to explore the potential of nanoparticle-based delivery systems for CRISPR/Cas9, highlighting their advantages and challenges in gene editing applications. The diverse range of nanoparticles further bolsters their potential. Polymeric nanoparticles, for instance, offer tunable properties for customization and controlled release of the CRISPR cargo. Lipid-based nanoparticles facilitate efficient cellular uptake and endosomal escape, ensuring the CRISPR components reach the target DNA. Even gold nanoparticles, known for their unique biocompatibility and photothermal properties, hold promise in light-activated editing strategies. Non-viral delivery systems, particularly those based on nanoparticles, stand out due to their inherent advantages. Collectively, the evidence paints a promising picture: nanoparticles are not merely passive carriers but active participants in the CRISPR/Cas9 delivery landscape. Their versatility, efficiency, and safety position them as key enablers of a future where gene editing can revolutionize drug development, offering personalized and targeted therapies for a wide range of diseases.},
}
@article {pmid40954078,
year = {2025},
author = {Bexte, T and Wagner, DL},
title = {Next-generation multiplex-edited CAR-NK cells: more edits, more power?.},
journal = {Journal for immunotherapy of cancer},
volume = {13},
number = {9},
pages = {},
doi = {10.1136/jitc-2025-012841},
pmid = {40954078},
issn = {2051-1426},
mesh = {*Killer Cells, Natural/immunology/metabolism/transplantation ; Humans ; *Gene Editing/methods ; Animals ; *Receptors, Chimeric Antigen/genetics/metabolism ; Mice ; *Immunotherapy, Adoptive/methods ; CRISPR-Cas Systems ; },
abstract = {First clinical trials demonstrated the safety of adoptive cell transfer with allogeneic natural killer (NK) cell products from healthy donors, making them an attractive candidate for 'off-the-shelf' chimeric antigen receptor (CAR)-immune cell therapy. However, reduced persistence and inactivation of NK cells by immunosuppressive cues likely limit the performance of CAR-redirected NK cells. Wang and colleagues demonstrate that multiplex CRISPR base editing allows optimization of the intrinsic functionality of CAR-NK cells improving their therapeutic potential. In contrast to conventional CRISPR-Cas nucleases, base editing avoided most double-stranded DNA breaks while enabling highly efficient editing at up to six sites simultaneously. The study further demonstrates the feasibility of a non-viral approach to integrate CAR transgene and multiplex base editing of several immune checkpoints in NK cells using a single electroporation. CAR-NK cells harboring up to three base edits demonstrate improved potency over unedited counterparts in vitro. Xenograft mouse models confirmed increased potency, but also indicated signs of organ toxicity - a phenomenon that will require future studies prior to clinical translation. The study demonstrates that CRISPR base editing is a powerful tool to unleash the full cytotoxic potential of NK cells, but it also warrants the question: How many internal breaks can be removed without hurting CAR-NK cell therapy's impeccable safety record?},
}
@article {pmid40952971,
year = {2025},
author = {Leonova, EI and Chirinskaite, AV and Akhmarov, II and Luganskaya, PS and Kirillov, OA and Kandina, DA and Romanovich, AE and Shkodenko, LA and Fedotov, SA and Rubel, AA and Sopova, YV},
title = {Improved Genome Editing via Oviductal Nucleic Acids Delivery-based In Vivo Electroporation Technique for Knockout Mice Generation.},
journal = {Journal of visualized experiments : JoVE},
volume = {},
number = {222},
pages = {},
doi = {10.3791/68704},
pmid = {40952971},
issn = {1940-087X},
mesh = {Animals ; Female ; *Electroporation/methods ; Mice ; Mice, Knockout ; Pregnancy ; *Gene Editing/methods ; *Fallopian Tubes ; Male ; CRISPR-Cas Systems ; *Nucleic Acids/administration &amp; dosage/genetics ; Oviducts ; RNA, Guide, CRISPR-Cas Systems/administration &amp; dosage/genetics ; *Gene Knockout Techniques/methods ; },
abstract = {Methods for creating knockout mice typically involve three main steps: (1) collecting embryos from donor females, (2) microinjecting genetic constructs into the zygotes ex vivo, and (3) surgically transferring them into the oviduct of pseudo-pregnant females. This process requires a significant number of animals, as it involves not only donor females but also vasectomized males and pseudo-pregnant females. Moreover, microinjections into the cytoplasm or pronucleus of mouse zygotes present challenges such as needle clogging, membrane permeability issues due to high elasticity, and potential embryo death. The development of advanced electroporators, such as the Nepa21, provides a unique opportunity to generate mice with targeted gene knockouts in a single step through a method known as Improved Genome Editing via Oviductal Nucleic Acids Delivery (I-GONAD). This technique involves microinjecting CRISPR-Cas components (Cas9 protein and guide RNA) into the oviducts of pregnant females at 0.7 days post-conception, followed by in vivo electroporation to deliver these components directly into the zygotes. Following the I-GONAD procedure, the pregnant mouse carries and gives birth to pups with the targeted gene knockout. This article provides a detailed, step-by-step protocol for implementing the I-GONAD method in mice, offering a more efficient and accessible alternative to traditional knockout mouse generation techniques.},
}
@article {pmid40952968,
year = {2025},
author = {Datta, A and Nelson, N and Orallo, GK and Krull, S},
title = {Targeted Corneal Sensory Nerve Depletion via Subconjunctival Injection: A Model for Investigating Bacterial Adhesion and Neuroimmune Interactions.},
journal = {Journal of visualized experiments : JoVE},
volume = {},
number = {222},
pages = {},
doi = {10.3791/68614},
pmid = {40952968},
issn = {1940-087X},
mesh = {Animals ; *Cornea/innervation/microbiology/immunology/drug effects ; *Bupivacaine/administration &amp; dosage/pharmacology ; *Bacterial Adhesion/drug effects/physiology ; Pseudomonas aeruginosa/physiology ; Conjunctiva/innervation/microbiology ; Staphylococcus aureus/physiology ; Staphylococcus epidermidis/physiology ; *Sensory Receptor Cells/drug effects/microbiology/immunology ; Mice ; },
abstract = {Corneal sensory nerves play a pivotal role in supporting ocular surface integrity and immune defense mechanisms. Loss of this innervation has been associated with increased vulnerability to microbial invasion, yet the precise contribution of nerve depletion to bacterial adhesion on the cornea remains insufficiently characterized. Here, we present a reproducible and temporally controlled method for selective corneal sensory nerve suppression using bupivacaine, a long-acting sodium channel blocker. By combining subconjunctival and topical delivery routes, this dual-application strategy achieves robust, sustained denervation, allowing for precise investigation of how altered sensory input influences corneal epithelial susceptibility to bacterial colonization. Using this model, we investigate how sensory denervation influences microbial adhesion dynamics for Staphylococcus aureus, Staphylococcus epidermidis, and Pseudomonas aeruginosa three clinically relevant pathogens with distinct adhesion mechanisms. Standardized bacterial inoculation via the laboratory wipe blotting method ensures uniform deposition on the corneal surface, followed by quantitative assessment of bacterial adhesion. Bupivacaine-induced nerve depletion correlates with reduced corneal nerve density and increased bacterial adhesion, confirming a functional link between sensory depletion and microbial susceptibility. By simulating neuropathic conditions such as diabetic neuropathy and neurotrophic keratitis, this approach provides a novel framework for studying neuroimmune interactions in ocular infections. Beyond infection models, this subconjunctival injection strategy offers a versatile platform for investigating ocular drug pharmacokinetics, neuroprotective interventions, and immune modulation. Furthermore, it can be adapted for gene modification studies, including subconjunctival delivery of CRISPR/Cas constructs or viral vectors, broadening its applications in ophthalmic research and therapeutics.},
}
@article {pmid40951304,
year = {2025},
author = {Chen, J and Wang, Y and Aikebaier, R and Liu, H and Li, Y and Yang, L and Haiyilati, A and Wang, L and Fu, Q and Shi, H},
title = {RAA-CRISPR/Cas12a-based visual field detection system for rapid and sensitive diagnosis of major viral pathogens in calf diarrhea.},
journal = {Frontiers in cellular and infection microbiology},
volume = {15},
number = {},
pages = {1616161},
pmid = {40951304},
issn = {2235-2988},
mesh = {Animals ; Cattle ; *Diarrhea/veterinary/virology/diagnosis ; Sensitivity and Specificity ; *Cattle Diseases/diagnosis/virology ; *CRISPR-Cas Systems ; *Molecular Diagnostic Techniques/methods ; China ; *Nucleic Acid Amplification Techniques/methods ; Feces/virology ; *Virus Diseases/diagnosis/veterinary/virology ; Recombinases/metabolism ; *Viruses/genetics/isolation &amp; purification/classification ; },
abstract = {Calf diarrhea is a complex digestive disorder in cattle that imposes significant economic losses in terms of calf mortality, growth impairment, and treatment costs. Both infectious and non-infectious agents contribute to its aetiology; however, most of the infectious cases are caused by viruses, often accompanied by severe co-infections. To identify viral culprits, we performed viral metagenomic sequencing on three pooled samples from the 150 diarrheal samples from Xinjiang, China, which helped with identification of the following four predominant agents: bovine nepovirus (BNeV), bovine coronavirus (BCoV), bovine viral diarrhea virus (BVDV) and bovine enterovirus (BEV). Currently, the process of diagnosing these pathogens involves time-consuming workflows, limited sensitivity, poor portability, and lack of field applicability. Keeping these diagnostic shortcomings in mind, an integrated platform called RAA-CRISPR/Cas12a system was developed by combining recombinase-aided amplification (RAA) at 37°C with CRISPR/Cas12a-mediated fluorescence detection, which achieved 100-100,000 times higher sensitivity than conventional polymerase chain reaction (PCR) (detection limits: 1-10 copies/μL) and demonstrated 100% specificity against non-target pathogens. Clinical validation of sensitivity and specificity of 252 samples revealed 1.6-4.9 times higher detection rates (239 positives) than PCR (81 positives), which was consistent with PCR-confirmed cases. The assay's 40-min. workflow enables rapid on-site deployment without specialized instrumentation, as it requires only a portable heat block and blue LED transilluminator. Hence, with its laboratory accuracy and field applicability, this method helps in early identification of pathogens, outbreak containment and mitigation of economic loss in the global cattle industry.},
}
@article {pmid40950114,
year = {2025},
author = {Saito, A and Tankou, S and Ishii, K and Sakao-Suzuki, M and Oh, EC and Murdoch, H and Namkung, H and Adelakun, S and Furukori, K and Fujimuro, M and Salomoni, P and Maul, GG and Hayward, GS and Tang, Q and Yolken, RH and Houslay, MD and Katsanis, N and Kosugi, I and Yang, K and Kamiya, A and Ishizuka, K and Sawa, A},
title = {DISC1-PML protein interaction for congenital CMV infection-induced cortical neural progenitor deficit: perturbance of host signaling via viral IE1.},
journal = {bioRxiv : the preprint server for biology},
volume = {},
number = {},
pages = {},
pmid = {40950114},
issn = {2692-8205},
abstract = {Congenital CMV infection is the most common perinatal infection, affecting up to 0.5% of infants. This elicits long-term disabilities that include neuropsychiatric manifestations, such as intellectual disability, microcephaly. Despite its high prevalence, the underlying mechanism of how congenitally acquired CMV infection causes brain pathology remains unknown. Here we discovered the molecular interplay of key host (DISC1 and PML) and viral (IE1) proteins within the neural progenitor cells, which underlay an attenuated neural progenitor proliferation. Abolishing the viral IE1 protein by delivering IE1-targeting CRISPR/Cas9 to fetal brain rescued this progenitor cell deficit, a key pathology in congenital CMV infection. A selective targeting to a viral-specific protein by the CRISPR/Cas9 system is minimal in off-target effects. Therefore, we believe that a pivotal role of IE1 in an attenuated neural progenitor proliferation in the developing cortex through its interfering with interaction between host DISC1 and PML proteins.},
}
@article {pmid40947195,
year = {2025},
author = {Zhou, G and Ding, J and Li, Y and Xu, Q and Zhang, Y and Tang, H and Qi, P and Deng, M and Ma, J and Chen, G and Wang, J and Lin, N and Wei, Y and Jiang, Q},
title = {Ethylene-responsive factor HvERF72 regulates starch synthesis and B-type starch granules initiation in barley.},
journal = {Carbohydrate polymers},
volume = {368},
number = {Pt 2},
pages = {124167},
doi = {10.1016/j.carbpol.2025.124167},
pmid = {40947195},
issn = {1879-1344},
mesh = {*Hordeum/metabolism/genetics ; *Starch/biosynthesis ; *Plant Proteins/genetics/metabolism ; Gene Expression Regulation, Plant ; *Transcription Factors/metabolism/genetics ; Promoter Regions, Genetic ; CRISPR-Cas Systems ; },
abstract = {Starch biosynthesis is a pivotal determinant of barley grain quality and yield, yet its regulatory mechanisms remain incompletely characterized. This study identifies HvERF72, an AP2-domain transcription factor, as a key regulator of starch biosynthesis and granule initiation in barley grains. Comparative analyses of CRISPR/Cas9-generated HvERF72 knockout mutants revealed enhanced B-type granule formation and elevated total starch content, whereas overexpression lines exhibited contrasting phenotypes, including reduced starch accumulation and suppressed B-type granule initiation. Transcriptional profiling at 15 DAF indicated significant upregulation of critical starch biosynthesis genes (HvAGPL1, HvAGPS1, HvSS2a, HvSBEI, HvSBEIIb, and HvGBSSI) in mutants, while overexpression lines showed downregulation of these genes. Mechanistic investigation demonstrated that HvERF72 directly binds to GCC-box motifs in the promoter regions of HvSS2a and HvSBEI, repressing their transcription. These findings establish HvERF72 as dual-function regulator that modulates starch biosynthesis and B-type granule initiation, providing novel molecular targets for optimizing starch yield and industrial quality in barley breeding programs.},
}
@article {pmid40943650,
year = {2025},
author = {Ulloa, D and Núñez, C and Matamala, R and San Martín, A and Páez-De Ávila, D and Mercado-Vides, J and Narváez, J and Aguirre, J and Effer, B and Iturrieta-González, I},
title = {CRISPR-Cas12 Application for the Detection of Pneumocystis jirovecii in Immunodepression Patients Through Fluorescent and Lateral Flow Colorimetric Assay.},
journal = {International journal of molecular sciences},
volume = {26},
number = {17},
pages = {},
pmid = {40943650},
issn = {1422-0067},
support = {21251211//ANID - Chile, Scholarship Programa de Doctorado Nacional 2025/ ; UDM010//FONCIENCIAS - Universidad del Magdalena/ ; },
mesh = {Humans ; *Pneumocystis carinii/genetics/isolation &amp; purification ; *CRISPR-Cas Systems ; *Colorimetry/methods ; *Pneumonia, Pneumocystis/diagnosis/microbiology/immunology ; *Immunocompromised Host ; Sensitivity and Specificity ; Tubulin/genetics ; },
abstract = {Pneumonia caused by Pneumocystis jirovecii poses a serious threat, particularly to immunocompromised patients such as those with HIV/AIDS, transplant recipients, or individuals undergoing chemotherapy. Its diagnosis is challenging because current methods, such as microscopy and certain molecular tests, have limitations in sensitivity and specificity, and require specialized equipment, which delays treatment initiation. In this context, CRISPR-Cas12-based methods offer a promising alternative: they are rapid, highly specific, sensitive, and low-cost, enabling more timely and accessible detection, even in resource-limited settings. We developed a simple and rapid detection platform based on the CRISPR-Cas12 coupled with lateral flow strips. A guide RNA was designed against DHPS, β-tubulin, and mtLSU rRNA genes. The guide corresponding to β-tubulin showed high sensitivity in the detection of P. jirovecii to produce a detectable fluorescence signal within the first 20-30 min. In addition, it demonstrated high specificity for P. jirovecii when DNA from other microorganisms was used. When coupled with lateral flow strips, high sensitivity and specificity were also observed for detecting positive samples, without the need for genetic amplification. CRISPR-Cas12 successfully detected P. jirovecii infection in an initial diagnostic application, demonstrating the potential of this method for integration into public health diagnostic systems, particularly in field, due to its adaptability, speed, and ease of use.},
}
@article {pmid40943640,
year = {2025},
author = {Yu, I and Jeong, J},
title = {Advancing Gene Therapy for Phenylketonuria: From Precision Editing to Clinical Translation.},
journal = {International journal of molecular sciences},
volume = {26},
number = {17},
pages = {},
pmid = {40943640},
issn = {1422-0067},
support = {RS-2023-00260529//National Research Foundation of Korea/ ; 2022R1A2C1002884//National Research Foundation of Korea/ ; 2024-0036//Seoul Women's University/ ; },
mesh = {*Phenylketonurias/therapy/genetics ; Humans ; *Genetic Therapy/methods ; *Gene Editing/methods ; Animals ; CRISPR-Cas Systems ; Phenylalanine Hydroxylase/genetics ; Gene Transfer Techniques ; Dependovirus/genetics ; Translational Research, Biomedical ; Nanoparticles/chemistry ; },
abstract = {Phenylketonuria (PKU) is an inherited disorder caused by mutations in the phenylalanine hydroxylase (PAH) gene that result in the amino acid phenylalanine (Phe) building up in the blood. Current therapies suggest low-Phe dietary management and (6R)-L-erythro-5,6,7,8-tetrahydrobiopterin (BH4) therapy, which are limited in efficacy and require lifelong treatment. Recent advances in gene therapy, including gene editing and viral-mediated gene delivery, produce therapeutic effects. Advancements in gene editing technologies, notably adenine base editors (ABEs) and CRISPR-based systems, in conjunction with enhanced delivery methods such as lipid nanoparticles (LNPs) and recombinant viruses, have demonstrated substantial promise in preclinical studies. This review details the pathophysiology of PKU treatment, and progress in preclinical and clinical gene therapy strategies. Emphasis is on adenine base editing using LNPs, recombinant adeno-associated virus (rAAV)-mediated gene transfer, and the translational challenges associated with these technologies. We also discuss future directions for therapeutic reach and ensuring long-term safety and efficacy.},
}
@article {pmid40943604,
year = {2025},
author = {Yamaji, M and Nakahara, T and Nakanishi, T and Aoyama-Kikawa, S and Yamaguchi, K and Furukawa, Y and Nakamura, M and Okada, T and Tabata, H and Fuse, R and Shimizu, E and Kasajima, R and Imoto, S and Kukimoto, I and Saito, I and Kiyono, T},
title = {Disruption of Human Papillomavirus 16 E6/E7 Genes Using All-in-One Adenovirus Vectors Expressing Eight Double-Nicking Guide RNAs.},
journal = {International journal of molecular sciences},
volume = {26},
number = {17},
pages = {},
pmid = {40943604},
issn = {1422-0067},
support = {JP21fk0108106//Japan Agency for Medical Research and Development/ ; JP22fk0310523//Japan Agency for Medical Research and Development/ ; JP25K02541//Japan Society for the Promotion of Science/ ; JP19K0647//Japan Society for the Promotion of Science/ ; JP23H02402//Japan Society for the Promotion of Science/ ; (JP22ym0126804//Japan Agency for Medical Research and Development/ ; 20jk0210009h0001//Japan Agency for Medical Research and Development/ ; },
mesh = {Humans ; *Oncogene Proteins, Viral/genetics ; *RNA, Guide, CRISPR-Cas Systems/genetics ; *Papillomavirus E7 Proteins/genetics ; *Adenoviridae/genetics ; *Genetic Vectors/genetics ; CRISPR-Cas Systems ; *Human papillomavirus 16/genetics ; Gene Editing/methods ; *Repressor Proteins/genetics ; Animals ; Cell Line, Tumor ; Papillomavirus Infections/virology/therapy ; Mice ; Female ; Genetic Therapy ; },
abstract = {Human papillomavirus (HPV) is a prime target for genome-editing therapy as its E6 and E7 oncogenes are crucial for cancer development and maintenance. A key challenge in CRISPR/Cas9 therapy is the off-target effects. This study utilized a double-nicking technique to introduce DNA breaks in the E6 and E7 regions of HPV16. From 146 gRNA candidates, 16 double-nicking pairs were selected. Multiple combinations of double-nicking (DN)-gRNA pairs were delivered to HPV16-positive cells via lentiviruses, followed by Cas9 nickase (Cas9n) expression. Combinations of 3-4 DN-gRNA pairs effectively killed HPV16-positive cells while sparing HPV-negative cells. Off-target effects were reduced by nearly three orders of magnitude. An "all-in-one" adenovirus (AdV) system expressing four gRNA pairs and Cas9n showed promise in inhibiting tumor growth in HPV16-positive cancer models, demonstrating its potential as a safe and effective treatment for HPV-induced tumors.},
}
@article {pmid40943194,
year = {2025},
author = {Qi, Y and Jia, X and Lin, C and Qian, W and Chen, H and Fang, D and Han, C},
title = {CRISPR/Cas9-Mediated Overexpression of HGF Potentiates Tarim Red Deer Antler MSCs into Osteogenic Differentiation.},
journal = {International journal of molecular sciences},
volume = {26},
number = {17},
pages = {},
pmid = {40943194},
issn = {1422-0067},
mesh = {Animals ; *Osteogenesis/genetics ; *Deer/genetics ; *Antlers/cytology/metabolism ; *Mesenchymal Stem Cells/metabolism/cytology ; *CRISPR-Cas Systems ; *Cell Differentiation/genetics ; *Hepatocyte Growth Factor/genetics/metabolism ; Signal Transduction ; Proto-Oncogene Proteins c-akt/metabolism ; Phosphatidylinositol 3-Kinases/metabolism ; Cells, Cultured ; },
abstract = {Previous studies conducted by our research groups have demonstrated that the HGF/c-Met signaling pathway promotes the proliferation and migration of MSCs in the antlers of Tarim red deer. However, the role and mechanism of this gene in the osteogenic differentiation of antler MSCs remain unclear. In this study, we used antler MSCs as experimental materials. CRISPR/Cas9 technology was employed to knock out the HGF gene, and lentivirus-mediated overexpression of the HGF gene was constructed in antler MSCs. Subsequently, antler MSCs were induced to undergo osteogenic differentiation in vitro. Alizarin Red staining was employed to identify calcium nodules, while the expression levels of various osteogenic differentiation marker genes were assessed using immunohistochemistry, RT-qPCR, and Western blotting techniques. The findings indicated that the HGF gene facilitates the osteogenic differentiation of antler MSCs. Analysis of genes associated with the PI3K/Akt and MEK/ERK signaling pathways demonstrated that in antler MSCs with HGF gene knockout, the expression levels of PI3K/Akt and MEK/ERK pathway genes were significantly downregulated on days 7 and 14 of osteogenic differentiation (p < 0.05). In contrast, antler MSCs with HGF gene overexpression exhibited a significant upregulation of the PI3K/Akt and MEK/ERK signaling pathways on days 4 and 6 of osteogenic differentiation (p < 0.01). These findings suggest that the HGF gene in antlers enhances the osteogenic differentiation of MSCs by activating the PI3K/Akt and MEK/ERK pathways.},
}
@article {pmid40943160,
year = {2025},
author = {Givi, S and Lohnes, BJ and Ebrahimi, S and Riedel, S and Khokhali, S and Khan, SA and Keller, M and Wölfel, C and Echchannaoui, H and Bockamp, E and Andre, MC and Abken, H and Theobald, M and Hartwig, UF},
title = {CRISPR/Cas9 TCR-Edited NKp30 CAR T Cells Exhibit Superior Anti-Tumor Immunity to B7H6-Expressing Leukemia and Melanoma.},
journal = {International journal of molecular sciences},
volume = {26},
number = {17},
pages = {},
pmid = {40943160},
issn = {1422-0067},
support = {2022.046.1//Wilhelm-Sander-Stiftung/ ; },
mesh = {Animals ; Humans ; *Melanoma/therapy/immunology/genetics ; Mice ; *CRISPR-Cas Systems ; *Immunotherapy, Adoptive/methods ; *B7 Antigens/genetics/metabolism/immunology ; *Natural Cytotoxicity Triggering Receptor 3/genetics/immunology/metabolism ; Cell Line, Tumor ; *Receptors, Chimeric Antigen/genetics/immunology ; *T-Lymphocytes/immunology/metabolism ; *Leukemia, Myeloid, Acute/therapy/immunology/genetics ; Xenograft Model Antitumor Assays ; *Receptors, Antigen, T-Cell/genetics ; },
abstract = {Chimeric antigen receptor (CAR) T-cell therapy directed to CD19 and B-cell maturation antigen has revolutionized treatment of B-cell leukemia and lymphoma, and multiple myeloma. However, identifying suitable targets for acute myeloid leukemia (AML) remains challenging due to concurrent expression of potential target antigens on normal hematopoietic stem cells or tissues. As the stress-induced B7H6 molecule is rarely found on normal tissues but expressed on many cancers including AML and melanoma, the NKp30-ligand B7H6 emerges as a promising target for NKp30-based CAR T therapy for these tumors. In this study, we report a comprehensive B7H6 expression analysis on primary AML and melanoma as well as on different tumor cell-lines examined by RT-qPCR and flow cytometry, and efficient anti-tumor reactivity of NKp30-CAR T cells to AML and melanoma. To overcome limitations of autologous CAR T-cell fitness-dependent efficacy and patient-tailored production, we generated CRISPR/Cas9-mediated TCR-knockout (TCR[KO]) NKp30-CAR T cells as an off-the-shelf approach for CAR T therapy. Functional studies comparing NKp30-CD28 CAR or NKp30-CD137 CAR TCR[+] and TCR[KO] T lymphocytes revealed superior anti-tumoral immunity of NKp30-CD28 CAR TCR[KO] T cells to AML and melanoma cell lines in vitro, and effective control of tumor burden in an NSG melanoma-xenograft mouse model. In conclusion, these findings highlight the therapeutic potential of NKp30 CAR TCR[KO] T cells for adoptive T-cell therapy to B7H6-expressing cancers, including melanoma and AML.},
}
@article {pmid40942109,
year = {2025},
author = {Ferrara, F and Sepe, A and Sguizzato, M and Marconi, P and Cortesi, R},
title = {A Pre-Formulation Study for Delivering Nucleic Acids as a Possible Gene Therapy Approach for Spinocerebellar Ataxia Disorders.},
journal = {Molecules (Basel, Switzerland)},
volume = {30},
number = {17},
pages = {},
pmid = {40942109},
issn = {1420-3049},
mesh = {Humans ; *Genetic Therapy/methods ; Liposomes/chemistry ; *Spinocerebellar Ataxias/therapy/genetics ; Cell Line, Tumor ; Gene Transfer Techniques ; *Nucleic Acids/chemistry/genetics ; Transfection ; CRISPR-Cas Systems ; RNA, Messenger/genetics ; Green Fluorescent Proteins/genetics ; Plasmids/genetics ; DNA/genetics/chemistry ; },
abstract = {Liposomes are lipid bilayer vesicles that are highly biocompatible, able to interact with the cell membrane, and able to release their cargo easily. The improvement of the physicochemical properties of liposomes, such as surface charge, lipid composition, and functionalization, makes these vesicles eligible delivery nanosystems for the gene therapy of many pathological conditions. In the present study, pre-formulation analysis was conducted to develop liposomes that facilitate the delivery of nucleic acids to neuronal cells, with the aim of future delivery of a CRISPR/Cas9 system designed to silence genes responsible for autosomal dominant neurodegenerative disorders. To this aim, different nucleic acid cargo models, including λ phage DNA, plasmid DNA, and mRNA encoding GFP, were considered. Liposomes with varying lipid compositions were produced using the ethanol injection method and analyzed for their dimensional stability and ability to interact with DNA. The selected formulations were tested in vitro using a neuroblastoma cell line (SH-SY5Y) to evaluate their potential toxicity and the ability to transfect cells with a DNA encoding the green fluorescent protein (pCMV-GFP). Among all formulations, the one containing phosphatidylcholine, phosphatidylethanolamine, pegylated 1,2-distearoyl-sn-glycero-3-phosphethanolamine, cholesterol, and dioctadecyl-dimethyl ammonium chloride (in the molar ratio 1:2:4:2:2) demonstrated the highest efficiency in mRNA delivery. Although this study was designed with the goal of ultimately enabling the delivery of a CRISPR/Cas9 system for treating autosomal dominant neurodegenerative disorders such as polyglutamine spinocerebellar ataxias (SCAs), CRISPR/Cas9 components were not delivered in the present work, and their application remains the objective of future investigations.},
}
@article {pmid40940741,
year = {2025},
author = {Yao, X and Feng, M and Sun, C and Yang, S and Yuan, Z and Liu, X and Li, Q and Jiang, C and Weng, X and Song, J and Mu, Y},
title = {Establishment of a CRISPR/dCas9 Activation Library for Screening Transcription Factors Co-Regulating OCT4 with GATA4 in Pig Cells.},
journal = {Cells},
volume = {14},
number = {17},
pages = {},
pmid = {40940741},
issn = {2073-4409},
mesh = {Animals ; *GATA4 Transcription Factor/metabolism/genetics ; *Octamer Transcription Factor-3/metabolism/genetics ; Swine ; *CRISPR-Cas Systems/genetics ; *Transcription Factors/metabolism/genetics ; Cell Line ; Promoter Regions, Genetic/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; Gene Library ; },
abstract = {OCT4 is a critical transcription factor for early embryonic development and pluripotency. Previous studies have shown that the regulation of OCT4 by the transcription factor GATA4 is species-specific in pigs. This study aimed to further investigate whether there are other transcription factors that co-regulate the transcription of OCT4 with GATA4 in pigs. A CRISPR activation (CRISPRa) sgRNA library was designed and constructed, containing 5056 sgRNAs targeting the promoter region of 1264 transcription factors in pigs. Then, a pig PK15 cell line was engineered with a single-copy OCT4 promoter-driven EGFP reporter at the ROSA26 locus, combined with the dCas9-SAM system for transcriptional activation. The CRISPRa sgRNA lentiviral library was used to screen for transcription factors, with or without GATA4 overexpression. Flow cytometry combined with high-throughput sequencing identified MYC, SOX2, and PRDM14 as activators and OTX2 and CDX2 as repressors of OCT4. In the presence of GATA4, transcription factors such as SALL4 and STAT3 showed synergistic activation. Functional validation confirmed that HOXD13 upregulates OCT4, while OTX2 inhibits it. GATA4 and SALL4 synergistically enhance OCT4 expression. These findings provide new insights into combinatorial mechanisms that control the transcriptional regulation of OCT4 in pigs.},
}
@article {pmid40940375,
year = {2025},
author = {Berger, T and Borisova, E and Gamerschlag, A and Terheyden-Keighley, D and Martins, S and Greber, B},
title = {Sequential factor delivery enables efficient workflow for universal gene editing in clinical grade iPS cells.},
journal = {Scientific reports},
volume = {15},
number = {1},
pages = {32514},
pmid = {40940375},
issn = {2045-2322},
mesh = {*Induced Pluripotent Stem Cells/metabolism/cytology ; Humans ; *Gene Editing/methods ; CRISPR-Cas Systems ; Workflow ; },
abstract = {Human induced pluripotent stem cells (iPSCs) are gaining momentum as a powerful starting material in cell therapy. To fully harness their potential, CRISPR technology permits endogenous gene modifications as well as the introduction of advanced features, to increase the immune compatibility of the cells or insert suicide genes for enhancing therapeutic safety, for instance. However, genetic manipulation of iPSCs, in particular the generation of knock-in lines, remains relatively inefficient. Conventional mitigation strategies, such as enriching for positive cells using antibiotic selection or complex instrumentation, may, however, cause conflicts with good manufacturing practice (GMP) requirements. To address this challenge, we have systematically optimized a basic gene editing procedure using both Cas9 and Cas12a-based ribonucleoprotein (RNP) complexes. Based on the sequential delivery of RNPs and donor plasmids as a critical hallmark, this virus-free approach permits knock-ins of full-length transgenes at above 30% efficiency, while readily identifying positive clones through random screening at small scale. We exemplify these advances by creating and characterizing homozygous iPSC lines depleted of HLA class I and carrying an inducible caspase-9 suicide gene. Isolated clones from independent GMP iPSC lines retained genomic integrity, differentiation capability, and functionality of the safety switch in the differentiated state. This improved methodology will form a flexible platform for custom gene editing universally applicable both in basic iPSC research and therapy.},
}
@article {pmid40897805,
year = {2025},
author = {Xu, N and Cho, HS and Hackland, JOS and Benito-Kwiecinski, S and Saurat, N and Harschnitz, O and Russo, MV and Garippa, R and Ciceri, G and Studer, L},
title = {Genome-wide CRISPR screen identifies Menin and SUZ12 as regulators of human developmental timing.},
journal = {Nature cell biology},
volume = {27},
number = {9},
pages = {1411-1421},
pmid = {40897805},
issn = {1476-4679},
support = {UM1 HG012654/HG/NHGRI NIH HHS/United States ; R01NS128087//U.S. Department of Health &amp; Human Services | NIH | National Institute of Neurological Disorders and Stroke (NINDS)/ ; R01 NS128087/NS/NINDS NIH HHS/United States ; P30 CA008748/CA/NCI NIH HHS/United States ; UM1HG012654//U.S. Department of Health &amp; Human Services | NIH | National Human Genome Research Institute (NHGRI)/ ; P30 CA08748//U.S. Department of Health &amp; Human Services | NIH | NCI | Division of Cancer Epidemiology and Genetics, National Cancer Institute (National Cancer Institute Division of Cancer Epidemiology and Genetics)/ ; },
mesh = {Humans ; Gene Expression Regulation, Developmental ; Cell Differentiation/genetics ; *Proto-Oncogene Proteins/genetics/metabolism ; *CRISPR-Cas Systems ; *Polycomb Repressive Complex 2/genetics/metabolism ; *Human Embryonic Stem Cells/metabolism/cytology ; Histones/metabolism/genetics ; PAX6 Transcription Factor/genetics/metabolism ; Promoter Regions, Genetic ; Epigenesis, Genetic ; Neurons/metabolism/cytology ; *Clustered Regularly Interspaced Short Palindromic Repeats ; *Embryonic Development/genetics ; Neoplasm Proteins ; Transcription Factors ; },
abstract = {Embryonic development follows a conserved sequence of events across species, yet the pace of development is highly variable and particularly slow in humans. Species-specific developmental timing is largely recapitulated in stem cell models, suggesting a cell-intrinsic clock. Here we use directed differentiation of human embryonic stem cells into neuroectoderm to perform a whole-genome CRISPR-Cas9 knockout screen and show that the epigenetic factors Menin and SUZ12 modulate the speed of PAX6 expression during neural differentiation. Genetic and pharmacological loss-of-function of Menin or SUZ12 accelerate cell fate acquisition by shifting the balance of H3K4me3 and H3K27me3 at bivalent promoters, thereby priming key developmental genes for faster activation upon differentiation. We further reveal a synergistic interaction of Menin and SUZ12 in modulating differentiation speed. The acceleration effects were observed in definitive endoderm, cardiomyocyte and neuronal differentiation paradigms, pointing to chromatin bivalency as a general driver of timing across germ layers and developmental stages.},
}
@article {pmid40890563,
year = {2025},
author = {Li, X and Liu, C and Guo, G and Xu, Q and Ren, X and Tuerhongjiang, A and Liu, J and Zeng, J and Wen, CY},
title = {CRISPR-Driven Portable Piezoresistive Biosensor with Cascaded Signal Amplification for Ultrasensitive Osteocalcin Detection.},
journal = {Analytical chemistry},
volume = {97},
number = {36},
pages = {19552-19559},
doi = {10.1021/acs.analchem.5c02678},
pmid = {40890563},
issn = {1520-6882},
mesh = {*Biosensing Techniques/methods ; Humans ; *Osteocalcin/analysis/blood ; Limit of Detection ; Platinum/chemistry ; *CRISPR-Cas Systems ; Metal Nanoparticles/chemistry ; *Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {Low-turnover osteoporosis diagnosis urgently requires sensitive detection of low-abundance osteocalcin (OC), yet conventional methods remain constrained by insufficient sensitivity, cumbersome instrumentation, and laborious operations. We devise a CRISPR-driven pressure bioassay that synergistically integrates molecular recognition, enzymatic amplification, and signal transduction for dual-amplification-enhanced OC quantification. The system features an engineered "locked-to-activated" molecular switch, where target binding liberates CRISPR-activating DNA strands, initiating Cas14a-catalyzed cleavage of ssDNA tethers on Fe3O4-ssDNA-Pt nanoassemblies. This cascade releases a multitude of platinum nanoparticles (the first amplification stage). Subsequently, the liberated platinum nanoparticles drive the catalytic decomposition of H2O2 within sealed microchambers, generating a massive flux of oxygen gas molecules (O2) (second amplification stage). Coupled with a laboratory-fabricated nanostructured piezoresistive sensor (20 Pa resolution), this two-stage amplification strategy achieves high sensitivity with a 7.31 pg/mL detection limit, 124-fold lower than commercial ELISA, while completing analysis within 60 min. The platform demonstrates remarkable specificity (spike recovery of 113%, 112%, and 110% in human serum), operational robustness across varying environmental temperatures (15-40 °C), and compatibility with miniaturized instrumentation. Clinical validation through serum matrix analysis reveals excellent correlation (R[2] = 0.982) with reference values. By integrating CRISPR programmability, nanozyme-amplified signaling, and portable piezoresistive sensing, this work provides a sensitive point-of-care osteoporosis screening method for resource-limited settings.},
}
@article {pmid40887819,
year = {2025},
author = {Pérez-Maroto, J and Sepp-Lorenzino, L and Castaño-Esteban, D and Palacios, D and Sot, B},
title = {Advancements in Nonviral Gene Editing Strategies for Rare Diseases.},
journal = {Human gene therapy},
volume = {36},
number = {17-18},
pages = {1118-1137},
doi = {10.1177/10430342251372056},
pmid = {40887819},
issn = {1557-7422},
mesh = {Humans ; *Gene Editing/methods ; *Rare Diseases/therapy/genetics ; *Genetic Therapy/methods ; Genetic Vectors/genetics ; Gene Transfer Techniques ; CRISPR-Cas Systems ; Animals ; Nanoparticles/chemistry ; Nanomedicine ; },
abstract = {Rare diseases are serious and often chronic conditions that affect a small number of individuals. However, with over 7,000 rare diseases identified, their cumulative global numbers and impact are substantial. A considerable proportion of these conditions is caused by genetic abnormalities. Among these, monogenic disorders are of particular relevance, as they are caused by mutations in specific genes. The development of gene therapy, and more specifically, gene editing, offers innovative approaches to treat these rare diseases. A significant challenge associated with the implementation of such strategies concerns the delivery of gene editing tools. Nonviral vectors based on nanomaterials have demonstrated considerable potential as promising alternatives to viral vectors, thereby overcoming their disadvantages. The biocompatibility and tunability of nanoparticles, along with their potential capacity to target diverse tissues, positions them as a promising therapeutic approach for the treatment of a wide range of organ-specific rare diseases. Here, we review current progress in the development and evaluation of novel nanomedicine strategies for gene editing in rare diseases, highlighting new gene editing approaches, delivery systems, and potential targets.},
}
@article {pmid40848918,
year = {2025},
author = {Coratella, E and Bohnert, R and Fischer, B and Lemmens, M and Alpert, T and Beibel, M and Oertli, M and Naumann, U and Elhajouji, A and Odermatt, A and Libertini, S},
title = {SACF and GILA assays on AML12 cells show limited predictive value for mouse liver genotoxicity.},
journal = {Toxicology and applied pharmacology},
volume = {504},
number = {},
pages = {117529},
doi = {10.1016/j.taap.2025.117529},
pmid = {40848918},
issn = {1096-0333},
mesh = {Animals ; Mice ; Gene Editing ; *Liver Neoplasms/genetics/pathology ; CRISPR-Cas Systems ; *Carcinoma, Hepatocellular/genetics/pathology ; Dependovirus/genetics ; *Liver/pathology/metabolism/drug effects ; Predictive Value of Tests ; Mutagenicity Tests/methods ; Humans ; },
abstract = {Hepatocellular carcinoma (HCC) has been observed in neonatal mice following the integration of recombinant Adeno-Associated Viruses (rAAV) into the Rian locus. rAAV-related oncogenic risk for patients remains unclear, and the lack of relevant in vitro methods hinders its proper assessment. The soft agar colony-forming (SACF) assay and the growth in low attachment assay (GILA) monitor anchorage-independent growth, a hallmark of transformed adherent cells, and have been previously proposed to assess the tumorigenicity of CRISPR/Cas9-edited human MCF10A cells. Here, we introduce murine versions of SACF and GILA as surrogate in vitro systems to evaluate the risk of HCC development following genome editing or rAAV induced insertional mutagenesis. Selected tumor suppressors linked to HCC onset in vivo were edited through CRISPR/Cas9 in the hepatic murine cell line AML12. The knockout of neurofibromin (Nf2) and the dual inactivation of tumor protein p53 (Tp53) and phosphatase and tensin homolog (Pten) induced anchorage-independence, while the editing of Axin1, Ctnnb1 (coding for β-catenin), and tuberous sclerosis complex 1 (Tsc1) did not promote growth in anchorage-free conditions. Additionally, we generated stable AML12 and MCF10A clones with the rAAV genome respectively integrated into Rian and MEG8, the human homolog of Rian; however, these clones did not show anchorage independence when seeded in SACF and GILA. Overall, the murine SACF and GILA exhibit low predictive value for HCC development, failing to detect rAAV- and tumor-suppressors-associated oncogenicity. While further optimization may improve assays performance, these results highlight the need for more appropriate in vitro methodologies to accurately evaluate rAAV genotoxicity.},
}
@article {pmid40845730,
year = {2025},
author = {Lai, B and Guo, H and Zhang, K and Wang, H and Qiu, X and Lin, L and Shi, R and Sedibe, M and Zou, Y and Norvienyeku, J},
title = {Putative SET domain-containing proteins play significant roles in regulating sporulation and pathogenicity in Phytophthora capsici.},
journal = {Microbiological research},
volume = {301},
number = {},
pages = {128316},
doi = {10.1016/j.micres.2025.128316},
pmid = {40845730},
issn = {1618-0623},
mesh = {*Phytophthora/pathogenicity/genetics/growth &amp; development ; *Plant Diseases/microbiology/parasitology ; Virulence/genetics ; Gene Expression Profiling ; Capsicum/microbiology ; CRISPR-Cas Systems ; *Fungal Proteins/genetics/metabolism ; },
abstract = {Phytophthora capsici is a filamentous oomycete responsible for root rot, fruit rot, leaf blight, and other economically destructive diseases in multiple plant species, including pepper (Capsicum annuum), tomato (Solanum lycopersicum), squash (Cucurbita pepo), eggplant (Solanum melongena), faba bean (Vicia faba), and lima bean (Phaseolus lunatus), among others. The pathogen causes significant yield losses in fruit and vegetable crops globally. Multiple molecular parameters, including effector proteins and epigenetic modulators, play vital roles in modulating the physio pathological development of P. capsici. Here, we identified 56 SET domain-containing proteins in P. capsici, with 35 predicted as potential effectors. Transcriptomic analysis revealed the upregulation of 13 candidate effector genes during infection, suggesting their roles in pathogenicity. We successfully deleted one effector, PcSET-C (DVH05_022087), and two non-effector proteins, PcSET-B (DVH05_004260), and PcSET-A (DVH05_000194), using CRISPR-Cas9 and homologous recombination strategies. Phenotypic analysis showed that targeted deletion of the PcSET-A, and PcSET-B gene significantly impaired vegetative growth, while ΔPcset-C strains displayed growth like wild-type strains. Additionally, we showed that targeted disruption of all three genes resulted in reduced asexual sporulation and almost completely abolished the pathogenicity or virulence of ΔPcset-A, ΔPcset-B, and ΔPcset-C strains on different P. capsici-susceptible plants. However, targeted replacement of the three SET domain-containing genes investigated in this study has no significant adverse effects on zoospore release in P. capsici. These findings provide insights into the crucial role of SET domain-containing proteins in both morphological and pathological development of P. capsici and underscore PcSETs as potential targets for disease control.},
}
@article {pmid40834922,
year = {2025},
author = {Zhao, JJ and Tian, SN and Peng, ZY and Ren, JX and Zhang, YY and Li, GH and Deng, DH and Zhang, JP and Zhang, XB},
title = {Biomembrane-inspired lipid nanoparticles enhance CRISPR-Cas9 editing for hemophilia A.},
journal = {Journal of controlled release : official journal of the Controlled Release Society},
volume = {386},
number = {},
pages = {114141},
doi = {10.1016/j.jconrel.2025.114141},
pmid = {40834922},
issn = {1873-4995},
mesh = {Animals ; *CRISPR-Cas Systems ; *Hemophilia A/therapy/genetics ; *Gene Editing/methods ; *Nanoparticles/administration &amp; dosage/chemistry ; Humans ; *Factor VIII/genetics ; Mice ; Mice, Inbred C57BL ; Genetic Therapy/methods ; Dependovirus/genetics ; Male ; *Lipids/chemistry/administration &amp; dosage ; Liposomes ; },
abstract = {Lipid nanoparticles (LNPs) have substantially advanced RNA-based therapies; however, their use for CRISPR-Cas9 remains limited by sub-optimal endosomal escape, innate immune activation, transient nuclease expression, and restricted tissue specificity. Here, we engineered biomembrane-inspired LNPs containing sphingomyelin and C18-galactosyl ceramide (C18-GalCer) to improve liver-targeted CRISPR delivery. The optimized formulation increased in vivo editing efficiency 2.3-fold relative to a benchmark ALC-0315 LNP. Compared with adeno-associated virus (AAV) vectors, transient mRNA delivery reduced the theoretical risk of insertional mutagenesis, lowered innate immune readouts, and allowed dose titration. By pairing AAV-mediated delivery of a therapeutic Factor VIII donor with LNP-mediated CRISPR-Cas9, we achieved efficient and durable gene correction in a hemophilia A mouse model. A single LNP dose restored plasma Factor VIII activity to >50 % of wild-type levels and maintained this correction for >12 weeks, with low systemic cytokine induction, undetectable off-target insertions, and no overt toxicity. The LNPs retained physicochemical properties and editing potency after prolonged storage and multiple freeze-thaw cycles, supporting their translational potential. Collectively, our findings position biomembrane-inspired LNPs as a safe and efficient non-viral CRISPR platform with potential applicability to other gene-editing therapies.},
}
@article {pmid40749819,
year = {2026},
author = {Gaur, A and Bhakhri, H and Mishra, N and Sharma, S and Bansal, T and Kalaivani, M and Brijwal, M and Das, BK and Lodha, R and Sinha, S and Luthra, K},
title = {A rapid CRISPR/Cas12a-based assay for the detection of HIV-1 Clade C in infants.},
journal = {Clinica chimica acta; international journal of clinical chemistry},
volume = {578},
number = {},
pages = {120518},
doi = {10.1016/j.cca.2025.120518},
pmid = {40749819},
issn = {1873-3492},
mesh = {Humans ; *HIV-1/genetics/isolation &amp; purification ; *CRISPR-Cas Systems/genetics ; Infant ; *HIV Infections/diagnosis/virology ; Nucleic Acid Amplification Techniques ; },
abstract = {BACKGROUND: Early detection of HIV-1 infection is essential for initiating antiretroviral therapy (ART) to suppress viremia and prevent disease progression. Timely diagnosis, especially in infants, is critical as rapid antibody-based serology tests are ineffective due to the presence of maternal antibodies.<br><br>METHODS: We developed a CRISPR/Cas12a-based HIV-1 detection assay by optimizing components for coupled isothermal preamplification using recombinase polymerase amplification (RPA). The assay targeted the conserved region in the pol gene specific to HIV-1 with the designed CRISPR RNA (crRNA). CRISPR/Cas12a-mediated cleavage of viral cDNA was visualized through the collateral cleavage of a single-stranded DNA-FAM-BQ reporter, enabling rapid and visually detectable outcomes. The performance of the assay was evaluated using plasma from 41 HIV-1 Clade C (HIV-1C) seropositive individuals, including 28 HIV-1C infected infant samples, HIV-1 Indian Clade C and Clade B genome plasmids, viral disease control DNA/RNA samples (Influenza, RSV, Parvovirus, HPIV, CMV, and HBV), and 31 healthy donor plasma samples. Sensitivity and specificity were assessed, and detection was performed using fluorescence, visual readout, and lateral flow dipsticks.<br><br>RESULTS: The CRISPR/Cas12a-based HIV-1 Clade C detection assay achieved a sensitivity of 96&#xa0;% and a specificity of 92.65&#xa0;%. The assay successfully provided results through both fluorescence and visual readouts and was compatible with lateral flow dipstick formats, facilitating easy and rapid detection.<br><br>CONCLUSIONS: The developed CRISPR/Cas12a-based HIV-1C detection assay demonstrates high sensitivity and specificity for Clade C, indicating its potential as a robust point-of-care molecular diagnostic tool for HIV-1C. Additionally, it may serve as a rapid nucleic acid test alternative for detecting mother-to-child transmission of HIV-1C in infants under two years of age, where traditional antibody-based tests are ineffective. This assay holds promise for improving early HIV-1 diagnosis and timely initiation of ART, ultimately contributing to better disease management and outcomes.},
}
@article {pmid40709654,
year = {2025},
author = {Chang, MM},
title = {An Undergraduate Laboratory on Recombineering and CRISPR/Cas9-Assisted Gene Editing in Escherichia coli.},
journal = {Biochemistry and molecular biology education : a bimonthly publication of the International Union of Biochemistry and Molecular Biology},
volume = {53},
number = {5},
pages = {555-562},
doi = {10.1002/bmb.70002},
pmid = {40709654},
issn = {1539-3429},
mesh = {*Escherichia coli/genetics ; *Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; *Laboratories ; Humans ; Students ; Universities ; *Recombination, Genetic ; *Genetic Engineering ; },
abstract = {Laboratory experience is vital to undergraduate science education. It allows students to observe and conduct engaging experiments to enhance their skills and literacy, helps them retain knowledge, and deepens their understanding of related content covered in lectures. This paper reports a 4-week undergraduate laboratory exercise on Escherichia coli gene editing by recombineering, recombination-mediated genetic engineering, with or without clustered regularly interspaced short palindromic repeats and their associated protein 9 (CRISPR/Cas9). Gene editing makes precise modifications to the DNA of living organisms that influence their development and functions. As technology evolves, recombineering and CRISPR/Cas9 have replaced methods that use restriction enzymes and DNA ligase and are applied to a wide variety of research and applications. It is necessary to introduce undergraduates to these two rapidly growing technologies. Student results obtained from the lab indicate that antisense single-stranded oligodeoxynucleotide (ssODN) has a 15-20 times higher recombineering efficiency than the sense strand. Treatment with a plasmid containing the crRNA target of CRISPR/Cas9 increased recombineering efficiency. Instructional assessments, based on student feedback, revealed that the lab had clear objectives, instructions, and explicit protocols, with sufficient time to complete them, and was found to be interesting and worthwhile. Student learning outcomes, assessed by comparing pre-lab questions and post-lab tests, suggested that they learned the underlying principles and detailed molecular mechanisms. Besides learning the technologies and acquiring basic laboratory skills, students practiced key components of scientific research, such as data collection, analysis, and scientific communication.},
}
@article {pmid40694540,
year = {2025},
author = {Knoll, N and Masser, S and Bordas, B and Ebright, RY and Li, G and Kesar, D and Destefanis, E and Kania, N and Rodriguez, DJ and Jen, J and Zagar, SE and Mensah, C and Chen, Z and Moffitt, SJ and Enakireru, EM and He, Y and Feng, B and Chokshi, MK and Jin, CY and Raghavan, S and Sellers, WR and Mulvaney, KM},
title = {CRISPR-Drug Combinatorial Screening Identifies Effective Combination Treatments for MTAP-Deleted Cancer.},
journal = {Cancer research},
volume = {85},
number = {18},
pages = {3518-3539},
doi = {10.1158/0008-5472.CAN-25-1464},
pmid = {40694540},
issn = {1538-7445},
support = {R35GM154987//National Institute of General Medical Sciences (NIGMS)/ ; 1R01CA233626//Center for Cancer Research (CCR)/ ; CA232543//Basic Research Laboratory (BRL)/ ; V Scholar Grant//V Foundation for Cancer Research (VFCR)/ ; Promos Scholarship//German Academic Exchange Service (DAAD)/ ; //Austrian Marshall Plan Scholarship/ ; Travel Scholarship//BioTechMed-Graz (BIOTECHMEDGRAZ)/ ; CA260442//Center for Cancer Research (CCR)/ ; //Hale Family Center for Pancreatic Cancer Research (Hale Family Research Center At Dana-Farber Cancer Institute)/ ; Claudia Adams Barr Program//Dana-Farber Cancer Institute (DFCI)/ ; },
mesh = {Humans ; *Purine-Nucleoside Phosphorylase/genetics ; Animals ; Mice ; *Protein-Arginine N-Methyltransferases/antagonists &amp; inhibitors/genetics ; *CRISPR-Cas Systems ; *Cyclin-Dependent Kinase Inhibitor p16/genetics ; Cell Line, Tumor ; Xenograft Model Antitumor Assays ; *Lung Neoplasms/genetics/drug therapy/pathology ; *Antineoplastic Combined Chemotherapy Protocols/pharmacology/therapeutic use ; },
abstract = {UNLABELLED: Cyclin-dependent kinase inhibitor 2A (CDKN2A)/methylthioadenosine phosphorylase (MTAP) codeletion occurs frequently in non-small cell lung cancer and other solid tumors, including glioblastoma and pancreatic ductal adenocarcinoma. Lung cancer remains the leading cause of cancer-related mortality, and fewer than 15% of patients with glioblastoma or pancreatic cancer survive 5 years, underscoring the need for more effective therapies. Protein arginine methyltransferase 5 (PRMT5) is a synthetic-lethal dependency in MTAP-null tumors and an attractive therapeutic target for CDKN2A/MTAP-deleted cancers. A new revolutionary class of inhibitors, referred to as methylthioadenosine (MTA)-cooperative PRMT5 inhibitors (PRMT5i), has shown promising results in ongoing early-phase clinical trials. Nonetheless, effective cancer treatment typically requires therapeutic combinations to improve response rates and defeat emergent resistant clones. Thus, we sought to determine whether perturbation of other pathways could improve the efficacy of MTA-cooperative PRMT5is (MTAC-PRMT5i). Using a paralog and single gene targeting CRISPR library, we screened MTAP-deleted cancers in the presence or absence of MTAC-PRMT5is. Loss of several genes sensitized cells to PRMT5 inhibition, including members of the MAPK pathway. Chemical inhibition of MAPK pathway members using KRAS, MEK, ERK, and RAF inhibitors synergized with PRMT5 inhibition to kill CDKN2A/MTAP-null, RAS-active tumors. Furthermore, MTAC-PRMT5is combined with either KRAS or RAF inhibitors led to complete responses in vivo, emphasizing the potential benefit for patients. Lastly, cell lines resistant to KRAS inhibition were not resistant to MTAC-PRMT5is and vice versa, suggesting noncross-reactive mechanisms of resistance. Overall, this study identifies therapeutic combinations with MTAC-PRMT5is that may offer significant benefits to patients.<br><br>SIGNIFICANCE: Combining PRMT5 and MAPK pathway inhibitors leads to complete, durable responses in lung cancer models, providing an effective therapeutic strategy for the 4-5% of cancer patients harboring CDKN2A/MTAP deletion and MAPK alterations. See related article by Drizyte-Miller et al., p. 3540.},
}
@article {pmid40684990,
year = {2025},
author = {Gustafsson, O and Krishna, S and Borate, S and Ghaeidamini, M and Liang, X and Saher, O and Cuellar, R and Birdsong, BK and Roudi, S and Estupiñán, HY and Alici, E and Smith, CIE and Esbjörner, EK and Spuler, S and de Jong, OG and Escobar, H and Nordin, JZ and Andaloussi, SEL},
title = {Advanced peptide nanoparticles enable robust and efficient delivery of gene editors across cell types.},
journal = {Journal of controlled release : official journal of the Controlled Release Society},
volume = {386},
number = {},
pages = {114038},
doi = {10.1016/j.jconrel.2025.114038},
pmid = {40684990},
issn = {1873-4995},
mesh = {Humans ; *Gene Editing/methods ; *Nanoparticles/chemistry/administration &amp; dosage ; HEK293 Cells ; CRISPR-Cas Systems ; *Cell-Penetrating Peptides/chemistry/administration &amp; dosage ; Silicon Dioxide/chemistry ; Induced Pluripotent Stem Cells/metabolism ; *Gene Transfer Techniques ; Integrases/genetics/administration &amp; dosage ; CRISPR-Associated Protein 9/genetics ; },
abstract = {Efficient delivery of the CRISPR/Cas9 system and its larger derivatives, base editors, and prime editors remain a major challenge, particularly in tissue-specific stem cells and induced pluripotent stem cells (iPSCs). This study optimized a novel family of cell-penetrating peptides, hPep, to deliver gene-editing ribonucleoproteins. The hPep-based nanoparticles enable highly efficient and biocompatible delivery of Cre recombinase, Cas9, base-, and prime editors. Using base editors, robust and nearly complete genome editing was achieved in the human cells: HEK293T (96%), iPSCs (74%), and muscle stem cells (80%). This strategy opens promising avenues for ex vivo and, potentially, in vivo applications. Incorporating silica particles enhanced the system's versatility, facilitating cargo-agnostic delivery. Notably, the nanoparticles can be synthesized quickly on a benchtop and stored as lyophilized powder without compromising functionality. This represents an important advancement in the feasibility and scalability of gene-editing delivery technologies.},
}
@article {pmid40680607,
year = {2025},
author = {Eladl, O},
title = {CRISPR interference-mediated silencing of pro-inflammatory genes in autoimmune models: functional and biophysical evaluation with sustained effects in primary human PBMCs.},
journal = {International immunopharmacology},
volume = {163},
number = {},
pages = {115257},
doi = {10.1016/j.intimp.2025.115257},
pmid = {40680607},
issn = {1878-1705},
mesh = {Humans ; *Leukocytes, Mononuclear/immunology ; *Gene Silencing ; CRISPR-Cas Systems ; *Autoimmune Diseases/genetics/immunology/therapy ; CD40 Antigens/genetics ; Interleukin-6/genetics ; Interferon-gamma/genetics ; *Inflammation/genetics ; Cells, Cultured ; Cytokines/genetics ; },
abstract = {Autoimmune diseases are caused by unregulated immune stimulation and overproduction of inflammatory cytokines, leading to chronic tissue injury and disability. Current treatments are broad-acting and short-lived, highlighting the need for targeted molecular interventions with prolonged immunomodulatory effects. Here, we demonstrate CRISPR interference (CRISPRi), employing a catalytically inactive Cas9 fused to a KRAB repression domain, to potently and selectively silence major inflammatory genes-IL-6, CD40, and IFN-γ-in human immune cell lines. Utilizing biophysical methods such as surface plasmon resonance and fluorescence anisotropy, complemented by flow cytometric analysis, we validated functional interference with inflammatory signaling cascades and significant downregulation of immune activation markers. These findings indicate that CRISPRi not only represses gene expression but also significantly affects downstream cytokine-receptor interactions and immune cell activation. Compared to other gene silencing approaches, like siRNA and antisense oligonucleotides, CRISPRi showed greater specificity and longer duration, with sustained repression lasting up to 72 h post-treatment. Importantly, we confirmed these effects in primary human peripheral blood mononuclear cells (PBMCs), where CRISPRi significantly suppressed pro-inflammatory cytokine release and modulated immune cell phenotypes. These results highlight the potent, programmable, and sustained immunomodulatory potential of CRISPRi as a therapeutic for autoimmune diseases characterized by inflammation and immune dysregulation.},
}
@article {pmid40673842,
year = {2025},
author = {Johnson, MJ and Sumstad, D and Folsom, TD and Slipek, NJ and DeFeo, AP and Growe, M and Kadidlo, D and Thyagarajan, B and Starr, TK and Lou, E and Choudhry, M and Moriarity, BS and Webber, BR and McKenna, DH},
title = {Clinical manufacture of CRISPR/Cas9-based cytokine-induced SH2 protein knock-out tumor-infiltrating lymphocytes for gastrointestinal cancers.},
journal = {Cytotherapy},
volume = {27},
number = {10},
pages = {1229-1239},
doi = {10.1016/j.jcyt.2025.06.007},
pmid = {40673842},
issn = {1477-2566},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Lymphocytes, Tumor-Infiltrating/immunology/metabolism ; *Gastrointestinal Neoplasms/immunology/therapy/pathology/genetics ; *Cytokines ; Male ; Female ; Gene Knockout Techniques ; },
abstract = {INTRODUCTION: The prognosis of stage IV gastrointestinal (GI) carcinomas is poor with a 15% five-year survival rate for colorectal carcinomas. To improve efficacy of tumor infiltrating lymphocytes (TIL), we isolated mutation-reactive autologous TIL and employed CRISPR/Cas9 to knockout (KO) the intracellular checkpoint protein CISH, which has been shown to enhance T cell expansion, functional avidity, and cytokine polyfunctionality, with consequent durable regression of established tumors in an animal model.<br><br>MATERIALS &amp; METHODS: TIL cultures were initiated from resected tumor fragments and maintained for six weeks before harvest and cryopreservation. Candidate neoantigens were nominated by exome sequencing and peptides were used to identify mutation reactive (MR) TIL. Selected MR TIL were thawed and allowed to recover for 24-36 h in media with 10% AB serum, 6000 IU/mL IL-2, and 5 ng/mL IL-7 and IL-15 followed by stimulation with plate-bound anti-CD3/soluble anti-CD28 for 4 days. CISH KO was performed by electroporation of Cas9 mRNA and chemically modified single guide RNA. Between 5 -7.5 million viable cells were added to each 100 cm[2] G-Rex vessel containing 600 mL expansion media (with allogeneic feeder MNC:TIL = 100:1) and incubated for 6-8 days. Cultures were evaluated and split according to cell concentration criteria (and dose cohort) and incubated for an additional 6-8 days. On day 14, all of the cells were harvested, washed with buffer and cryopreserved (5% DMSO). Lot release testing included: viability, %CD3+, cytology review, Gram stain, sterility, endotoxin, mycoplasma, and interferon gamma (IFN-&#x3b3;) production. Additional testing included DNA sequencing to determine genomic CISH editing efficiency and a Western blot for determination of CISH protein loss.<br><br>RESULTS: Patients with GI cancers (colon [10], rectal [8], pancreatic [1], and esophageal [1]) underwent tumor collection. Nineteen of 22 tumor biopsies sampled from 20 patients total proceeded to KO/expansion. Final TIL product results (mean [SD], median [range]) were: viable count (x 10[10]) -3.25 (3.67), 1.95 (0.018-12.40); viable TIL fold expansion -327.1 (364.8), 153.1 (8-1454); % viability - 76 (13), 78 (43-92); % CD3 -94.4 (5.4), 95.8 (78.6-99.4); % CISH KO efficiency - 75 (29), 87 (0-96); % editing efficiency - 59.9 (24.8), 66.9 (0.4-86). Viability fell below 70% for five TIL products. All other lot release testing has met specification. Thirteen patients have received TIL; six patients were not treated due to disease progression prior to anticipated infusion.<br><br>CONCLUSION: The translation of CRISPR/Cas9-based CISH KO MR TIL from the basic research lab to current good manufacturing practices The (cGMP) facility was successful, allowing for optimized, large-scale expansion in support of a first-in-human clinical trial to treat patients with metastatic GI cancers (ClinicalTrials.gov Identifier: NCT04426669).},
}
@article {pmid40641457,
year = {2025},
author = {Su, P and Wu, L and Li, D and Song, W and Tao, D and Liu, L and Wang, Q and Gao, M and Xu, T and Liu, X and Xie, S and Zhang, X and Zhou, J and Miao, YL},
title = {CRISPR Screening Reveals a Novel Role for FOXH1 in Regulating Pluripotency of Porcine Embryonic Stem Cells.},
journal = {Advanced science (Weinheim, Baden-Wurttemberg, Germany)},
volume = {12},
number = {34},
pages = {e09495},
doi = {10.1002/advs.202509495},
pmid = {40641457},
issn = {2198-3844},
support = {2024YFA1802400//National Key R&amp;D Program of China/ ; 2022YFD1302200//National Key R&amp;D Program of China/ ; 32425051//National Natural Science Foundation of China/ ; 2662023DKPY001//Fundamental Research Funds for the Central Universities/ ; 2024040801020291//Knowledge Innovation Program of Wuhan-Basic Research/ ; },
mesh = {Animals ; Swine ; Cell Differentiation/genetics ; *CRISPR-Cas Systems/genetics ; *Embryonic Stem Cells/metabolism/cytology ; *Pluripotent Stem Cells/metabolism/cytology ; *Forkhead Transcription Factors/genetics/metabolism ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; },
abstract = {Porcine extended potential stem cells (pEPSCs), which exhibit both self-renewal and pluripotency, are promising for application in both agricultural biotechnology and regenerative medicine. However, the molecular mechanisms governing these two interconnected properties remain elusive. Here, two types of CRISPR-Cas9 screenings are conducted in pEPSCs. This fitness screening identified several genes essential for cell viability, including PRMT1, MYBL2, and NASP. Concurrently, FACS-based screenings revealed genes crucial for pluripotency, such as SOX2, ZFP42, and FOXH1. Notably, it is demonstrated that FOXH1 is required for maintaining pluripotency in pEPSCs, which complements the understanding of its role in mesendoderm specification. pEPSCs lacking FOXH1 exhibited a flatter and more dispersed clonal morphology, accompanied by downregulation of pluripotency genes and upregulation of lineage-specific genes. Additionally, FOXH1 knockdown significantly impaired blastocyst formation during early pig embryogenesis. Functionally, the dual role of FOXH1 in pluripotency maintenance and cell differentiation is validated: FOXH1 preserves pluripotency by enhancing chromatin accessibility at pluripotency gene loci, while also influencing lineage specification through H3K4me3 modification at developmental related genes. Thus, these findings uncover a novel role of FOXH1 involved in the core regulatory network that orchestrates gene expression programs to maintain the pluripotency state of pEPSCs and provide valuable insights into categorizing gene function.},
}
@article {pmid40586425,
year = {2025},
author = {Han, Z and Wang, X and Yu, D and Wang, J and Sun, K and Wang, S and Zhang, Y and Feng, G and Li, W and Hai, T and Ren, J},
title = {Achieving scalable expansion of therapeutic porcine hepatocytes in vivo through serial transplantation.},
journal = {Animal models and experimental medicine},
volume = {8},
number = {7},
pages = {1337-1344},
pmid = {40586425},
issn = {2576-2095},
support = {2021YFA0805905//National Key Research and Development Program of China/ ; 2023YFC3404305//National Key Research and Development Program of China/ ; 2024YFA1107900//National Key Research and Development Program of China/ ; XDB1150000//the Strategic Priority Research Program of the Chinese Academy of Sciences/ ; YSBR-012//the CAS Project for Young Scientists in Basic Research/ ; NYHXGG2023AA01//Bingtuan Science and Technology Project/ ; },
mesh = {*Hepatocytes/transplantation/physiology ; Animals ; Swine ; Liver Regeneration ; CRISPR-Cas Systems ; Liver ; },
abstract = {The clinical application of hepatocyte transplantation has been significantly hindered by the scarcity of primary hepatocytes and the functional immaturity of in vitro-produced hepatocytes. By performing serial allogeneic hepatocyte transplantation in CRISPR/Cas9-mediated Fah-knockout pigs, we successfully achieved large-scale expansion of hepatocytes while maintaining their authentic biological characteristics. Particularly, the established model enables sustained in vivo liver reconstruction, concurrently ameliorating hepatic fibrosis and demonstrating functional microenvironmental remodeling. Moreover, through comprehensive single-cell transcriptomic profiling of 52 418 hepatocytes across transplant generations (F0-F2), we discovered that the cellular composition of these transplanted hepatocytes is similar to that of wild-type hepatocytes. The regenerated liver exhibits all six major hepatic cell types identical to the wild-type counterparts, with the characteristic lobular zonation patterns well preserved. Our research provides valuable insights into the large-scale expansion of physiologically functional hepatocytes in vivo without compromising their biological properties. This finding holds great promise for advancing the clinical application of human hepatocyte transplantation, potentially offering more effective treatment options for patients with liver diseases.},
}
@article {pmid40432605,
year = {2025},
author = {López-Manzaneda, S and Mencía, &#xc1; and Bonafont, J and Bassons-Bascuñana, A and García, M and Nyström, A and Duarte, B and Llames, S and Murillas, R and Modamio-Hoybjor, S and Morín, M and Soletto, L and Escamez, MJ and Moreno-Pelayo, MA and Rio, MD and Larcher, F},
title = {Safe and Efficacious Permanent Removal of Large COL7A1 Exons for Gene Reframing as a Reliable Therapeutic Strategy for Recessive Dystrophic Epidermolysis Bullosa.},
journal = {Human gene therapy},
volume = {36},
number = {17-18},
pages = {1211-1221},
doi = {10.1089/hum.2024.238},
pmid = {40432605},
issn = {1557-7422},
mesh = {*Collagen Type VII/genetics/metabolism ; *Epidermolysis Bullosa Dystrophica/genetics/therapy/pathology ; *Exons ; Humans ; *Gene Editing/methods ; Animals ; CRISPR-Cas Systems ; *Genetic Therapy/methods ; Mice ; DNA End-Joining Repair ; },
abstract = {Mutations leading to premature termination codons in COL7A1 are commonly associated with severe generalized recessive dystrophic epidermolysis bullosa (RDEB). Previous research, including our own, has indicated that removing mutated COL7A1 exons along with the consequent reframing of COL7A1 may not pose noticeable impact on protein function, offering a potential therapeutic strategy. However, investigations into the long-term in vivo effects of genome editing-mediated removal of mutant exons have only focused on the small exon 80 thus far. Hence, this study focuses on exons 73 and 105 of COL7A1 to explore whether targeted exon removal, through a CRISPR/Cas9-assisted, Non-homologous end joining (NHEJ)-mediated approach, could be extended to other larger exons. Introducing ribonucleoprotein complexes carrying Cas9 and optimized sgRNA guide pairs for each exon (73 and 105) through electroporation efficiently led to their removal, consequently restoring type VII collagen (C7) synthesis in RDEB primary patient cells carrying frameshift mutations in these exons. In vitro tests indicated the normal stability of the resulting C7 variants expressed at physiological levels, while in vivo analyses of regenerated skin grafted onto immunodeficient mice using E73 or E105 RDEB edited cells demonstrated the proper deposition of C7 at the basement membrane zone, thereby restoring normal dermo-epidermal adherence. This study enhances the broader potential of the exon deletion approach in the treatment of RDEB.},
}
@article {pmid40252393,
year = {2025},
author = {Almasoudi, HH},
title = {Therapeutic promise of CRISPR-Cas9 gene editing in sickle cell disease and β-thalassemia: A current review.},
journal = {Current research in translational medicine},
volume = {73},
number = {3},
pages = {103513},
doi = {10.1016/j.retram.2025.103513},
pmid = {40252393},
issn = {2452-3186},
mesh = {Humans ; *Anemia, Sickle Cell/therapy/genetics ; *beta-Thalassemia/therapy/genetics ; *Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; *Genetic Therapy/methods ; Animals ; Mutation ; beta-Globins/genetics ; },
abstract = {Sickle cell disease (SCD and β-thalassemia (BT) affects millions of people worldwide. In addition, around 500,000 infants are born with SCD and 60,000 people are diagnosed with BT every year. Mutations in the hemoglobin subunit beta (HBB) gene are responsible for causing both BT and SCD. Indeed, the diversity of potential mutations in the HBB gene elucidates the diversity in clinical severity observed in individuals with BT and related morbidities. On the other hand, SCD takes place because of the alteration in a single amino acid at position 6 in the beta-globin chain, where a base substitution occurs from glutamic acid to valine, which eventually results in abnormal sickle hemoglobin. Conventional therapies for BT and SCD including pharmaceutical drugs and blood transfusion might temporarily improve the clinical severity of these diseases, however these therapies cannot cure the diseases. CRISPR-Cas9 (CC9) is revolutionizing genome engineering, offering promising therapeutic avenues for genetic diseases. Therefore, CC9-mediated gene therapy provides great hope in the treatment of both BT and SCD. CC9-mediated gene therapy has already demonstrated its effectiveness in correcting both SCD and BT-causing mutations. Moreover, CC9-mediated gene editing was found to be effective in reactivating the expression of hemoglobin F (HbF) and regulating LRF and BCL11A. A number of clinical trials with CC9 gene-edited therapies are being carried out to elucidate their potential in treating BT and SCD. Genetics and pathophysiological mechanisms of SCD and BT, the mechanism of CC9-mediated gene editing, and common delivery methods of the CC9 system have been discussed in this review. Moreover, an in-depth discussion on applications and the current status of CC9-mediated gene editing in SCD and BT along with current challenges and future perspectives have been provided.},
}
@article {pmid39500392,
year = {2025},
author = {Huang, M and Wang, K and Li, A and Zhu, X and Zhou, Z and Yang, C and Bi, C and Zhang, X},
title = {Mini and enhanced CRISPR activators for cancer therapies.},
journal = {Journal of advanced research},
volume = {75},
number = {},
pages = {151-161},
doi = {10.1016/j.jare.2024.10.027},
pmid = {39500392},
issn = {2090-1224},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Neoplasms/therapy/genetics ; Animals ; *Gene Editing/methods ; *Genetic Therapy/methods ; Cell Line, Tumor ; Caenorhabditis elegans/genetics ; Cell Proliferation/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Transcriptional Activation/genetics ; },
abstract = {INTRODUCTION: The RNA-guided nuclease Cas9 can be used as a programmable transcription activator, but there is still room for improvement in its effectiveness in eukaryotes, and its potential in cancer genetic therapy has been poorly investigated.<br><br>OBJECTIVES: We aim to construct optimized CRISPRa tools and detect their potential role in cancer therapy by screening 9aa-TAD.<br><br>METHODS: We selected a range of transcriptional coactivators for programmable activation and analyzed their effects on the expression of multiple endogenous genes using Flow cytometry and qRT-PCR. In order to improve the activation capacity of the CRISPRa tool, we fused the coactivators with the efficient dCas9-VPR system to construct a new activation system. Utilize RNA-seq to assess the activation specificity of genome-wide. To evaluate the value of the newly constructed activation system in cancer gene therapy, we activated the expression of the tumor suppressor genes PER2 and ZNF382, and performed changes in cancer cell proliferation qRT-PCR and clonal formation analysis.<br><br>RESULTS: In this study, we screened the NHR module from C. elegans, which demonstrated a high transcription activation capacity with a compact size compared to VP64. We successfully demonstrated its efficiency in activating endogenous genes in mammalian cells. Furthermore, we developed an enhanced fused variant called NHR-VP64-p65-Rta (NVPR), which showed even higher efficiency compared to the previously established VPR module, making it an effective CRISPRa tool. The dCas9-NVPR complex also exhibited high specificity on a genome-wide scale. Finally, we utilized the dCas9-NVPR tool to restore the expression of tumor suppressor genes PER2 and ZNF382, effectively inhibiting the malignant phenotype of cancer cells.<br><br>CONCLUSION: We have successfully developed and demonstrated a breakthrough CRISPRa tool with promising implications for cancer genetic therapy. This innovation expands the range of available gene editing tools and further validates the immense potential of CRISPR-based approaches in precision medicine.},
}
@article {pmid39487265,
year = {2025},
author = {Tycko, J and Van, MV and Aradhana, and DelRosso, N and Ye, H and Yao, D and Valbuena, R and Vaughan-Jackson, A and Xu, X and Ludwig, C and Spees, K and Liu, K and Gu, M and Khare, V and Mukund, AX and Suzuki, PH and Arana, S and Zhang, C and Du, PP and Ornstein, TS and Hess, GT and Kamber, RA and Qi, LS and Khalil, AS and Bintu, L and Bassik, MC},
title = {Development of compact transcriptional effectors using high-throughput measurements in diverse contexts.},
journal = {Nature biotechnology},
volume = {43},
number = {9},
pages = {1525-1538},
pmid = {39487265},
issn = {1546-1696},
support = {K00 DK126120/DK/NIDDK NIH HHS/United States ; R01 EB029483/EB/NIBIB NIH HHS/United States ; R01 HG011866/HG/NHGRI NIH HHS/United States ; },
mesh = {Humans ; *Transcription Factors/genetics/metabolism ; CRISPR-Cas Systems/genetics ; *High-Throughput Screening Assays/methods ; HEK293 Cells ; Protein Domains ; *Transcription, Genetic ; Gene Expression Regulation ; },
abstract = {Transcriptional effectors are protein domains known to activate or repress gene expression; however, a systematic understanding of which effector domains regulate transcription across genomic, cell type and DNA-binding domain (DBD) contexts is lacking. Here we develop dCas9-mediated high-throughput recruitment (HT-recruit), a pooled screening method for quantifying effector function at endogenous target genes and test effector function for a library containing 5,092 nuclear protein Pfam domains across varied contexts. We also map context dependencies of effectors drawn from unannotated protein regions using a larger library tiling chromatin regulators and transcription factors. We find that many effectors depend on target and DBD contexts, such as HLH domains that can act as either activators or repressors. To enable efficient perturbations, we select context-robust domains, including ZNF705 KRAB, that improve CRISPRi tools to silence promoters and enhancers. We engineer a compact human activator called NFZ, by combining NCOA3, FOXO3 and ZNF473 domains, which enables efficient CRISPRa with better viral delivery and inducible control of chimeric antigen receptor T cells.},
}
@article {pmid39415058,
year = {2025},
author = {Chen, K and Han, H and Zhao, S and Xu, B and Yin, B and Lawanprasert, A and Trinidad, M and Burgstone, BW and Murthy, N and Doudna, JA},
title = {Lung and liver editing by lipid nanoparticle delivery of a stable CRISPR-Cas9 ribonucleoprotein.},
journal = {Nature biotechnology},
volume = {43},
number = {9},
pages = {1445-1457},
pmid = {39415058},
issn = {1546-1696},
support = {RM1HG009490//U.S. Department of Health &amp; Human Services | National Institutes of Health (NIH)/ ; UH3 NS115599/NS/NINDS NIH HHS/United States ; R01MH125979-01//U.S. Department of Health &amp; Human Services | National Institutes of Health (NIH)/ ; R01 MH125979/MH/NIMH NIH HHS/United States ; DISC2-14097//California Institute for Regenerative Medicine (CIRM)/ ; DISC2-14045//California Institute for Regenerative Medicine (CIRM)/ ; UG3 NS115599/NS/NINDS NIH HHS/United States ; UM1AI164559//U.S. Department of Health &amp; Human Services | NIH | National Institute of Allergy and Infectious Diseases (NIAID)/ ; },
mesh = {*Gene Editing/methods ; Animals ; *CRISPR-Cas Systems/genetics ; *Ribonucleoproteins/genetics/metabolism ; Mice ; *Nanoparticles/chemistry ; *Liver/metabolism ; *Lung/metabolism ; *CRISPR-Associated Protein 9/genetics/metabolism ; Humans ; Lipids/chemistry ; Liposomes ; },
abstract = {Lipid nanoparticle (LNP) delivery of clustered regularly interspaced short palindromic repeat (CRISPR) ribonucleoproteins (RNPs) could enable high-efficiency, low-toxicity and scalable in vivo genome editing if efficacious RNP-LNP complexes can be reliably produced. Here we engineer a thermostable Cas9 from Geobacillus stearothermophilus (GeoCas9) to generate iGeoCas9 variants capable of >100× more genome editing of cells and organs compared with the native GeoCas9 enzyme. Furthermore, iGeoCas9 RNP-LNP complexes edit a variety of cell types and induce homology-directed repair in cells receiving codelivered single-stranded DNA templates. Using tissue-selective LNP formulations, we observe genome-editing levels of 16‒37% in the liver and lungs of reporter mice that receive single intravenous injections of iGeoCas9 RNP-LNPs. In addition, iGeoCas9 RNPs complexed to biodegradable LNPs edit the disease-causing SFTPC gene in lung tissue with 19% average efficiency, representing a major improvement over genome-editing levels observed previously using viral or nonviral delivery strategies. These results show that thermostable Cas9 RNP-LNP complexes can expand the therapeutic potential of genome editing.},
}
@article {pmid39385008,
year = {2025},
author = {Wang, Y and Liu, KI and Liu, MM and Ooi, KH and Nguyen, TA and Chee, JE and Teo, SXD and He, S and Tay, JWD and Teo, SY and Liew, KS and Ge, XY and Ng, ZJ and Avagyan, H and Liu, H and Yi, Z and Chang, K and Kok, EPL and Chen, R and Yau, CE and Koh, JW and Wan, Y and Tan, MH},
title = {A circularly permuted CasRx platform for efficient, site-specific RNA editing.},
journal = {Nature biotechnology},
volume = {43},
number = {9},
pages = {1548-1561},
pmid = {39385008},
issn = {1546-1696},
support = {NRF2017-NRF-ISF002-2673//National Research Foundation Singapore (National Research Foundation-Prime Minister's office, Republic of Singapore)/ ; SSG 2021-03//National Research Foundation Singapore (National Research Foundation-Prime Minister's office, Republic of Singapore)/ ; T2EP30223-0028//Ministry of Education - Singapore (MOE)/ ; RG50/17//Ministry of Education - Singapore (MOE)/ ; NMRC/OFIRG/0017/2016//MOH | National Medical Research Council (NMRC)/ ; },
mesh = {*RNA Editing/genetics ; Humans ; Adenosine Deaminase/genetics/metabolism ; *Protein Engineering/methods ; *CRISPR-Cas Systems/genetics ; RNA-Binding Proteins/genetics ; *CRISPR-Associated Proteins/genetics ; HEK293 Cells ; },
abstract = {Inactive Cas13 orthologs have been fused to a mutant human ADAR2 deaminase domain at the C terminus to enable programmable adenosine-to-inosine (A-to-I) RNA editing in selected transcripts. Although promising, existing RNA-editing tools generally suffer from a trade-off between efficacy and specificity, and off-target editing remains an unsolved problem. Here we describe the development of an optimized RNA-editing platform by rational protein engineering, CasRx-based Programmable Editing of RNA Technology (xPERT). We demonstrate that the topological rearrangement of a CasRx K940L mutant by circular permutation results in a robust scaffold for the tethering of a deaminase domain. We benchmark our tool against the REPAIR system and show that xPERT exhibits strong on-target activity like REPAIRv1 but low off-target editing like REPAIRv2. Our xPERT platform can be used to alter RNA sequence information without risking genome damage, effect temporary cellular changes and customize protein function.},
}
@article {pmid39375445,
year = {2025},
author = {Song, J and Cho, MH and Cho, H and Song, Y and Lee, SW and Nam, HC and Yoon, TH and Shin, JC and Hong, JS and Kim, Y and Ekanayake, E and Jeon, J and You, DG and Im, SG and Choi, GS and Park, JS and Carter, BC and Balaj, L and Seo, AN and Miller, MA and Park, SY and Kang, T and Castro, CM and Lee, H},
title = {Amplifying mutational profiling of extracellular vesicle mRNA with SCOPE.},
journal = {Nature biotechnology},
volume = {43},
number = {9},
pages = {1485-1495},
pmid = {39375445},
issn = {1546-1696},
support = {HR22C1832//Korea Health Industry Development Institute (KHIDI)/ ; U01CA284982//U.S. Department of Health &amp; Human Services | NIH | National Cancer Institute (NCI)/ ; DP2CA259675//U.S. Department of Health &amp; Human Services | NIH | NIH Office of the Director (OD)/ ; 2021M3E5E3080379//National Research Foundation of Korea (NRF)/ ; U01 CA230697/CA/NCI NIH HHS/United States ; 2023R1A2C2005185//National Research Foundation of Korea (NRF)/ ; 1U01CA279858//U.S. Department of Health &amp; Human Services | NIH | National Cancer Institute (NCI)/ ; 2021R1A2B5B03001416//National Research Foundation of Korea (NRF)/ ; 2021R1A2C1005342//National Research Foundation of Korea (NRF)/ ; R01 CA237500/CA/NCI NIH HHS/United States ; R01 CA239078/CA/NCI NIH HHS/United States ; R01CA264363//U.S. Department of Health &amp; Human Services | NIH | National Cancer Institute (NCI)/ ; },
mesh = {*Extracellular Vesicles/genetics/metabolism ; Humans ; *RNA, Messenger/genetics ; *Mutation/genetics ; Animals ; Mice ; CRISPR-Cas Systems/genetics ; Colorectal Neoplasms/genetics ; Glioblastoma/genetics ; Lung Neoplasms/genetics ; Cell Line, Tumor ; Liquid Biopsy ; },
abstract = {Sequencing of messenger RNA (mRNA) found in extracellular vesicles (EVs) in liquid biopsies can provide clinical information such as somatic mutations, resistance profiles and tumor recurrence. Despite this, EV mRNA remains underused due to its low abundance in liquid biopsies, and large sample volumes or specialized techniques for analysis are required. Here we introduce Self-amplified and CRISPR-aided Operation to Profile EVs (SCOPE), a platform for EV mRNA detection. SCOPE leverages CRISPR-mediated recognition of target RNA using Cas13 to initiate replication and signal amplification, achieving a sub-attomolar detection limit while maintaining single-nucleotide resolution. As a proof of concept, we designed probes for key mutations in KRAS, BRAF, EGFR and IDH1 genes, optimized protocols for single-pot assays and implemented an automated device for multi-sample detection. We validated SCOPE's ability to detect early-stage lung cancer in animal models, monitored tumor mutational burden in patients with colorectal cancer and stratified patients with glioblastoma. SCOPE can expedite readouts, augmenting the clinical use of EVs in precision oncology.},
}
@article {pmid40950596,
year = {2025},
author = {Hadian, S and Smith, DL and Suproniene, S},
title = {Genomic and phenotypic profiling of an Artemisia endophyte: dual biostimulant and biocontrol activities in pea under Fusarium stress.},
journal = {Frontiers in microbiology},
volume = {16},
number = {},
pages = {1643204},
pmid = {40950596},
issn = {1664-302X},
abstract = {OBJECTIVE: To evaluate the plant growth-promoting and disease-suppressing potential of Serratia sp. AR11, an endophytic bacterium isolated from Artemisia absinthium, through phenotypic assessment and whole-genome analysis in pea (Pisum sativum) under normal and Fusarium-stress conditions.<br><br>MATERIALS AND METHODS: Greenhouse experiments were conducted to assess the effects of AR11 inoculation on shoot and root growth, biomass, chlorophyll content, and Fusarium-induced stunting. Whole-genome sequencing was performed using the PacBio SMRT platform, followed by functional annotation to identify genes related to nutrient metabolism, secondary metabolite biosynthesis, and stress adaptation. Biosafety assessment included screening for virulence and antibiotic resistance genes.<br><br>RESULTS: AR11 inoculation significantly increased shoot and root length and biomass, while reducing Fusarium-induced stunting by over 70%. Under pathogen stress, treated plants showed a 67% increase in SPAD index compared to controls. Genome analysis revealed a complete 5.49 Mb circular genome with 5,175 protein-coding genes, including those for nitrogen metabolism, phosphate solubilization, siderophore production, and antifungal secondary metabolite biosynthesis. Additional features included stress-responsive regulatory systems, CRISPR-Cas loci, and DNA methylation patterns. No high-risk virulence or acquired antibiotic resistance genes were detected.<br><br>CONCLUSION: Serratia sp. AR11 is a safe, multifunctional endophytic bacterium with strong genomic and phenotypic evidence for enhancing pea growth and suppressing Fusarium-related diseases, making it a promising candidate for sustainable low-input agriculture.},
}
@article {pmid40946761,
year = {2025},
author = {Uddin, N and Ullah, MW and Li, K and Liu, F and Xin, X},
title = {Plant cell wall biosynthesis: Immune signaling, genome editing, and physiological implications for biomass valorization.},
journal = {Biotechnology advances},
volume = {},
number = {},
pages = {108714},
doi = {10.1016/j.biotechadv.2025.108714},
pmid = {40946761},
issn = {1873-1899},
abstract = {Plants continuously face biotic stress from pathogens, pests, and environmental challenges that threaten their survival and productivity. In response, plants have developed complex immune systems, with the cell wall playing a central role in defense. The plant cell wall not only provides mechanical strength but also acts as a dynamic barrier against pathogens, influencing both plant growth and immune responses. This review discusses the molecular mechanisms of cell wall biosynthesis, facilitated by multi-omics technologies, particularly the synthesis and regulation of lignin and other polysaccharides, which contribute to cell wall integrity and plant immunity. It explores the interplay between cell wall modifications and immune signaling pathways, highlighting the role of pattern recognition receptors in pathogen detection and defense activation. Additionally, the potential of genome editing, especially CRISPR-Cas, in enhancing cell wall characteristics to improve pathogen resistance and biomass utilization is discussed. With growing interest in lignocellulosic biomass as a renewable resource for biofuels and bioproducts, this review also addresses the challenges of biomass recalcitrance, focusing on biotechnological advancements to improve saccharification efficiency. Finally, the review proposes integrated strategies combining genetic modifications, biotechnological innovations, and sustainable practices to optimize lignocellulosic biomass for a bio-based economy, contributing to both agricultural resilience and sustainable energy production. As climate change accelerates, these technologies hold the promise of developing resilient crops and enhancing the capacity of the bioeconomy to mitigate environmental impacts.},
}
@article {pmid40946164,
year = {2025},
author = {Sun, J and Liu, K and Sheng, Y and Zhang, H and Wang, J and Wang, Y and Tian, R and Zhu, X and Tian, SS and Wang, P and Zhao, X and Liu, Y},
title = {High-Fidelity AaCas12b[Max] Enables the Development of an Engineered T Cell Therapy with Enhanced Safety and Functional Fitness.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.ymthe.2025.09.009},
pmid = {40946164},
issn = {1525-0024},
abstract = {CRISPR-Cas systems have transformed genome editing, yet the commonly used Streptococcus pyogenes Cas9 (SpCas9) is limited by off-target effects and chromosomal instability. Here, we characterize AaCas12b[MAX], an engineered Alicyclobacillus acidiphilus Cas12b variant, as a high-precision editing platform optimized for tumor infiltrating lymphocyte (TIL) therapy. Using an FDA-compliant safety assessment framework, we systemically compared AaCas12b[MAX]- and SpCas9-edited TIL products in terms of on-target efficiency, genome-wide off-target activity, and structural variants (SVs) formation. AaCas12b[MAX] achieved >80% on-target editing efficiency with undetectable off-target events and a 3.3-fold reduction in SVs relative to SpCas9. Mechanistic studies revealed different DNA repair kinetics in AaCas12b[MAX]-edited cells, reducing sustained DNA damage responses and chromosomal instability. Structural modeling suggested a more stable enzyme-sgRNA-DNA ternary complex, enabling stringent PAM specificity and minimal mismatch tolerance. Functionally, AaCas12b[MAX]-edited TILs exhibited superior therapeutic potential, including enhanced cellular fitness, a twofold increase in expansion capacity, and enrichment of stem-like tumor-reactive CD39[-]CD69[-]CD8[+] subsets. Together, these results establish AaCas12b[MAX] as a robust, clinically translatable platform that improves the safety and functional limitations of SpCas9, enabling the development of next-generation T cell therapies.},
}
@article {pmid40945731,
year = {2025},
author = {Jain, M and Pattnayak, AK and Aggarwal, S and Rai, P and Kavya, J and Chandrayan, S and Goel, M and Gaur, V},
title = {Branched DNA processing by a thermostable CAS-Cas4 from Thermococcus onnurineus: expanding biochemical landscape of nuclease activity.},
journal = {The Journal of biological chemistry},
volume = {},
number = {},
pages = {110701},
doi = {10.1016/j.jbc.2025.110701},
pmid = {40945731},
issn = {1083-351X},
abstract = {The adaptive immune function of CRISPR-Cas systems in bacteria and archaea is mediated through CRISPR-Associated Proteins (Cas). The adaptation module, typically involving Cas1, Cas2, and Cas4, helps integrate viral "spacer" sequences into the host genome. Cas4 proteins are classified into two types based on neighboring genes: CAS-Cas4, flanked by other cas genes, and Solo-Cas4, which exist independently. While CAS-Cas4 proteins are implicated in adaptation, they remain biochemically uncharacterized in archaea, unlike archaeal Solo-Cas4 proteins. This study biochemically characterizes TON_0321, a CAS-Cas4 protein from the Type IV-C CRISPR cassette of Thermococcus onnurineus. TON_0321 exhibits 5' to 3' exonuclease activity and unique structure-dependent endonuclease activity, shedding light on CAS-Cas4 functional diversity. A distinct spatial organization of the catalytic site, angled with the positively charged patch on the protein surface, enables TON_0321 to recognize branching points in DNA substrates. Furthermore, this spatial arrangement facilitates cleavage 2 to 3 nucleotides away from the branch point in the 5' direction, demonstrating structure-specific endonuclease activity.},
}
@article {pmid40943511,
year = {2025},
author = {Ma, Z and Ren, J and Liu, Q and Li, J and Zhao, H and Tibesigwa, DG and Matola, SH and Gulfam, T and Yang, J and Wang, F},
title = {Integrating Traditional Breeding and Modern Biotechnology for Advanced Forest Tree Improvement.},
journal = {International journal of molecular sciences},
volume = {26},
number = {17},
pages = {},
pmid = {40943511},
issn = {1422-0067},
abstract = {In the context of global climate change and efforts toward "carbon peak and carbon neutrality," forest resource protection and restoration have become fundamental to ecological civilization. The genetic improvement of trees, as the primary component of forest ecosystems, holds strategic importance for ecological security, resource supply, and carbon neutrality. Traditional tree breeding techniques, including selective and hybrid breeding, have established robust technical systems through extensive practice. However, these methods face limitations such as extended cycles, reduced efficiency, and constrained genetic gains in meeting contemporary requirements. Modern biotechnologies, including genomic selection (GS), gene editing (CRISPR/Cas9), and marker-assisted selection (MAS), substantially enhance the precision and efficiency of genetic improvement. Nevertheless, exclusive reliance on either traditional or modern methods proves insufficient for addressing complex environmental adaptation and rapid breeding requirements. Consequently, the integration of traditional breeding with modern biotechnology to develop intelligent, sustainable, and efficient breeding strategies has emerged as a central focus in tree genetics and breeding. An integrated "step-by-step" approach warrants promotion, supported by a multi-source data sharing platform, an optimized core germplasm repository, and a "climate-soil-genotype" matching model to facilitate the region-specific deployment of improved varieties.},
}
@article {pmid40943296,
year = {2025},
author = {Sheveleva, O and Butorina, N and Protasova, E and Medvedev, S and Grigor'eva, E and Melnikova, V and Kuziaeva, V and Minzhenkova, M and Tatarenko, Y and Lyadova, I},
title = {The Generation of iPSCs Expressing Interferon-Beta Under Doxycycline-Inducible Control.},
journal = {International journal of molecular sciences},
volume = {26},
number = {17},
pages = {},
pmid = {40943296},
issn = {1422-0067},
support = {&#x2116; 0088-2024-0013//government basic research program at the Koltzov Institute of Developmental Biology of the Russian Academy of Sciences in 2024/ ; },
mesh = {*Induced Pluripotent Stem Cells/metabolism/cytology/drug effects ; Humans ; *Doxycycline/pharmacology ; *Interferon-beta/genetics/metabolism ; Cell Differentiation/drug effects ; CRISPR-Cas Systems ; Cell Line ; },
abstract = {Type 1 interferons (IFN-Is) exhibit significant antiviral, antitumor, and immunoregulatory properties, demonstrating substantial therapeutic potential. However, IFN-Is are pleiotropic cytokines, and the available data on their effect under specific pathological conditions are inconclusive. Furthermore, the systemic administration of IFN-Is can result in side effects. Generating cells that can migrate to the pathological focus and provide regulated local production of IFN-Is could overcome this limitation and provide a model for an in-depth analysis of the biological and therapeutic effects of IFN-Is. Induced pluripotent stem cells (iPSCs) are a valuable source of various differentiated cell types, including human immune cells. In this study, we describe the generation of genetically modified human iPSCs with doxycycline-controlled overexpression of interferon β (IFNB1). Three IFNB1-overexpressing iPSC lines (IFNB-iPSCs) and one control line expressing the transactivator M2rtTA (TA-iPSCs) were generated using the CRISPR/Cas9 technology. The pluripotency of the generated cell lines has been confirmed by the following: (i) cell morphology; (ii) the expression of the pluripotency markers OCT4, SOX2, TRA 1-60, and NANOG; and (iii) the ability to spontaneously differentiate into the derivatives of the three germ layers. Upon the addition of doxycycline, all IFNB-iPSCs upregulated IFNB1 expression at RNA (depending on the iPSC line, 126-816-fold) and protein levels. The IFNB-iPSCs and TA-iPSCs generated here represent a valuable cellular model for studying the effects of IFN-β on the activity and differentiation trajectories of different cell types, as well as for generating different types of cells with controllable IFN-β expression.},
}
@article {pmid40938447,
year = {2025},
author = {Kang, X and Tian, F and Liu, X and Xiao, G and Cai, Y},
title = {CRISPR/Cas13 system-based entropy-driven DNAzyme switch powered DNA walking system for sensitive and direct rotavirus detection.},
journal = {Mikrochimica acta},
volume = {192},
number = {10},
pages = {663},
pmid = {40938447},
issn = {1436-5073},
support = {No. 2018QN035//Sichuan Provincial Administration of Traditional Chinese Medicine Medical Research Special 2018/ ; },
mesh = {*DNA, Catalytic/chemistry/metabolism/genetics ; *Rotavirus/isolation &amp; purification/genetics ; Entropy ; *CRISPR-Cas Systems ; Limit of Detection ; Metal Nanoparticles/chemistry ; *Biosensing Techniques/methods ; Gold/chemistry ; DNA Probes/chemistry/genetics ; Nucleic Acid Amplification Techniques/methods ; Humans ; },
abstract = {DNA walker-based strategies are confronted with significant challenges in harmonizing design complexity, sequence dependence, and amplification efficiency. This study describes the innovative design of a double-stranded DNA probe, named the "LW probe," which integrates a locked DNAzyme segment, enabling the coupling of the entropy-driven amplification (EDA) process with a DNAzyme-powered DNA walker. In the absence of the target, the "LW probe" remains in an inactive ("OFF") state. Upon encountering target rotavirus sequences, the LW probe receives the trans-cleavage activity of Cas13a/crRNA and undergoes a conformational change, transforming into an activated structure. This structural transition initiates the EDA process continuously, leading to the release of the DNAzyme segment. Subsequently, the released DNAzyme segment acts on the surface of gold nanoparticles (AuNPs), cleaving the "Substrate probe" and consequently liberating fluorescence signals. Distinct from traditional DNA walkers that rely exclusively on the EDA for product amplification, the proposed approach synergistically combines the high-precision target recognition capacity of the EDA process with the potent signal amplification efficiency of DNA walkers. This integration results in remarkable enhancements in both specificity, demonstrated by the ability to discriminate single-base mismatched sequences, and sensitivity, with a detection limit as low as 2.7 fM. By synergizing EDA with the DNAzyme-driven DNA walker, our method achieves high sensitivity, with a detection limit of 2.7 fM, outperforming or matching the performance of previous DNA walker-based systems. This system enables highly sensitive and specific detection of low-abundance rotavirus with robust stability, offering a promising platform for disease diagnosis and biomedical research.},
}
@article {pmid40938388,
year = {2025},
author = {Higa, L and Blank, M and Hampson, E and Matsuyama, J and Wilkes, K and Uehara, A and Bouwman, T and Lee, K and Wang, K and Muszynski, M and Du, ZY},
title = {Rapid assessment of CRISPR gRNAs with optimized protoplast transformation in Maize.},
journal = {Plant cell reports},
volume = {44},
number = {10},
pages = {212},
pmid = {40938388},
issn = {1432-203X},
support = {2121410//National Science Foundation/ ; },
mesh = {*Zea mays/genetics ; *Protoplasts/metabolism ; *Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; *RNA, Guide, CRISPR-Cas Systems/genetics ; *Transformation, Genetic ; Plants, Genetically Modified ; Transfection ; },
abstract = {We developed an optimized CRISPR/Cas9 gene editing system using maize mesophyll protoplasts to enable rapid evaluation of guide RNA (gRNA) activity. Using the tropical inbred line Tzi8, we improved protoplast isolation and transfection protocols, achieving high yields of 17.88 × 10[6] viable protoplasts per gram fresh weight while extending post-transfection viability. Etiolated seedlings and vertical leaf cutting significantly enhanced protoplast recovery and viability. A transfection efficiency of ~ 50% was achieved using 10 µg of plasmid DNA; higher DNA inputs did not result in significant gains, resulting in a more resource-efficient approach. Protoplast viability was maintained for up to seven days post-transfection, allowing for downstream applications that require extended incubation. This optimized system was used to assess the editing efficiency of nine gRNAs targeting three key floral repressors (ZmCCT9, ZmCCT10, and ZmRap2.7) across four maize genotypes (Tzi8, CML277, B73, and B104). These floral repressor genes are involved in the photoperiod sensitivity of tropical maize, a major challenge in the effort to introduce tropical maize germplasm into temperate breeding programs. Editing efficiencies ranged from 0.4% to 23.7%, with some variation observed between gRNAs and genotypes. Although protoplast-based assays do not currently enable plant regeneration, this platform offers a rapid method for in vivo gRNA validation, reducing assay time from months to days. This work expands the gene editing toolkit for tropical maize, supporting efforts to overcome breeding barriers through gene editing.},
}
@article {pmid40934206,
year = {2025},
author = {Lima, DA and Costa-Silva, HM and Albergaria, KSS and Ribeiro, JM and Resende, DM and Santarossa, BA and Liarte, DB and Calderano, SG and Murta, SMF},
title = {Glycosomal ABC transporter 3 (GAT3) deletion enhances the oxidative stress responses and reduces the infectivity of Trypanosoma cruzi.},
journal = {PLoS neglected tropical diseases},
volume = {19},
number = {9},
pages = {e0013479},
pmid = {40934206},
issn = {1935-2735},
mesh = {*Trypanosoma cruzi/genetics/pathogenicity/drug effects/metabolism ; *Oxidative Stress ; *ATP-Binding Cassette Transporters/genetics/metabolism ; Animals ; *Protozoan Proteins/genetics/metabolism ; CRISPR-Cas Systems ; Mice ; Chagas Disease/parasitology ; Nitroimidazoles/pharmacology ; *Microbodies/metabolism/genetics ; Trypanocidal Agents/pharmacology ; Gene Knockout Techniques ; Gene Deletion ; },
abstract = {Glycosomes, peroxisome-like organelles in Trypanosoma cruzi, contain enzymes involved in various metabolic processes, including glycolysis. Glycosomal ABC transporters (GATs) play a vital role in maintaining metabolic homeostasis by facilitating metabolite exchange between glycosomes and the cytoplasm. GAT3 is a member of the GAT family, which also includes GAT1 and GAT2. GAT3 transcript levels are downregulated in benznidazole-resistant T. cruzi populations; however, its specific functions remain unknown. Therefore, in this study, we generated GAT3 single-knockout and null mutant lines of the T. cruzi Dm28c strain using the CRISPR/Cas9 system to investigate GAT3 roles in parasite biology. RT-qPCR revealed increased GAT2 transcript levels in the GAT3 null mutant line, without any changes in GAT1 levels. Our findings suggest that GAT3 is not essential for T. cruzi survival, as null mutant parasites showed no growth difference compared to the Cas9-expressing controls. Moreover, the GAT3 single-knockout line exhibited increased resistance to benznidazole, whereas the null mutant line exhibited benznidazole susceptibility similar to the control. Furthermore, both GAT3 single-knockout and null mutant lines showed increased tolerance to hydrogen peroxide-induced oxidative stress. In vitro infection assay of L929 murine fibroblasts revealed that the GAT3 null parasites exhibited a significantly lower infection rate and fewer intracellular amastigotes than the controls. Overall, GAT3 is crucial for T. cruzi infectivity and the regulation of oxidative stress responses, playing key roles in the metabolic regulation and pathogenicity of this parasite.},
}
@article {pmid40934013,
year = {2025},
author = {Zhang, Y and Liu, T and Zhang, P and Ni, B and Wang, X and Bai, L and Sun, W and Guan, Y and Xia, X and Cao, H and Gu, J},
title = {A rapid and accurate method for Helicobacter pylori detection via integrating LAMP assay with CRISPR/Cas12b detection by one-step in one-pot.},
journal = {Frontiers in cellular and infection microbiology},
volume = {15},
number = {},
pages = {1611134},
pmid = {40934013},
issn = {2235-2988},
mesh = {*Helicobacter pylori/genetics/isolation &amp; purification ; Humans ; *Molecular Diagnostic Techniques/methods ; *Nucleic Acid Amplification Techniques/methods ; *Helicobacter Infections/diagnosis/microbiology ; *CRISPR-Cas Systems ; Sensitivity and Specificity ; Limit of Detection ; },
abstract = {INTRODUCTION: Accurate and timely detection of Helicobacter pylori (HP) is crucial for the diagnosis and management of gastritis and other HP-associated gastrointestinal disorders. Conventional diagnostic methods, such as PCR and culture, require specialized equipment and expertise, limiting their applicability in resource-limited settings. There is a pressing need for a rapid, cost-effective, and user-friendly diagnostic platform for HP detection, particularly in point-of-care settings.<br><br>METHODS: We developed an integrated detection platform combining loop-mediated isothermal amplification (LAMP) with the CRISPR/Cas12b system in a single, one-step, one-pot reaction. The assay was optimized to function at a constant temperature of 58 &#xb0;C and provides results within 45 minutes. The clinical performance of the system was evaluated using 22 clinical samples, and its diagnostic accuracy was compared with conventional PCR.<br><br>RESULTS: The LAMP-CRISPR/Cas12b assay demonstrated a limit of detection (LOD) of 14.77 copies per test, with no cross-reactivity observed against potential interfering nucleic acids, ensuring 100% specificity for HP. Clinical validation revealed a concordance rate of 90.91% (20/22) between the LAMP-CRISPR/Cas12b platform and conventional PCR, supporting the diagnostic reliability of the system.<br><br>DISCUSSION: The integrated LAMP-CRISPR/Cas12b platform represents a promising alternative for the rapid and sensitive detection of HP. It combines the simplicity and rapidity of LAMP with the specificity of CRISPR/Cas12b, offering a robust, cost-effective, and high\-sensitivity diagnostic tool without the need for complex instrumentation. The method shows great potential for use in point-of-care testing (POCT) and could significantly enhance clinical practice by facilitating timely diagnosis and treatment of HP-related diseases.},
}
@article {pmid40825449,
year = {2025},
author = {Chowdhury, R and Roure, A and Darras, S},
title = {Towards functional genetics in the European amphioxus: Efficient CRISPR/Cas9 editing reveals Ascl1/2.1 requirement for peripheral nervous system development.},
journal = {Developmental biology},
volume = {527},
number = {},
pages = {218-225},
doi = {10.1016/j.ydbio.2025.08.012},
pmid = {40825449},
issn = {1095-564X},
mesh = {Animals ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; *Basic Helix-Loop-Helix Transcription Factors/genetics/metabolism ; *Lancelets/genetics/embryology ; *Peripheral Nervous System/embryology/metabolism ; Gene Expression Regulation, Developmental ; },
abstract = {Amphioxus, or cephalochordates, have a key phylogenetic position among chordates and serve as pivotal invertebrate models for investigating the evolutionary origins of vertebrate traits. Although functional genetic tools have recently been developed, their application has been limited to the Floridian and Asian species, Branchiostoma floridae and Branchiostoma belcheri, respectively. In this study, we established a CRISPR/Cas9-based genome editing protocol to generate F0 mosaic mutants (crispants) in the European amphioxus B. lanceolatum. As a proof of concept, we targeted the Bl-Ascl1/2.1 gene, a putative regulator of epidermal sensory neuron (ESN) development in the peripheral nervous system coding for a bHLH transcription factor. Using a novel microinjection method of the sgRNA/Cas9 complex in fertilized eggs and two-cell stage embryos, we demonstrated the disruption of Bl-Ascl1/2.1 that resulted in a partial to complete loss of ESNs. Importantly, this phenotype could be rescued by Bl-Ascl1/2.1 mRNA microinjection. These findings demonstrate the efficiency of CRISPR/Cas9-mediated gene editing in B. lanceolatum and establish a foundation for future functional studies in this emerging EvoDevo model.},
}
@article {pmid40770575,
year = {2025},
author = {Southard, KM and Ardy, RC and Tang, A and O'Sullivan, DD and Metzner, E and Guruvayurappan, K and Norman, TM},
title = {Comprehensive transcription factor perturbations recapitulate fibroblast transcriptional states.},
journal = {Nature genetics},
volume = {57},
number = {9},
pages = {2323-2334},
pmid = {40770575},
issn = {1546-1718},
support = {GM132083//U.S. Department of Health &amp; Human Services | NIH | National Institute of General Medical Sciences (NIGMS)/ ; DRG-2462-22//Damon Runyon Cancer Research Foundation (Cancer Research Fund of the Damon Runyon-Walter Winchell Foundation)/ ; DP2 GM140925/GM/NIGMS NIH HHS/United States ; AWD-GC-259296//Damon Runyon Cancer Research Foundation (Cancer Research Fund of the Damon Runyon-Walter Winchell Foundation)/ ; GM140925//U.S. Department of Health &amp; Human Services | NIH | NIH Office of the Director (OD)/ ; HG012103//U.S. Department of Health &amp; Human Services | NIH | National Human Genome Research Institute (NHGRI)/ ; },
mesh = {*Fibroblasts/metabolism ; Humans ; *Transcription Factors/genetics/metabolism ; Kruppel-Like Factor 4 ; *Transcription, Genetic ; Animals ; Mice ; Gene Expression Regulation ; Kruppel-Like Transcription Factors/genetics ; CRISPR-Cas Systems ; Cell Differentiation/genetics ; Chromatin/genetics ; },
abstract = {Cell atlas projects have revealed that common cell types often comprise distinct, recurrent transcriptional states, but the function and regulation of these states remain poorly understood. Here, we show that systematic activation of transcription factors can recreate such states in vitro, providing tractable models for mechanistic and functional studies. Using a scalable CRISPR activation (CRISPRa) Perturb-seq platform, we activated 1,836 transcription factors in two cell types. CRISPRa induced gene expression within physiological ranges, with chromatin features predicting responsiveness. Comparisons with atlas datasets showed that transcription factor perturbations recapitulated key fibroblast states and identified their regulators, including KLF2 and KLF4 for a universal state present in many tissues, and PLAGL1 for a disease-associated inflammatory state. Inducing the universal state suppressed the inflammatory state, suggesting therapeutic potential. These findings position CRISPRa as a nuanced tool for perturbing differentiated cells and establish a general strategy for studying clinically relevant transcriptional states ex vivo.},
}
@article {pmid40748247,
year = {2025},
author = {Zhang, Y and Yang, X and Wang, Y and Chen, F and Liu, Y and Jiang, H and Wang, Y and Hu, Y and Li, S},
title = {Establishment of a CRISPR/Cas12b-Based Multiple Cross Displacement Amplification Assay for the Rapid, Sensitive, and Specific Detection of Brucella ovis.},
journal = {ACS infectious diseases},
volume = {11},
number = {9},
pages = {2446-2456},
doi = {10.1021/acsinfecdis.5c00287},
pmid = {40748247},
issn = {2373-8227},
mesh = {*Brucella ovis/genetics/isolation &amp; purification ; *CRISPR-Cas Systems ; *Brucellosis/diagnosis/veterinary/microbiology ; Animals ; Sheep ; *Nucleic Acid Amplification Techniques/methods ; Sensitivity and Specificity ; *Sheep Diseases/diagnosis/microbiology ; Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {Brucella ovis (B. ovis), a major pathogenic species within the Brucella genus, causes ovine epididymitis. Although the isolation and identification of B. ovis remain the gold standard for diagnosis, these methods are unsuitable for early detection. The traditional polymerase chain reaction (PCR) offers faster detection but requires specialized equipment such as PCR thermal cyclers and gel electrophoresis imagers, limiting its use in basic laboratories. Thus, developing rapid, sensitive, and specific diagnostic strategies is vital for preventing and controlling the spread of ovine brucellosis. In this study, we developed a diagnostic assay combining clustered regularly interspaced short palindromic repeats (CRISPR)/Cas12b with multiple cross displacement amplification (MCDA)─termed CRISPR/Cas12b-MCDA─for rapid, sensitive, and specific identification of B. ovis. In the CRISPR/Cas12b-MCDA system, MCDA amplicons containing protospacer adjacent motif (PAM) sites are recognized by the Cas12b/gRNA complex, which binds the target region and triggers trans-cleavage of a single-stranded DNA (ssDNA) reporter. The CRISPR/Cas12b-MCDA assay demonstrated a detection limit of 10 fg/μL for synthetic genomic DNA and exhibited 100% specificity for B. ovis, with no cross-reactivity against other Brucella or non-Brucella species. The preamplification for template extraction takes 20 min, then 5 min for uracil DNA glycosylase (UDG) digestion, and 45 min for MCDA amplification. The total detection time was 75 min using real-time fluorescence analysis and 90 min with a lateral flow biosensor (LFB). Additionally, the results were validated using UV visualization to confirm the CRISPR/Cas12b-MCDA results. Notably, both LFB and UV analyses are instrument-free, enhancing their accessibility. In conclusion, the CRISPR/Cas12b-MCDA assay is a simple, rapid, sensitive, specific, and reliable method for detecting B. ovis.},
}
@article {pmid40437754,
year = {2025},
author = {Xu, Y and Li, J and Wang, Z and Lu, R and Liu, Y and Wang, M and Li, H and Zhao, R and Feng, W},
title = {Ablation of dysmorphic neurons is a safe and effective treatment for focal cortical dysplasia II.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {33},
number = {9},
pages = {4414-4430},
pmid = {40437754},
issn = {1525-0024},
mesh = {*Epilepsy/genetics/pathology/therapy ; *Malformations of Cortical Development, Group I/genetics/pathology/therapy ; Proto-Oncogene Proteins c-akt/genetics ; Disease Models, Animal ; Animals ; Mice ; *Diphtheria Toxin/therapeutic use ; *Ablation Techniques ; *Heparin-binding EGF-like Growth Factor/genetics ; Humans ; HEK293 Cells ; TOR Serine-Threonine Kinases/genetics ; *Neurons/pathology ; CRISPR-Cas Systems ; *Gene Editing/methods ; },
abstract = {Focal cortical dysplasia type II (FCDII) is a leading cause of refractory epilepsy in children, yet treatment options remain limited. The most frequent genetic cause of FCDII is mosaic and somatic variants in genes of the phosphatidylinositol 3-kinase (PI3K)-protein kinase B (AKT)-mammalian target of rapamycin (mTOR) pathway, leading to hyperactivation of mTOR signaling. The presence of dysmorphic neurons (DNs) resulting from hyperactive mTOR signaling is critical for the development of epilepsy in FCDII. One critical therapeutic challenge and opportunity for FCDII is to selectively eliminate DNs. Here, we developed two strategies to specifically ablate DNs in FCDII mouse models, and the results demonstrate that DN ablation is sufficient to both prevent and eliminate epilepsy in mice. Moreover, the associated neurobehavioral abnormalities were also reversed following treatment. Therefore, our study provides proof-of-concept evidence that DN ablation is a highly promising approach for curing FCDII in the future.},
}
@article {pmid40943207,
year = {2025},
author = {Gilyazova, I and Korytina, G and Kochetova, O and Savelieva, O and Mikhaylova, E and Vershinina, Z and Chumakova, A and Markelov, V and Abdeeva, G and Karunas, A and Khusnutdinova, E and Gusev, O},
title = {Advances in Genomics and Postgenomics in Poultry Science: Current Achievements and Future Directions.},
journal = {International journal of molecular sciences},
volume = {26},
number = {17},
pages = {},
pmid = {40943207},
issn = {1422-0067},
support = {&#x2116; 075-15-2025-484//Government of Russian Federation/ ; },
abstract = {The poultry industry, a globally fast growing agricultural sector, provides affordable animal protein due to high efficiency. Gallus gallus domesticus are the most common domestic birds. Hybrid chicken breeds (crosses) are widely used to achieve high productivity. Maintaining industry competitiveness requires constant genetic selection of parent stock to improve performance traits. Genetic studies, which are essential in modern breeding programs, help identify genome variants linked to economically important traits and preserve population health. Next-generation sequencing (NGS) has identified millions of single nucleotide polymorphisms (SNPs) and insertions/deletions (INDELs), enabling detection of genome-wide regions associated with selection traits. Recent studies have pinpointed such regions using broiler lines, laying hen lines, or pooled genomic data. This review discusses advances in chicken genomic and transcriptomic research focused on traits enhancing meat breed performance and reproductive abilities. Special attention is given to transcriptome studies revealing regulatory mechanisms and key signaling pathways involved in artificial molting, as well as metagenome studies investigating resistance to infectious diseases and climate adaptation. Finally, a dedicated section highlights CRISPR/Cas genomic editing techniques for targeted genome modification in chicken genomics.},
}
@article {pmid40943047,
year = {2025},
author = {Huang, C and Cheng, L},
title = {Unlocking Casein Bioactivity: Lactic Acid Bacteria and Molecular Strategies for Peptide Release.},
journal = {International journal of molecular sciences},
volume = {26},
number = {17},
pages = {},
pmid = {40943047},
issn = {1422-0067},
abstract = {Bioactive peptides encrypted in bovine β-casein display diverse physiological functions, including antihypertensive, antioxidative, antimicrobial, and immunomodulatory activities. These peptides are normally released during gastrointestinal digestion or microbial fermentation, especially by proteolytic systems of lactic acid bacteria (LAB). However, peptide yields vary widely among LAB strains, reflecting strain-specific protease repertoires. To overcome these limitations, the scientific goal of this study is to provide a comprehensive synthesis of how synthetic biology, molecular biotechnology, and systems-level approaches can be leveraged to enhance the targeted discovery and production of β-casein-derived bioactive peptides. Genome engineering tools such as clustered regularly interspaced short palindromic repeats associated system (CRISPR/Cas) systems have been applied to modulate gene expression and metabolic flux in LAB, while inducible expression platforms allow on-demand peptide production. Additionally, cell-free systems based on LAB lysates further provide rapid prototyping for high-throughput screening. Finally, multi-omics approaches, including genomics, transcriptomics, proteomics, and metabolomics, further help pinpoint regulatory bottlenecks and facilitate rational strain optimization. This review provides a comprehensive overview of bioactive peptides derived from bovine β-casein and highlights recent progress in LAB-based strategies-both natural and engineered-for their efficient release. These advances pave the way for developing next-generation functional fermented foods enriched with targeted bioactivities.},
}
@article {pmid40825952,
year = {2025},
author = {Vats, P and Kumar, R and Kumar, R and Kaushik, JK and Mohanty, AK and Kumar, S},
title = {Deciphering the role of MFGE8 in lactation using CRISPR-CAS9 based gene editing in Buffalo mammary epithelial cells.},
journal = {Scientific reports},
volume = {15},
number = {1},
pages = {30194},
pmid = {40825952},
issn = {2045-2322},
support = {EMR/2017/000152//DST-SERB/ ; },
mesh = {Animals ; *CRISPR-Cas Systems ; *Gene Editing/methods ; Female ; *Lactation/genetics ; *Epithelial Cells/metabolism ; *Buffaloes/genetics ; *Mammary Glands, Animal/cytology/metabolism ; *Milk Proteins/genetics/metabolism ; Proteomics ; Gene Knockout Techniques ; *Glycoproteins/genetics/metabolism ; Proteome ; },
abstract = {Milk fat globule EGF factor 8 (MFGE8) is a glycoprotein which plays a crucial role in mammary gland remodeling. Our group previously identified MFGE8 as a marker associated with high milk yielding cows. Here, we generated MFGE8 knock-out buffalo mammary epithelial cells (BuMEC) via CRISPR-cas9 technology to decipher its role in lactation. gRNA3 reduced MFGE8 expression with good efficiency which was confirmed at transcriptomic and proteomic level and the stable knock-out cells obtained were named mfge8-/-/gRNA3. The amplicon sequencing of the edited region using next generation sequencing (NGS) showed that 54% of total reads showed indels, 3-4 bp upstream to PAM site in 2nd exon. A total 4282 proteins were identified when proteome level changes were examined and 178 were found to be differentially expressed above and below a threshold of ≥ 1.5 and ≤ 0.6. Major DEPs were found to be associated with regulation of hydrolase activity, endopeptidase activity and cytoskeletal organization and some DEPs including FABP3, FABP4, FABP5, KNG1, MT2A, CD82, SLC7A1 and SERPINH1 belonged to genes associated with milk synthesis. To the best of our knowledge, this is the first study which provides a comprehensive proteome profile of MFGE8 knockout BuMEC and explores downstream effects of disruption of MFGE8 gene.},
}
@article {pmid40825942,
year = {2025},
author = {Countryman, AD and Doherty, CA and Herrera-Perez, RM and Kasza, KE},
title = {Endogenous OptoRhoGEFs reveal biophysical principles of epithelial tissue furrowing.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {7665},
pmid = {40825942},
issn = {2041-1723},
support = {F31 HD118793/HD/NICHD NIH HHS/United States ; F31HD118793//U.S. Department of Health &amp; Human Services | NIH | Eunice Kennedy Shriver National Institute of Child Health and Human Development (NICHD)/ ; P40 OD018537/OD/NIH HHS/United States ; R35GM138380//U.S. Department of Health &amp; Human Services | NIH | National Institute of General Medical Sciences (NIGMS)/ ; R35 GM138380/GM/NIGMS NIH HHS/United States ; Sloan Fellowship//Alfred P. Sloan Foundation/ ; Packard Fellowship//David and Lucile Packard Foundation (David &amp; Lucile Packard Foundation)/ ; CAREER Award//National Science Foundation (NSF)/ ; },
mesh = {Animals ; *Drosophila Proteins/metabolism/genetics ; Epithelium/embryology/metabolism ; *Drosophila melanogaster/embryology/genetics/metabolism ; Optogenetics/methods ; Morphogenesis ; Actomyosin/metabolism ; Animals, Genetically Modified ; *Rho Guanine Nucleotide Exchange Factors/metabolism/genetics ; Gastrulation ; Embryo, Nonmammalian/metabolism ; CRISPR-Cas Systems ; Signal Transduction ; Gene Expression Regulation, Developmental ; },
abstract = {During development, epithelia function as malleable sheets that undergo extensive remodeling to shape developing embryos. Optogenetic control of Rho signaling provides an avenue to investigate mechanisms of epithelial morphogenesis, but transgenic optogenetic tools can be limited by variability in expression levels and deleterious effects of transgenic overexpression on development. Here, we use CRISPR/Cas9 to tag Drosophila RhoGEF2 and Cysts/Dp114RhoGEF with components of the iLID/SspB optogenetic heterodimer, permitting light-dependent control over endogenous protein activities. Using quantitative optogenetic perturbations, we uncover a dose-dependence of tissue furrow depth and bending behavior on RhoGEF recruitment, revealing mechanisms by which developing embryos can shape tissues into particular morphologies. We show that at the onset of gastrulation, furrows formed by cell lateral contraction are oriented and size-constrained by basal actomyosin. Our findings demonstrate the use of quantitative, 3D-patterned perturbations of cell contractility to precisely shape tissue structures and interrogate developmental mechanics.},
}
@article {pmid40825812,
year = {2025},
author = {Netsawang, C and Tongbaen, M and Jearawiriyapaisarn, N and Leecharoenkiat, K},
title = {Precise correction of G6PD Viangchan mutation in iPSCs by prime editing strategy.},
journal = {Scientific reports},
volume = {15},
number = {1},
pages = {30192},
pmid = {40825812},
issn = {2045-2322},
support = {B05F640126//The NSRF via the Program Management Unit for Human Resources &amp; Institutional Development, Research and Innovation/ ; 196807//Thailand Science research and Innovation Fund Chulalongkorn University/ ; },
mesh = {Humans ; *Glucosephosphate Dehydrogenase/genetics ; *Induced Pluripotent Stem Cells/metabolism ; *Gene Editing/methods ; HEK293 Cells ; *Glucosephosphate Dehydrogenase Deficiency/genetics/therapy ; *Mutation ; CRISPR-Cas Systems ; },
abstract = {Individuals with glucose-6-phosphate dehydrogenase (G6PD) deficiency hold a significant risk of severe hemolytic crises under oxidative stress. Currently, the definitive and curative treatment for the disorder has not been developed. Among over 200 G6PD variants, G6PD Viangchan (c.871 G > A) is the most prevalent and has been extensively studied in Southeast Asia. This study assessed the effectiveness of prime editing for correcting the G6PD Viangchan mutation in an established mutant HEK293T cell line and G6PD-deficient induced pluripotent stem cells (iPSCs). Using optimized modalities, prime editing achieved a high correction efficiency of over 25% in the HEK293T cells. In iPSCs, this gene editing tool yielded satisfactory correction outcomes, with approximately 5% corrected alleles. Our findings indicate that prime editing provides high precision, producing minimal by-products below baseline and showing undetectable off-target effects. Overall, prime editing has the potential to correct the G6PD Viangchan mutation, providing a valuable approach for future therapeutic strategies and the generation of isogenic cell lines to promote extensive studies in drug discovery and the pathogenesis of the G6PD variant.},
}
@article {pmid40825783,
year = {2025},
author = {Calvo-Villamañán, A and Sastre-Dominguez, J and Barrera-Martín, &#xc1; and Costas, C and San Millan, &#xc1;},
title = {Dissecting pOXA-48 fitness effects in clinical Enterobacterales using plasmid-wide CRISPRi screens.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {7700},
pmid = {40825783},
issn = {2041-1723},
support = {ALTF 322-2022//European Molecular Biology Organization (EMBO)/ ; },
mesh = {*Plasmids/genetics ; *beta-Lactamases/genetics/metabolism ; *Bacterial Proteins/genetics/metabolism ; *Enterobacteriaceae/genetics/drug effects ; Humans ; Enterobacteriaceae Infections/microbiology/drug therapy ; *Genetic Fitness ; CRISPR-Cas Systems/genetics ; Drug Resistance, Multiple, Bacterial/genetics ; Anti-Bacterial Agents/pharmacology ; Carbapenems/pharmacology ; },
abstract = {Conjugative plasmids are the main vehicle for the spread of antimicrobial resistance (AMR) genes in clinical bacteria. AMR plasmids allow bacteria to survive antibiotic treatments, but they also produce physiological alterations in their hosts that commonly translate into fitness costs. Despite the key role of plasmid-associated fitness effects in AMR evolution, their origin and molecular bases remain poorly understood. In this study, we introduce plasmid-wide CRISPR interference (CRISPRi) screens as a tool to dissect plasmid-associated fitness effects. We design and perform CRISPRi screens targeting the globally distributed carbapenem resistance plasmid pOXA-48 in 13 different multidrug resistant clinical Enterobacterales. Our results reveal that pOXA-48 gene-level effects are conserved across clinical strains, and expose the key role of the carbapenemase-encoding gene, blaOXA-48, as the main culprit for pOXA-48 fitness costs. Moreover, our results highlight the relevance of postsegregational killing systems in pOXA-48 vertical transmission, and uncover new genes implicated in pOXA-48 stability (pri, korC, DNDJGHEP_13 and 14 and H-NS). This study sheds new light on the biology and evolution of carbapenem resistant Enterobacterales and endorses CRISPRi screens as a powerful method for studying plasmid-mediated AMR.},
}
@article {pmid40824957,
year = {2025},
author = {Naes, SM and Ab-Rahim, S and Mazlan, M and Syafruddin, SE and Mohtar, MA and Abuhamad, AY and Abdul Rahman, A},
title = {CRISPR/Cas9 mediated ENT2 gene knockout altered purine catabolic pathway and induced apoptosis in colorectal cell lines.},
journal = {PloS one},
volume = {20},
number = {8},
pages = {e0329501},
pmid = {40824957},
issn = {1932-6203},
mesh = {Humans ; *Apoptosis/genetics ; *CRISPR-Cas Systems ; *Purines/metabolism ; *Colorectal Neoplasms/genetics/metabolism/pathology ; Gene Knockout Techniques ; Cell Line, Tumor ; *Equilibrative-Nucleoside Transporter 2/genetics/metabolism ; Reactive Oxygen Species/metabolism ; Xanthine Oxidase/metabolism ; HT29 Cells ; Hypoxanthine/metabolism ; Gene Expression Regulation, Neoplastic ; },
abstract = {Although purine metabolism is one of the most impacted pathways in colorectal cancer (CRC), little is known about the role of equilibrative nucleoside transporter 2 (ENT2) in CRC development and its association with the altered purine metabolism pathway. This study aimed to determine the role of ENT2 in altered purine metabolism in the early and late stages of CRC using CRISPR/Cas9 gene editing tools and a variety of functional experiments. The expression of ENT2 was significantly higher (P < 0.001) in all CRC cell lines as compared to the normal colon cells. The two CRC cell lines with the highest ENT2 expression, the early stage HT29 cells and the late stage DLD1 cells, were knocked out (KO) using the CRISPR/Cas9 tool. The hypoxanthine (HPX) level and the xanthine oxidase (XO) activity were significantly higher in both HT29/KO and DLD1/KO single cell-derived clones (P < 0.01). The increase in HPX level and XO activity were associated with an elevation in the reactive oxygen species (ROS) level. These data suggest that the ENT2 KO elevated the ROS levels induced apoptosis and impaired the cell proliferation of the early stage of CRC cell line, i.e., HT29/KO clonal cells. In this context, targeting ENT2 gene might be a potential strategy in CRC treatment by increasing the production of ROS and hence, inducing the apoptosis pathway.},
}
@article {pmid40823809,
year = {2025},
author = {Migliori, V and Bruntraeger, MB and Gyulev, IS and Lichou, F and Burgold, T and Gitterman, DP and Iwama, S and Trinh, AL and Washer, SJ and Jones, CP and Trynka, G and Bassett, AR},
title = {ONE-STEP tagging: a versatile method for rapid site-specific integration by simultaneous reagent delivery.},
journal = {Nucleic acids research},
volume = {53},
number = {15},
pages = {},
pmid = {40823809},
issn = {1362-4962},
support = {220540/Z/20/A/WT_/Wellcome Trust/United Kingdom ; //Sanger Translation Committee/ ; },
mesh = {Humans ; *CRISPR-Cas Systems ; *Gene Editing/methods ; Induced Pluripotent Stem Cells/metabolism ; T-Lymphocytes/metabolism ; Integrases/genetics/metabolism ; },
abstract = {We present a novel, versatile genome editing method termed ONE-STEP tagging, which combines CRISPR-Cas9-mediated targeting with Bxb1 integrase-based site-specific integration for efficient, precise, and scalable protein tagging. Applied in human-induced pluripotent stem cells (hiPSCs), cancer cells and primary T cells, this system enables rapid generation of endogenously tagged proteins. By enhancing the nuclear localization signal of the catalytically superior eeBxb1 integrase and co-delivering a DNA-PK inhibitor, we achieved up to ∼90% integration efficiency at the ACTR10 locus in hiPSCs. ONE-STEP tagging is robust across loci and cell types and supports large DNA cargo integration, with efficiencies reaching 16.6% for a 14.4 kb construct. The method also enables multiplexed tagging of multiple proteins within the same cell and simultaneous CRISPR-based editing at secondary loci, such as gene knockouts or homology-directed repair. Importantly, we demonstrate successful application in primary T cells by targeting the T cell receptor locus while simultaneously knocking out B2M, a key step towards generating immune-evasive, off-the-shelf chimeric antigen receptor T cells. Additionally, we introduce a dual-cassette version of the method compatible with universal donor plasmids, allowing use of entirely off-the-shelf reagents. Together, these advances establish ONE-STEP tagging as a powerful tool for both basic and therapeutic genome engineering.},
}
@article {pmid40814254,
year = {2025},
author = {Nevot, G and Güell, M and Santos-Moreno, J},
title = {Critical Analysis of Preprints and Inquiry-Based Lessons Improve the Synthetic Biology Learning Experience.},
journal = {ACS synthetic biology},
volume = {14},
number = {8},
pages = {2878-2884},
pmid = {40814254},
issn = {2161-5063},
mesh = {*Synthetic Biology/education ; Humans ; *Problem-Based Learning/methods ; Students ; Universities ; CRISPR-Cas Systems/genetics ; },
abstract = {Synthetic biology is a transformative field crucial to address global challenges. It is highly interdisciplinary, integrating different subjects beyond biology. Therefore, traditional lecture-based teaching methods often fall short in effectively covering the diverse and rapidly evolving advancements in synthetic biology. We developed active learning workflows for complementing classic theoretical lectures in universities to improve the synthetic biology learning experience. We used preprints as an educational resource for the students to critically analyze differences comparing manuscripts and the final published work. In addition, we designed a practical laboratory session where students had to infer the logic behind CRISPRi-based gene circuits that they assembled, thus engaging with every step of the design-build-test-learn cycle. Following these activities, 90% of the students reported having improved critical analysis skills and 80% felt that they had learned a wide range of synthetic biology concepts. These approaches demonstrate the potential of innovative teaching for synthetic biology, which helps students with both technical and soft skills at the same time and has the potential to be adapted to other fields.},
}
@article {pmid40736249,
year = {2025},
author = {Zhao, Y and Guo, G and Sun, Y and Zhang, M and Yang, G and Liu, Z and Song, Y and Ghonaim, AH and Ma, N and Zhang, M and Jongkaewwattana, A and He, Q and Li, W},
title = {Membrane protein CRISPR screen identifies RPSA as an essential host factor for porcine epidemic diarrhea virus replication.},
journal = {Journal of virology},
volume = {99},
number = {8},
pages = {e0064925},
pmid = {40736249},
issn = {1098-5514},
mesh = {Animals ; *Porcine epidemic diarrhea virus/physiology ; *Virus Replication ; Swine ; *Coronavirus Infections/virology/veterinary/metabolism ; CRISPR-Cas Systems ; *Swine Diseases/virology/metabolism/genetics ; Host-Pathogen Interactions ; *Membrane Proteins/genetics/metabolism ; MAP Kinase Signaling System ; Gene Knockout Techniques ; Chlorocebus aethiops ; },
abstract = {UNLABELLED: Porcine epidemic diarrhea, caused by porcine epidemic diarrhea virus (PEDV), is one of the most devastating diseases in the global pig industry due to its high mortality rate in piglets. The host factors required for PEDV replication, including receptors, remain poorly understood. Here, we developed a porcine membrane-protein-scale CRISPR/Cas9 knockout (PigMpCKO) library and performed two rounds of PEDV infection. Ribosomal protein SA (RPSA), the known receptor of dengue virus, was found to be a potent host factor. Moreover, our research revealed that RPSA is involved in the replication stage of PEDV and not in the entry stage. Inhibitor and activator experiments demonstrated that knockout (KO) of RPSA downregulates the ERK1/2 signaling pathway to impair PEDV infection. Additionally, RNA sequencing data indicated that cellular lipid biosynthesis and lipid transport processes were significantly inhibited in the absence of RPSA during PEDV infection. Mechanistic studies revealed that the reduction in total cholesterol and triglyceride levels, resulting from RPSA KO, was partially mediated by the ERK1/2 pathway, leading to impaired lipid accumulation during PEDV replication. Interestingly, RPSA KO also significantly downregulated the expression of aminopeptidase N (APN) and inhibited infection by transmissible gastroenteritis virus (TGEV) and porcine deltacoronavirus (PDCoV), both of which belong to the swine enteric coronavirus group. In summary, our results establish RPSA as a novel host factor that is critical for coronavirus replication. This provides new insights into the mechanisms of virus-host interactions and paves the way for the development of broad-spectrum antiviral therapies.<br><br>IMPORTANCE: Swine enteric coronaviruses (SeCoVs) cause severe economic losses to the global swine industry and pose a potential threat to public health. Identification of receptors required for PEDV infection could develop novel targets for drug therapy and disease-resistant breeding. We conducted a CRISPR/Cas9 screen targeting membrane proteins in porcine kidney cells infected with PEDV to identify possible receptors and discovered numerous novel candidate host factors. Considering RPSA's known role as a receptor for multiple viruses, we focused on investigating its potential in coronavirus infection. Our results revealed that RPSA does not contribute to the entry stage but to the replication stage of coronavirus infection. We first reported the role that RPSA plays in the regulation of APN expression and lipid metabolism. RPSA is essential for PEDV and other SeCoVs replication, providing a novel insight into the search for the receptor of PEDV and identifying potential therapeutic targets for coronaviruses.},
}
@article {pmid40707359,
year = {2025},
author = {Campbell, RR and Green, M and Choi, EY and Wulff, AB and Siclair, AN and Khatri, S and Virata, G and Barrett, C and Key, S and Patel, S and Rowell, MB and Franco, D and Ganapathy-Kanniappan, S and Mathur, BN and Lobo, MK},
title = {Dopamine Receptor 1 Specific CRISPRa Mice Exhibit Disrupted Behaviors and Striatal Baseline Cellular Activity.},
journal = {eNeuro},
volume = {12},
number = {8},
pages = {},
pmid = {40707359},
issn = {2373-2822},
support = {R01 DA038613/DA/NIDA NIH HHS/United States ; R33 DA052101/DA/NIDA NIH HHS/United States ; },
mesh = {Animals ; *Receptors, Dopamine D1/genetics/metabolism ; Mice, Transgenic ; *Corpus Striatum/metabolism ; Mice ; Male ; Reward ; *Behavior, Animal/physiology ; CRISPR-Cas Systems ; Mice, Inbred C57BL ; Motor Activity/physiology ; },
abstract = {The two main cell types in the striatum, dopamine receptor 1 and adenosine receptor 2a spiny projection neurons (D1-SPNs and A2A-SPNs), have distinct roles in regulating motor- and reward-related behaviors. Cre-selective CRISPR-dCas9 systems allow for cell-type specific, epigenomic-based manipulation of gene expression with gene-specific single guide RNAs (sgRNAs) and have potential to elucidate molecular mechanisms underlying striatal subtype mediated behaviors. Conditional transgenic Rosa26:LSL-dCas9-p300 mice were recently generated to allow for robust expression of dCas9-p300 expression with Cre-driven cell-type specificity. This system utilizes p300, a histone acetyltransferase which regulates gene expression by unwinding chromatin and making that region of the genome more accessible for transcription. Rosa26-LSL-dCas9-p300 mice were paired with Drd1-Cre and Ador2a-Cre mice to generate Drd1-Cre:dCas9-p300 and Ador2a-Cre:dCas9-p300 mouse lines and underwent behavioral phenotyping when sgRNAs were not present. Both Drd1-Cre:dCas9-p300 and Ador2a-Cre:dCas9-p300 have cell-type-specific expression of spCas9 mRNA. Baseline behavioral assessments revealed that, under a sgRNA absent nontargeted state, Drd1-Cre:dCas9-p300 mice display repetitive spinning behavior, hyperlocomotion, and enhanced acquisition of reward learning in comparison with all genotypic littermates. In contrast, Ador2a-Cre:dCas9-p300 do not exhibit any changes in behavior in comparison with their littermates. Electrophysiological recordings of dorsal striatum D1-SPNs revealed that Drd1-Cre:dCas9-p300 mice have increased input resistance and increased spontaneous excitatory postsynaptic current amplitude, together suggesting greater excitatory drive of D1-SPNs. Overall, these data demonstrate the necessity to validate CRISPR-dCas9 lines for research investigations. Additionally, the Drd1-Cre:dCas9-p300 line has the potential to be used to study underlying mechanisms of stereotypy and reward learning.},
}
@article {pmid40941236,
year = {2025},
author = {Liu, Y and Xie, Y and Wang, Z and Gai, Z and Zhang, X and Chen, J and Lei, H and Xu, Z and Shen, X},
title = {A Simple, Rapid, and Contamination-Free Ultra-Sensitive Cronobacter sakazakii Visual Diagnostic Platform Based on RPA Combined with CRISPR/Cas12a.},
journal = {Foods (Basel, Switzerland)},
volume = {14},
number = {17},
pages = {},
doi = {10.3390/foods14173120},
pmid = {40941236},
issn = {2304-8158},
support = {2023YFF1105103//National Key Research and Development Program of China/ ; },
abstract = {CRISPR/Cas systems have made significant progress in the field of molecular diagnostics in recent years. To overcome the aerosol contamination problem brought on by amplicon transfer in the common two-step procedure, the "one-pot method" has become a major research hotspot in this field. However, these methods usually rely on specially designed devices or additional chemical modifications. In this study, a novel "one-pot" strategy was developed to detect the foodborne pathogen Cronobacter sakazakii (C. sakazakii). A specific sequence was screened out from the virulence gene ompA of C. sakazakii as the detection target. Combining with the recombinase polymerase amplification (RPA), a rapid detection platform for C. sakazakii based on the CRISPR/Cas12a system was established for the first time. The sensitivity of this method was determined from three different levels, which are 10[-4] ng/μL for genomic DNA (gDNA), 1.43 copies/μL for target DNA, and 6 CFU/mL for pure bacterial culture. Without any microbial enrichment, the detection limits for artificially contaminated cow and goat milk powder samples were 4.65 CFU/mL and 4.35 CFU/mL, respectively. To address the problem brought on by aerosol contamination in the common RPA-CRISPR/Cas12a two-step method, a novel pipette tip-in-tube (PTIT) method for simple and sensitive one-pot nucleic acid detection was further developed under the inspiration of the capillary principle. The RPA and CRISPR/Cas systems were isolated from each other by the force balance of the solution in a pipette tip before amplification. The detection limits of the PTIT method in pure bacterial culture and the spiked samples were exactly the same as that of the two-step method, but with no false positive cases caused by aerosol contamination at all. Compared with other existing one-pot methods, the PTIT method requires no additional or specially designed devices, or any chemical modifications on crRNA and nucleic acid probes. Therefore, the PTIT method developed in this study provides a novel strategy for realizing one-pot CRISPR/Cas detection easily and holds significant potential for the rapid point-on-care testing (POCT) application.},
}
@article {pmid40940734,
year = {2025},
author = {Cattin, E and Schena, E and Mattioli, E and Marcuzzo, S and Bonanno, S and Cavalcante, P and Corradi, F and Benati, D and Farinazzo, G and Cattaneo, M and De Sanctis, V and Bertorelli, R and Maggi, L and Giannotta, M and Pini, A and Vattemi, G and Cassandrini, D and Cavallo, M and Manferdini, C and Lisignoli, G and Fontana, B and Pace, I and Bruno, C and Roncarati, R and Fiorillo, C and Ferracin, M and Schirmer, EC and Recchia, A and Lattanzi, G},
title = {Profibrotic Molecules Are Reduced in CRISPR-Edited Emery-Dreifuss Muscular Dystrophy Fibroblasts.},
journal = {Cells},
volume = {14},
number = {17},
pages = {},
doi = {10.3390/cells14171321},
pmid = {40940734},
issn = {2073-4409},
support = {20223WFJJ3//Ministero dell'universit&#xe0; e della ricerca/ ; CUP B33C22001640007//Associazione Italiana DIstrofia Muscolare di Emery-Dreifuss - AIDMED/ ; CUP B33C22001640007//Associazione Alessandra Proietti OdV/ ; ECOSISTER Project. cod. ECS_00000033-CUP B89I22000650001//EU-funded PNRR/ ; ECOSISTER project ECS_00000033 - CUP E93C22001100001//EU-funded PNRR/ ; TREAT-LMNA 2019-004426-24//AIFA/ ; T3-AN-03 CUP: B53C22002520006 REGINA//Ministero della Salute/ ; RRC//The Italian Ministry of Health/ ; },
abstract = {Emery-Dreifuss muscular dystrophy (EDMD) is caused by mutations in EMD, LMNA, SYNE1, SYNE2, and other related genes. The disease is characterized by joint contractures, muscle weakening and wasting, and heart conduction defects associated with dilated cardiomyopathy. Previous studies demonstrated the activation of fibrogenic molecules such as TGFbeta 2 and CTGF in preclinical models of EDMD2 and increased secretion of TGFbeta 2 in patient serum. A wide screening of patient cells suggested fibrosis, metabolism, and myogenic signaling as the most affected pathways in various EDMD forms. In this study, we show that alpha-smooth muscle actin-positive myofibroblasts are overrepresented in patient fibroblast cultures carrying EMD, LMNA, or SYNE2 mutations, and profibrotic miRNA-21 is upregulated. Upon CRISPR/Cas correction of the mutated EMD or LMNA sequence in EDMD1 or EDMD2 fibroblasts, respectively, we observe a reduced expression of fibrogenic molecules. However, in patient myoblasts, neither fibrogenic proteins nor miRNA-21 were upregulated; instead, miRNA-21-5p was downregulated along with muscle-specific miRNA-133b and miRNA-206, which have a crucial role in muscle cell homeostasis. These observations suggest that the conversion of laminopathic fibroblasts into a profibrotic phenotype is a determinant of EDMD-associated muscle fibrosis, while miRNA-206-dependent defects of laminopathic myoblasts, including altered regulation of VEGF levels, contribute to muscle cell deterioration. Notably, our study provides a proof-of-principle for the application of gene correction to EDMD1 and EDMD2 and presents EDMD1 isogenic cells that exhibit an almost complete rescue of a disease-specific miRNA signature. These cells can be used as experimental models for studying muscular laminopathies.},
}
@article {pmid40938972,
year = {2025},
author = {Abdirassilova, AA and Yessimseit, DT and Kassenova, AK and Abdeliyev, BZ and Zhumadilova, ZB and Tokmurziyeva, GZ and Kovaleva, GG and Abdel, ZZ and Meka-Mechenko, TV and Umarova, SK and Begimbayeva, EZ and Agzam, SD and Motin, VL and Reva, ON and Rysbekova, AK},
title = {Whole genome sequencing of Yersinia pestis isolates from Central Asian natural plague foci revealed the role of adaptation to different hosts and environmental conditions in shaping specific genotypes.},
journal = {PLoS neglected tropical diseases},
volume = {19},
number = {9},
pages = {e0013533},
doi = {10.1371/journal.pntd.0013533},
pmid = {40938972},
issn = {1935-2735},
abstract = {The genetic diversity and biovar classification of Yersinia isolates from Central Asia were investigated using whole-genome sequencing. In total, 98 isolates from natural plague foci were sequenced using the MiSeq platform. Computational pipelines were developed for accurate assembly of Y. pestis replicons, including small cryptic plasmids, and for identifying genetic polymorphisms. A panel of 99 diagnostic polymorphisms was established, enabling the distinction of dominant Medievalis isolates derived from desert and upland regions. Evidence of convergent evolution was observed in polymorphic allele distributions across genetically distinct Y. pestis biovars, Y. pseudotuberculosis, and other Y. pestis strains, likely driven by adaptation to similar environmental conditions. Genetic polymorphisms in the napA, araC, ssuA, and rhaS genes, along with transposon and CRISPR-Cas insertion patterns, were confirmed as suitable tools for identifying Y. pestis biovars, although their homoplasy suggests limited utility for phylogenetic inference. Notably, a novel cryptic plasmid, pCKF, previously associated with the strain of the population 2.MED0 from the Central-Caucasus high-altitude autonomous plague focus, was detected in a genetically distinct isolate of 2.MED1 population from the Ural-Embi region, indicating potential plasmid transfer across the 2.MED lineage. These findings emphasize the need for ongoing genomic surveillance to monitor the spread of virulence-associated genetic elements and to improve our understanding of Y. pestis evolution and ecology.},
}
@article {pmid40938680,
year = {2025},
author = {Yang, K and Wang, T and Zhu, Q and Shen, C},
title = {CRISPR/Cas-based detection strategies for tumor biomarker detection.},
journal = {Analytical methods : advancing methods and applications},
volume = {},
number = {},
pages = {},
doi = {10.1039/d5ay01150g},
pmid = {40938680},
issn = {1759-9679},
abstract = {Tumor biomarkers, such as nucleic acids, proteins, extracellular vesicles (EVs) and circulating tumor cells (CTCs), can provide valuable information for tumor risk assessment, diagnosis, prognosis and recurrence monitoring. Currently, polymerase chain reaction (PCR)-based approaches and enzyme linked immunosorbent assay (ELISA) are typically used for detecting tumor biomarkers in clinics. However, PCR-based methods have limits in sensitivity and detection channels. Besides, ELISA suffers from cumbersome operation and limited sensitivity. Clustered regularly interspaced short palindromic repeats/CRISPR-associated protein (CRISPR/Cas) systems are adopted for developing novel detection strategies due to the characteristics of high sensitivity, high specificity, simple operability and flexible programmability. Besides, CRISPR/Cas systems are amenable to combination with isothermal amplification techniques, primarily attributed to their compatibility and stability. Moreover, the combination of different CRISPR/Cas systems enables multiplex target detection. Therefore, CRISPR/Cas-based detection strategies have emerged as highly promising approaches for the sensitive, specific and multiplex detection of tumor biomarkers. In this review, we at first introduced the classification and working mechanisms of CRISPR/Cas systems. And then, we comprehensively summarized recently developed CRISPR/Cas-based detection strategies for tumor biomarkers. Besides, we reviewed detection strategies based on CRISPR/Cas systems for multiplex tumor biomarker detection. Furthermore, the challenges and prospects of existing CRISPR/Cas-based detection strategies were thoroughly discussed.},
}
@article {pmid40935887,
year = {2025},
author = {Alves, CRR and Das, S and Krishnan, V and Ha, LL and Fox, LR and Stutzman, HE and Shamber, CE and Kalailingam, P and McCarthy, S and Lino Cardenas, CL and Fong, CE and Imai, T and Mitra, S and Yun, S and Wood, RK and Benning, FMC and Roh, K and Lawton, J and Kim, N and Silverstein, RA and Ferreira da Silva, J and de la Cruz, D and Richa, R and Xie, J and Gray-Edwards, HL and Malhotra, R and Chung, DY and Chao, LH and Tsai, SQ and Maguire, CA and Lindsay, ME and Kleinstiver, BP and Musolino, PL},
title = {Treatment of a severe vascular disease using a bespoke CRISPR-Cas9 base editor in mice.},
journal = {Nature biomedical engineering},
volume = {},
number = {},
pages = {},
pmid = {40935887},
issn = {2157-846X},
support = {P01HL142494//U.S. Department of Health &amp; Human Services | National Institutes of Health (NIH)/ ; R01NS125353//U.S. Department of Health &amp; Human Services | National Institutes of Health (NIH)/ ; DP2CA281401//U.S. Department of Health &amp; Human Services | National Institutes of Health (NIH)/ ; K01NS134784//U.S. Department of Health &amp; Human Services | National Institutes of Health (NIH)/ ; R01NS125353//U.S. Department of Health &amp; Human Services | National Institutes of Health (NIH)/ ; R01NS125353//U.S. Department of Health &amp; Human Services | National Institutes of Health (NIH)/ ; },
abstract = {Pathogenic missense mutations in the alpha actin isotype 2 (ACTA2) gene cause multisystemic smooth muscle dysfunction syndrome (MSMDS), a genetic vasculopathy that is associated with stroke, aortic dissection and death in childhood. Here we perform mutation-specific protein engineering to develop a bespoke CRISPR-Cas9 enzyme with enhanced on-target activity against the most common MSMDS-causative mutation ACTA2 R179H. To directly correct the R179H mutation, we screened dozens of configurations of base editors to develop a highly precise corrective A-to-G edit with minimal deleterious bystander editing that is otherwise prevalent when using wild-type SpCas9 base editors. We create a murine model of MSMDS that shows phenotypes consistent with human patients, including vasculopathy and premature death, to explore the in vivo therapeutic potential of this strategy. Delivery of the customized base editor via an engineered smooth muscle-tropic adeno-associated virus (AAV-PR) vector substantially prolongs survival and rescues systemic phenotypes across the lifespan of MSMDS mice, including in the vasculature, aorta and brain. Our results highlight how bespoke mutant-specific CRISPR-Cas9 enzymes can improve mutation correction with base editors.},
}
@article {pmid40934884,
year = {2025},
author = {Gast, K and Barrangou, R},
title = {All systems go: CRISPR crosstalk for enhanced immunity.},
journal = {Cell host &amp; microbe},
volume = {33},
number = {9},
pages = {1470-1472},
doi = {10.1016/j.chom.2025.08.002},
pmid = {40934884},
issn = {1934-6069},
abstract = {In this issue of Cell Host &amp; Microbe, companion manuscripts from Margolis &amp; Meeske[1] and Smith &amp; Fineran[2] demonstrate that CRISPR-Cas systems have an unprecedented level of cooperative crosstalk between different subtypes, which enables primed spacer acquisition. These studies illustrate how CRISPR-Cas systems cooperate to enhance adaptive immunity in bacteria.},
}
@article {pmid40641152,
year = {2025},
author = {Robertson, NR and Lenert-Mondou, C and Leonard, AC and Tafrishi, A and Carrera, S and Lee, S and Aguilar, Y and Sanchez Zamora, L and Nguyen, T and Beltrán, J and Li, M and Cutler, SR and Whitehead, TA and Wheeldon, I},
title = {PYR1 Biosensor-Driven Genome-Wide CRISPR Screens for Improved Monoterpene Production in Kluyveromyces marxianus.},
journal = {ACS synthetic biology},
volume = {14},
number = {8},
pages = {2972-2978},
pmid = {40641152},
issn = {2161-5063},
mesh = {*Biosensing Techniques/methods ; *CRISPR-Cas Systems/genetics ; *Kluyveromyces/genetics/metabolism ; *Monoterpenes/metabolism ; Metabolic Engineering/methods ; *Fungal Proteins/genetics/metabolism ; Acyclic Monoterpenes/metabolism ; Genome, Fungal ; },
abstract = {Monoterpenes are valued for their roles as flavors, fragrances, insecticides, and energy-dense fuels. Microorganisms provide sustainable biosynthesis routes for these important molecules, but production levels remain limited. Here, we introduce a biosensor-driven microbial engineering strategy to enhance monoterpene production, specifically targeting geraniol. Using mutagenized libraries of the PYR1 receptor─a versatile biosensor from plant ABA signaling pathways with a malleable binding pocket─we screened 24 monoterpenes and identified PYR1 variants responsive to eight, including geraniol. A low background, highly selective geraniol-sensitive PYR1 variant was expressed in the thermotolerant yeast Kluyveromyces marxianus as a growth-based biosensor circuit, allowing for rapid strain engineering. By coupling the geraniol-sensitive PYR1 sensor with a genome-wide CRISPR-Cas9 mutagenesis approach, we identified six gene knockouts that enhance geraniol production, achieving up to a 2-fold increase in titer. This study demonstrates the power of the PYR1 biosensor platform to enable rapid strain engineering and the identification of mutants that improve the titer of a desired metabolite.},
}
@article {pmid40580745,
year = {2025},
author = {Asadi-Sarabi, P and Rismani, E and Shabanpouremam, M and Hendi, Z and Nikoubin, B and Rahimi, S and Taleb, M and Khosravi, A and Zarrabi, A and Hassan, M and Vosough, M},
title = {Hypoimmunogenic pluripotent stem cells: A game-changer in cell-based regenerative medicine.},
journal = {International immunopharmacology},
volume = {162},
number = {},
pages = {115134},
doi = {10.1016/j.intimp.2025.115134},
pmid = {40580745},
issn = {1878-1705},
mesh = {Humans ; *Regenerative Medicine/methods ; *Pluripotent Stem Cells/immunology/transplantation ; Animals ; Gene Editing ; *Cell- and Tissue-Based Therapy/methods ; CRISPR-Cas Systems ; },
abstract = {Hypoimmunogenic pluripotent stem cells (hPSCs) represent a transformative innovation in regenerative medicine, offering solutions to the longstanding challenge of immune rejection in cell-based therapies. Through advanced gene-editing techniques, particularly CRISPR/Cas9, hPSCs are engineered to downregulate or eliminate the expression of major histocompatibility complex (MHC) molecules while upregulating immunomodulatory proteins such as HLA-G, PD-L1, and CD47. These modifications enhance immune evasion and create the foundation for universal donor cells. Compared to conventional cell therapies that rely on lifelong immunosuppression, hPSC-based strategies offer safer, more sustainable, and patient-friendly solutions by minimizing the risks of infection, malignancy, and drug toxicity. Beyond immune compatibility, critical challenges persist, including the risk of tumorigenicity, off-target genetic alterations, and ethical considerations surrounding genome editing. Recent advances, such as the integration of suicide gene systems and sensitive monitoring assays, offer promising strategies to enhance the safety and functional stability of hPSC-derived therapies. This review comprehensively discusses the molecular engineering of hPSCs, their biomedical applications, safety strategies, ethical implications, and the evolving regulatory frameworks needed for clinical translation. By addressing both the scientific and societal dimensions, hPSCs have the potential to revolutionize personalized and off-the-shelf regenerative treatments, provided that rigorous safeguards are implemented.},
}
@article {pmid40931807,
year = {2025},
author = {Wang, Y and Phelps, A and Godbehere, A and Evans, B and Takizawa, C and Chinen, G and Singh, H and Fang, Z and Du, ZY},
title = {Revolutionizing Agriculture With CRISPR Technology: Applications, Challenges, and Future Perspectives.},
journal = {Biotechnology journal},
volume = {20},
number = {9},
pages = {e70113},
doi = {10.1002/biot.70113},
pmid = {40931807},
issn = {1860-7314},
support = {//MBBE Molecular Biotechnology Lab/ ; HAW05047-H//USDA National Institute of Food and Agriculture/ ; 6114549//USDA REEU-Technology/ ; 5605280//CTAHR Agricultural Research and Extension Stations (CARES)/ ; //Undergraduate Research Opportunities Program (UROP)/ ; 5605280//U.S. Department of Agriculture/ ; 6114549//U.S. Department of Agriculture/ ; HATCH project HAW05047-H//U.S. Department of Agriculture/ ; },
mesh = {Animals ; *Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; *Agriculture/methods/trends ; Crops, Agricultural/genetics ; Livestock/genetics ; Aquaculture ; },
abstract = {CRISPR technologies are rapidly transforming agriculture by enabling precise and programmable modifications across a wide range of organisms. This review provides an overview of CRISPR applications in crops, livestock, aquaculture, and microbial systems, highlighting key advances in sustainable agriculture. In crops, CRISPR has accelerated the improvement of traits such as drought tolerance, nutrient efficiency, and pathogen resistance. In livestock and aquaculture, CRISPR has enabled disease-resistant pigs and poultry, hornless cattle, and fast-growing, stress-tolerant fish. Engineered microbes are also being leveraged to enhance nitrogen fixation and reduce input reliance. We examine the evolution of CRISPR tools, such as base and prime editing, multiplex editing, and epigenome modulation, that expand precision and control beyond traditional gene knockouts. These innovations offer significant advantages over conventional breeding, yet challenges remain, including off-target effects, delivery efficiency, and regulatory variability across countries. The review also explores emerging directions such as novel Cas variants and AI-integrated breeding platforms for high-throughput trait discovery. Together, these developments demonstrate the transformative potential of CRISPR technology to reshape agriculture, not only by enhancing productivity and resilience but also by reducing environmental impacts. With responsible implementation, CRISPR-enabled innovations are well-positioned to support global food security and sustainability targets by 2050.},
}
@article {pmid40931602,
year = {2025},
author = {Pan, L and Wang, P},
title = {DNA nanotechnology-enabled bioanalysis of extracellular vesicles.},
journal = {Nanoscale horizons},
volume = {},
number = {},
pages = {},
doi = {10.1039/d5nh00557d},
pmid = {40931602},
issn = {2055-6764},
abstract = {Extracellular vesicles (EVs) have emerged as valuable sources for liquid biopsy in disease diagnostics, given their protein and nucleic acid cargoes (e.g., miRNA, mRNA, glycoRNA) can serve as critical biomarkers. DNA nanotechnology, leveraging its inherent programmability, high specificity, and powerful signal amplification capability, offers a transformative approach for the bioanalysis of EVs. This review summarizes recent advances in DNA nanotechnology-based analytical methodologies for detecting EV-associated proteins and nucleic acids. We detail the underlying principles, applications, and performance of key strategies, including aptamer-based recognition, enzyme-free catalytic amplification circuits (e.g., HCR, CHA), enzyme catalytic amplification techniques (e.g., RCA, CRISPR-Cas systems), and DNA nanostructures-assisted amplification. The integration of these DNA tools into multiplexed detection platforms is also discussed. Finally, current challenges and future perspectives concerning clinical translation of EV detection are presented.},
}
@article {pmid40114032,
year = {2025},
author = {Tang, K and Zhou, L and Tian, X and Fang, SY and Vandenbulcke, E and Du, A and Shen, J and Cao, H and Zhou, J and Chen, K and Kim, HR and Luo, Z and Xin, S and Lin, SH and Park, D and Yang, L and Zhang, Y and Suzuki, K and Majety, M and Ling, X and Lam, SZ and Chow, RD and Ren, P and Tao, B and Li, K and Codina, A and Dai, X and Shang, X and Bai, S and Nottoli, T and Levchenko, A and Booth, CJ and Liu, C and Fan, R and Dong, MB and Zhou, X and Chen, S},
title = {Cas12a-knock-in mice for multiplexed genome editing, disease modelling and immune-cell engineering.},
journal = {Nature biomedical engineering},
volume = {9},
number = {8},
pages = {1290-1308},
pmid = {40114032},
issn = {2157-846X},
support = {RF1DA048811//U.S. Department of Health &amp; Human Services | National Institutes of Health (NIH)/ ; K99CA282989//U.S. Department of Health &amp; Human Services | National Institutes of Health (NIH)/ ; F30CA250249//U.S. Department of Health &amp; Human Services | National Institutes of Health (NIH)/ ; RF1 DA048811/DA/NIDA NIH HHS/United States ; R01 CA231112/CA/NCI NIH HHS/United States ; DP2 CA238295/CA/NCI NIH HHS/United States ; T32 GM007205/GM/NIGMS NIH HHS/United States ; F30 CA250249/CA/NCI NIH HHS/United States ; U54CA209992//U.S. Department of Health &amp; Human Services | National Institutes of Health (NIH)/ ; DP2CA238295//U.S. Department of Health &amp; Human Services | National Institutes of Health (NIH)/ ; K99 CA282989/CA/NCI NIH HHS/United States ; U54 CA209992/CA/NCI NIH HHS/United States ; R33 CA281702/CA/NCI NIH HHS/United States ; T32GM007205//U.S. Department of Health &amp; Human Services | National Institutes of Health (NIH)/ ; R33CA281702//U.S. Department of Health &amp; Human Services | National Institutes of Health (NIH)/ ; R33 CA225498/CA/NCI NIH HHS/United States ; R01CA231112//U.S. Department of Health &amp; Human Services | National Institutes of Health (NIH)/ ; },
mesh = {Animals ; *Gene Editing/methods ; Mice ; Mice, Inbred C57BL ; *Gene Knock-In Techniques/methods ; *CRISPR-Associated Proteins/genetics/metabolism ; Disease Models, Animal ; CRISPR-Cas Systems/genetics ; *Cell Engineering/methods ; Dependovirus/genetics ; Humans ; Dendritic Cells ; Bacterial Proteins ; Endodeoxyribonucleases ; },
abstract = {The pleiotropic effects of human disease and the complex nature of gene-interaction networks require knock-in mice allowing for multiplexed gene perturbations. Here we describe a series of knock-in mice with a C57BL/6 background and with the conditional or constitutive expression of LbCas12a or of high-fidelity enhanced AsCas12a, which were inserted at the Rosa26 locus. The constitutive expression of Cas12a in the mice did not lead to discernible pathology and enabled efficient multiplexed genome engineering. We used the mice for the retrovirus-based immune-cell engineering of CD4[+] and CD8[+] T cells, B cells and bone-marrow-derived dendritic cells, for autochthonous cancer modelling through the delivery of multiple CRISPR RNAs as a single array using adeno-associated viruses, and for the targeted genome editing of liver tissue using lipid nanoparticles. We also describe a system for simultaneous dual-gene activation and knockout (DAKO). The Cas12a-knock-in mice and the viral and non-viral delivery vehicles provide a versatile toolkit for ex vivo and in vivo applications in genome editing, disease modelling and immune-cell engineering, and for the deconvolution of complex gene interactions.},
}
@article {pmid40930534,
year = {2025},
author = {Lejars, M and Maeda, T and Guillier, M},
title = {EASY-edit: a toolbox for high-throughput single-step custom genetic editing in bacteria.},
journal = {Nucleic acids research},
volume = {53},
number = {17},
pages = {},
doi = {10.1093/nar/gkaf883},
pmid = {40930534},
issn = {1362-4962},
support = {/ERC_/European Research Council/International ; 818750//European Union's Horizon 2020 research and innovation/ ; //CNRS/ ; ANR-11-LABX-0011//Initiative d'Excellence/ ; },
mesh = {*Gene Editing/methods ; *Escherichia coli/genetics ; *CRISPR-Cas Systems ; Operon ; RNA, Guide, CRISPR-Cas Systems/genetics ; Genes, Reporter ; },
abstract = {Targeted gene editing can be achieved using CRISPR-Cas9-assisted recombineering. However, high-efficiency editing requires careful optimization for each locus to be modified, which can be tedious and time-consuming. In this work, we developed a simple, fast and cheap method: Engineered Assembly of SYnthetic operons for targeted editing (EASY-edit) in Escherichia coli. Highly efficient editing of the different constitutive elements of the operons can be achieved by using a set of optimized guide RNAs and single- or double-stranded DNA repair templates carrying relatively short homology arms. This facilitates the construction of multiple genetic tools, including mutant libraries or reporter genes. EASY-edit is also highly modular, as we provide alternative and complementary versions of the operon inserted in three loci which can be edited iteratively and easily combined. As a proof of concept, we report the construction of several fusions with reporter genes confirming known post-transcriptional regulation mechanisms and the construction of saturated and unbiased mutant libraries. In summary, the EASY-edit system provides a flexible genomic expression platform that can be used both for the understanding of biological processes and as a tool for bioengineering applications.},
}
@article {pmid40930528,
year = {2025},
author = {Jiang, Q and Jin, S and Qin, Z and Zhang, J and He, R and Chen, Z and Qiao, B and Qiao, J and Liu, Y},
title = {CRISPR/Cas12a DTR system: a topology-guided Cas12a assay for specific dual detection of RNA and DNA targets.},
journal = {Nucleic acids research},
volume = {53},
number = {17},
pages = {},
doi = {10.1093/nar/gkaf893},
pmid = {40930528},
issn = {1362-4962},
support = {2022YFC2304304//National Key Research and Development Program of China/ ; 2023DJC136//Science and Technology Innovation Talent Plan of Hubei Province/ ; 2025AFB825//Natural Science Foundation of Hubei Province/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; Humans ; *MicroRNAs/genetics/analysis ; *DNA/genetics/analysis ; *CRISPR-Associated Proteins/genetics/metabolism ; *Endodeoxyribonucleases/genetics/metabolism ; *RNA/genetics/analysis ; *Bacterial Proteins/genetics/metabolism ; RNA, Guide, CRISPR-Cas Systems/genetics ; },
abstract = {The CRISPR/Cas12a technology has revolutionized molecular diagnostics. However, existing Cas12a systems depend on continuous target DNA activation, which limits them to single-target detection. In this study, we developed a novel topology-guided Cas12a system, the double-target responsive (DTR) system, capable of being activated by noncontiguous dual RNA/DNA targets. The DTR system employs two split CRISPR RNA (crRNA) fragments and two Cas12a proteins that cooperatively reconstitute upon recognizing two nucleic acid activators. We demonstrated the DTR system's ability to specifically detect dual nucleic acid substrates in a single readout, achieving a detection limit of 78 fM for RNA and exceptional specificity for single-nucleotide variations. Additionally, we successfully applied the DTR system to clinical samples, enabling simultaneous detection of two oral squamous cell carcinoma-related microRNAs (miR-155 and miR-let-7a), thereby distinguishing healthy individuals from patients. This work establishes an efficient Cas12a-based platform for sensitive, simultaneous, and discriminative detection of RNA and DNA targets, enhancing the versatility of Cas12a in analytical detection and clinical diagnosis.},
}
@article {pmid40929845,
year = {2025},
author = {Chang, Y and Ding, J},
title = {CircRNA knockout/knockdown tools in molecular biology research.},
journal = {Biochemical and biophysical research communications},
volume = {783},
number = {},
pages = {152607},
doi = {10.1016/j.bbrc.2025.152607},
pmid = {40929845},
issn = {1090-2104},
abstract = {Circular RNAs (circRNAs), characterized by their covalently closed circular architecture, represent a unique class of endogenous RNA molecules that serve as pivotal regulators in post-transcriptional gene regulation in organisms. Accumulating evidence has established their potential as promising diagnostic biomarkers across various human pathologies, including but not limited to malignant neoplasms, neurodegenerative disorders, and metabolic dysregulation.By inhibiting circRNA expression, we can better understand their functions and their impact on related biological processes. Over the past decade, remarkable advancements have emerged in circRNAs manipulation technologies, including siRNA, DNAzyme, and CRISPR-Cas systems emerging as powerful tools for precise circRNAs editing in both experimental models and preclinical studies. In this review, we summarize the advantages and identification of these editing methods and discuss future challenges and prospects.},
}
@article {pmid40927181,
year = {2025},
author = {Ziemann, M and Mitrofanov, A and Stöckl, R and Alkhnbashi, OS and Backofen, R and Hess, WR},
title = {Analysis of tracrRNAs reveals subgroup V2 of type V-K CAST systems.},
journal = {microLife},
volume = {6},
number = {},
pages = {uqaf020},
pmid = {40927181},
issn = {2633-6693},
abstract = {Clustered regularly interspaced palindromic repeats (CRISPR)-associated transposons (CAST) consist of an integration between certain class 1 or class 2 CRISPR-Cas systems and Tn7-like transposons. Class 2 type V-K CAST systems are restricted to cyanobacteria. Here, we identified a unique subgroup of type V-K systems through phylogenetic analysis, classified as V-K_V2. Subgroup V-K_V2 CAST systems are characterized by an alternative tracrRNA, the exclusive use of Arc_2-type transcriptional regulators, and distinct differences in the length of protein domains in TnsB and TnsC. Although the occurrence of V-K_V2 CAST systems is restricted to Nostocales cyanobacteria, it shows signs of horizontal gene transfer, indicating its capability for genetic mobility. The predicted V-K_V2 tracrRNA secondary structure has been integrated into an updated version of the CRISPRtracrRNA program available on GitHub under https://github.com/BackofenLab/CRISPRtracrRNA/releases/tag/2.0.},
}
@article {pmid40678944,
year = {2025},
author = {Zhu, C and Huang, Q and Fu, R and Xun, Z and Ou, Q and Xianyu, Y and Liu, C},
title = {A Triple-Modal Biosensing Strategy for Hepatitis B Virus Based on Mg[2+]-Mediated Modulation of CRISPR/Cas12a and Au@Pt Nanoparticles.},
journal = {Small (Weinheim an der Bergstrasse, Germany)},
volume = {21},
number = {36},
pages = {e05341},
doi = {10.1002/smll.202505341},
pmid = {40678944},
issn = {1613-6829},
support = {82030063 82372317 82372316//National Natural Science Foundation of China/ ; 2023Y4004//University-Industry Research Cooperation Project of Science and Technology of Fujian Province/ ; 2025C02124//"Pioneer" and "Leading Goose" R&amp;D Program of Zhejiang/ ; 2023QH1102//Startup Fund for Scientific Research of Fujian Medical University/ ; 2024QH2043//Startup Fund for Scientific Research of Fujian Medical University/ ; },
mesh = {*Hepatitis B virus/genetics/isolation &amp; purification ; *Biosensing Techniques/methods ; *Metal Nanoparticles/chemistry/ultrastructure ; *Gold/chemistry ; *CRISPR-Cas Systems/genetics ; *Magnesium/chemistry ; DNA, Viral ; *Platinum/chemistry ; Nucleic Acid Amplification Techniques ; Humans ; },
abstract = {Hepatitis B virus (HBV) infection remains a significant global public health issue, and rapid detection of HBV DNA is crucial for disease prevention and control. However, traditional methods for HBV DNA detection are limited by their reliance on precise instruments and single readout, which can hardly meet the requirements of on-site detection. In this study, the Mg[2+]-enhanced trans-cleavage activity of clustered regularly interspaced short palindromic repeats/associated protein 12a (CRISPR/Cas12a) is reported and coupled with loop-mediated isothermal amplification (LAMP) and Au@Pt nanoparticles as a signaling reporter for on-site detection of HBV DNA. This triple-modal biosensing strategy enables multiple signal readouts including UV-vis spectrum, RGB value, and temperature with high sensitivity and accuracy. The linear detection ranges using UV-vis spectroscopy, RGB color recognition, and photothermal modes are from 50 to 10,000 copies µL[-1], with the limitation of detection of 24.07, 39.65, and 23.33 copies µL[-1], respectively. This biosensing strategy is further employed for the qualitative detection of HBV DNA in 48 serum samples, achieving sensitivities of 100%, 100%, and 95.24% for triple modes. This work offers a promising tool of next-generation LAMP-CRISPR/Cas12a for the rapid and portable detection of nucleic acids.},
}
@article {pmid40925176,
year = {2025},
author = {He, Z and Zhang, J and Kuang, S and Li, S and Wang, Y and Ding, J and Ma, Z and Zhang, B},
title = {Colloidal gold technology in viral diagnostics: Recent innovations, clinical applications, and future perspectives.},
journal = {Virology},
volume = {612},
number = {},
pages = {110686},
doi = {10.1016/j.virol.2025.110686},
pmid = {40925176},
issn = {1096-0341},
abstract = {Colloidal gold technology has revolutionized viral diagnostics through its rapid, cost-effective, and user-friendly applications, particularly in point-of-care testing (POCT). This review synthesizes recent advancements, focusing on its role in detecting respiratory viruses, hepatitis viruses, and emerging pathogens. The technology leverages the unique optical and physicochemical properties of gold nanoparticles (AuNPs), including localized surface plasmon resonance (LSPR) and high surface-to-volume ratios, to achieve rapid antigen-antibody recognition with visual readouts within 15 min. Innovations such as CRISPR-Cas-integrated lateral flow immunoassays (LFIAs), dual-mode plasmonic biosensors, and nanomaterials like CeO2-colloidal gold composites have enhanced sensitivity and multiplex capability, enabling simultaneous identification of co-circulating pathogens. Case studies highlight its efficacy in dengue serotyping, SARS-CoV-2 neutralizing antibody quantification, and HBV/HCV co-detection, demonstrating high clinical specificity. However, challenges persist, including the need for improved sensitivity; interference of sample matrix with immunity; false positives caused by cross-reactions; and limitations of semi-quantitative analysis. Recent progress in hybrid nanomaterial synthesis, surface functionalization, and device-level multiplexing-coupled with AI-driven data interpretation- promises to address these gaps. Future trends emphasize integration with surface-enhanced Raman scattering (SERS), microfluidics, and portable sensors to achieve sub-zeptomolar sensitivity and scalable deployment. By bridging nanotechnology with precision diagnostics, colloidal gold platforms are poised to redefine global viral surveillance, particularly in resource-limited settings, underscoring their indispensable role in pandemic preparedness.},
}
@article {pmid40922689,
year = {2025},
author = {Zhang, Q and Ren, J and Wu, S and Tan, Y and Wang, W and Feng, C and Zhao, L and Zhu, Z},
title = {Plasmid-Free CRISPR/Cpf1 Genome Editing With In Vivo T7 RNA Polymerase-Transcribed CRISPR RNA From Short Double-Stranded DNA.},
journal = {Biotechnology and bioengineering},
volume = {},
number = {},
pages = {},
doi = {10.1002/bit.70062},
pmid = {40922689},
issn = {1097-0290},
support = {//This study was funded by the National Key Research and Development Program (Grant Number: 2022YFA0911802); the National Natural Science Foundation of China (Grant Number: 22177018); the Liaoning Revitalization Talents Program (Grant Number: XLYC2203057) and the Fundamental Research Funds for the Central Universities (Grant Number: DUT23LAB104)./ ; },
abstract = {Plasmids are commonly employed in the delivery of clustered regularly interspaced shortpalindromic repeats (CRISPR)/CRISPR-associated (Cas) components for genome editing. However, the absence of heritable plasmids in numerous organisms limits the development of CRISPR/Cas genome editing tools. Moreover, cumbersome procedures for plasmid construction and curing render genome editing time-consuming. In this study, we developed a plasmid-free CRISPR/Cpf1 genome editing system for Saccharomyces cerevisiae and Starmerella bombicola. This system leveraged integrative expression of the Cpf1 nuclease and T7 RNA polymerase (T7RNAP), as well as the delivery of linear fragments including (i) a marker cassette for integration and selection, (ii) short double-stranded DNA (crDNA) for in vivo transcription of crRNA by T7RNAP, and (iii) donor DNA for homology-directed repair. We demonstrated that this editing system enabled efficient multiplexed and iterative genome editing without the need for marker recycling and plasmid curing. The use of short crDNA (87 bp) and donor DNA (≤ 120 bp), both readily prepared from ordered oligonucleotides via annealing or overlap extension, dramatically simplified the editing process. Successful implementation in S. bombicola, which lacks heritable plasmids for genetic engineering, highlighted the potential of this approach especially for genome engineering of genetically intractable organisms in a plasmid-free way.},
}
@article {pmid40920944,
year = {2025},
author = {Mangeot, PE and Ohlmann, T},
title = {[Harnessing retroviral engineering for genome reprogramming].},
journal = {Medecine sciences : M/S},
volume = {41},
number = {8-9},
pages = {647-656},
doi = {10.1051/medsci/2025098},
pmid = {40920944},
issn = {1958-5381},
support = {ANRS0516//ANRS/ ; },
mesh = {Humans ; *Retroviridae/genetics/physiology ; *Genetic Engineering/methods ; Genetic Vectors/genetics ; Animals ; CRISPR-Cas Systems ; Gene Editing/methods ; Genome, Human ; *Cellular Reprogramming/genetics ; },
abstract = {The accumulated knowledge on the biology of the HIV-1 virus has led to the emergence of technologies that exploit the architecture of retroviruses and their integration or vectorization properties. This field of study constitutes retroviral vectorology, democratized in laboratories by the use of lentiviral vectors. By hijacking retroviral assembly, other systems are emerging and are increasingly mentioned in recent literature. In particular, defective retroviral particles are capable of transiently delivering effectors that act on the genome: they thus appear to be more suitable tools for delivering genetic scalpels, whose persistence in the target cell or organism is not required. Since the description of the CRISPR Cas9 system in 2012, genome engineering techniques have continued to evolve in terms of capacity and reliability. Several derivatives of the CRISPR system can now modify the human genome with nucleotide-level precision. Introducing these effectors into the cell or organism remains a major technical challenge that vector scientists are striving to overcome. This review describes the major retroviral systems used for genome manipulation. Following an overview of genetic engineering techniques, we will see how researchers have developed a wide range of genomic tools by manipulating different processes in the retroviral architecture.},
}
@article {pmid40920796,
year = {2025},
author = {Nakagata, N and Nakao, S and Mikoda, N and Yamaga, K and Nakagawa, Y and Sakuma, T and Yamamoto, T and Takeo, T},
title = {Improved protocol for the vitrification and warming of rat zygotes by optimizing the warming solution and oocyte donor age.},
journal = {PloS one},
volume = {20},
number = {9},
pages = {e0328718},
pmid = {40920796},
issn = {1932-6203},
mesh = {Animals ; Female ; *Zygote/physiology/drug effects/cytology ; *Vitrification ; Rats ; *Oocytes/cytology ; *Cryopreservation/methods ; Rats, Sprague-Dawley ; Embryo Transfer ; Fertilization in Vitro/methods ; Rats, Inbred F344 ; Male ; CRISPR-Cas Systems ; Sucrose/pharmacology ; },
abstract = {Zygotes are used to create genetically modified animals by electroporation using the CRISPR-Cas9 system. Such zygotes in rats are obtained from superovulated female rats after mating. Recently, we reported that in vivo-fertilized zygotes had higher cryotolerance and developmental ability than in vitro-fertilized zygotes in Sprague Dawley (SD) and Fischer 344 rats. To apply the in vitro-fertilized zygotes in creating genetically modified rats, we need to address their low cryotolerance and developmental ability. Hence, we evaluated the effects of warming solutions containing different sucrose concentrations (0-0.3 M) and the oocyte donor's age (3-7-week-old SD rats) on the viability of vitrified-warmed zygotes after in vitro fertilization and on developmental ability by embryo transfer in SD rats. A warming solution containing 0.1 M sucrose enhanced the survival rate of vitrified-warmed zygotes and their rate of development to two-cell embryos. Additionally, zygotes derived from 6- and 7-week-old female rats had higher cryotolerance and developmental ability than those from 3-week-old ones. Next, vitrified-warmed rat zygotes produced using the optimized protocol underwent genome editing by electroporation with Cas9 ribonucleoprotein and gRNA introduced to disrupt the Tyr gene. We then found that 86.5% of the pups derived from zygotes demonstrated mutation of the targeted gene. Therefore, the improved protocol for vitrifying and warming rat zygotes is useful for preserving and producing genetically modified rats.},
}
@article {pmid40891143,
year = {2025},
author = {Zheng, X and Zhai, Y and Chathurika, HAW and Ni, X and Lv, R and Wu, C and Sun, Z and Shen, Y and Zhang, CY and Zheng, P and Sun, J},
title = {A Highly Efficient 5S rRNA-CRISPR/Cas9 Genome Editing Toolkit in Acremonium chrysogenum.},
journal = {Journal of agricultural and food chemistry},
volume = {73},
number = {36},
pages = {22607-22616},
doi = {10.1021/acs.jafc.5c06429},
pmid = {40891143},
issn = {1520-5118},
mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems ; *Acremonium/genetics/metabolism ; *RNA, Ribosomal, 5S/genetics/metabolism ; Cephalosporins/biosynthesis ; Fungal Proteins/genetics/metabolism ; },
abstract = {Acremonium chrysogenum is an important industrial producer of cephalosporin C (CPC), and efficient genome editing tools are critical for its exploitation and metabolic engineering. Currently, CRISPR/Cas9 systems for A. chrysogenum employ heterologous promoters, including Aspergillus nidulans PgpdA or Aspergillus fumigatus AfU6p, to drive sgRNA expression. These systems often required additional sgRNA processing elements such as ribozymes or tRNAs, which increased cloning complexity and experimental workload. Here, we developed a simplified and highly efficient CRISPR/Cas9 genome editing system using the endogenous 5S rRNA promoter for sgRNA transcription in A. chrysogenum. This system obviated the need for processing elements and achieved up to 100% gene disruption efficiency, as demonstrated by targeting the sorB gene. Furthermore, this platform enabled 100% single gene deletion and efficient large-scale chromosomal deletion, up to 66.17 kb within the sorbicillinoid biosynthetic gene cluster, without donor DNA. To our knowledge, this represents the largest chromosomal deletion reported in A. chrysogenum to date. Moreover, the system also facilitated precise and iterative gene editing through homologous recombination-mediated marker replacement at the kusA locus. Overall, this 5S rRNA-CRISPR/Cas9 system provides a versatile, powerful, and efficient genome editing toolkit for functional genomics and strain improvement in A. chrysogenum.},
}
@article {pmid40877754,
year = {2025},
author = {Singuru, MMR and Bhattacharyya, P and Sriramakrishnan, HP and You, M},
title = {Sensitive Detection of Intercellular Tensile Forces via Cas12a-Assisted Membrane Molecular Probes.},
journal = {Nano letters},
volume = {25},
number = {36},
pages = {13519-13525},
doi = {10.1021/acs.nanolett.5c02983},
pmid = {40877754},
issn = {1530-6992},
mesh = {Cadherins/metabolism ; Humans ; *Cell Membrane/chemistry/metabolism ; CRISPR-Cas Systems ; *CRISPR-Associated Proteins/metabolism/chemistry/genetics ; Tensile Strength ; Mechanotransduction, Cellular ; Epithelial-Mesenchymal Transition ; *Endodeoxyribonucleases/metabolism/chemistry/genetics ; *Molecular Probes/chemistry ; *Bacterial Proteins/metabolism/chemistry ; },
abstract = {Intercellular forces are critical for shaping cells, driving migration, and guiding tissue development and morphogenesis. However, these transient and low-intensity forces are still challenging to detect. Here, we developed a Force-Responsive Cas12a-assisted Tension Sensor (FRCTS), which leverages the clustered regularly interspaced short palindromic repeat (CRISPR)-Cas12a technology to enable more reliable detection of cumulative molecular force events generated at cell-cell junctions. FRCTS incorporates a lipid-modified DNA hairpin to spontaneously anchor onto live-cell membranes. The hairpin unfolds upon molecular tension exerted by neighboring cells through an integrin or cadherin receptor and reveals a hidden strand to activate Cas12a. Cas12a activation leads to an irreversible cleavage of a fluorogenic reporter on the cell surface, causing cumulative cell membrane fluorescence signals for recording intercellular force events. After systematic optimization, we applied FRCTS to quantify E-cadherin/N-cadherin mechanical correlations during the epithelial-mesenchymal transition. This modular and sensitive FRCTS platform can potentially be used for assessing various intercellular mechanotransduction processes.},
}
@article {pmid40816522,
year = {2025},
author = {Meng, XQ and Xu, XL and Gao, Y and Deng, SL},
title = {Establishment of CRISPR/Cas9 lineage tracking technology for pig embryos.},
journal = {Molecular and cellular probes},
volume = {83},
number = {},
pages = {102046},
doi = {10.1016/j.mcp.2025.102046},
pmid = {40816522},
issn = {1096-1194},
mesh = {Animals ; *CRISPR-Cas Systems/genetics ; Swine/genetics/embryology ; *Cell Lineage/genetics ; *Embryo, Mammalian/cytology/metabolism ; Single-Cell Analysis/methods ; },
abstract = {Understanding tissue development in pigs is critical for biomedical research and genetic engineering, particularly for modeling human disease. However, tracing developmental origins and reconstructing lineage trees for pig cells remains a significant challenge. Here, we present a high-resolution lineage tracing system that combines molecular barcoding with single-cell transcriptomics in pigs. Our system combines two key components: DNA barcodes (three CRISPR/Cas9 target sites and an 8-base pair intBC) integrated into the genome via piggyBac transposition, and a constitutive Cas9-EGFP cassette stably integrated at the Rosa26 locus using CRISPR/Cas12a. By combining lineage barcodes with single-cell RNA sequencing (scRNA-seq), we constructed an evolutionary lineage recorder that captures distinct cell states across developmental or differentiation trajectories. This system provides an essential tool for the subsequent construction of complete porcine cell fate maps. Our work provides a tool for studying porcine developmental biology, but also helps to optimize regenerative medicine strategies and improve the design of genetically engineered animal models.},
}
@article {pmid40815167,
year = {2025},
author = {Sunshine, S and Puschnik, A and Retallack, H and Laurie, MT and Liu, J and Peng, D and Knopp, K and Zinter, MS and Ye, CJ and DeRisi, JL},
title = {Defining the host dependencies and the transcriptional landscape of RSV infection.},
journal = {mBio},
volume = {16},
number = {9},
pages = {e0101025},
doi = {10.1128/mbio.01010-25},
pmid = {40815167},
issn = {2150-7511},
support = {//Chan Zuckerberg Biohub San Francisco/ ; F31AI150007//National Institute of Allergy and Infectious Diseases/ ; },
mesh = {Humans ; *Respiratory Syncytial Virus Infections/virology/genetics/immunology ; *Respiratory Syncytial Virus, Human/genetics/physiology ; *Host-Pathogen Interactions/genetics ; Single-Cell Analysis ; CRISPR-Cas Systems ; Gene Expression Profiling ; },
abstract = {Respiratory syncytial virus (RSV) is a globally prevalent pathogen, causes severe disease in older adults, and is the leading cause of bronchiolitis and pneumonia in the United States for children during their first year of life. Despite its prevalence worldwide, RSV-specific treatments remain unavailable for most infected patients. Here, we leveraged a combination of genome-wide CRISPR knockout screening and single-cell RNA sequencing to improve our understanding of the host determinants of RSV infection and the host response in both infected cells and uninfected bystanders. These data reveal temporal transcriptional patterns that are markedly different between RSV-infected and bystander-activated cells. Our data show that expression of interferon-stimulated genes is primarily observed in bystander activated cells, while genes implicated in the unfolded protein response and cellular stress are upregulated specifically in RSV-infected cells. Furthermore, genome-wide CRISPR screens identified multiple host factors important for viral infection, findings which we contextualize relative to 29 previously published screens across 17 additional viruses. These unique data complement and extend prior studies that investigate the proinflammatory response to RSV infection, and juxtaposed to other viral infections, provide a rich resource for further hypothesis testing.IMPORTANCERespiratory syncytial virus (RSV) is a leading cause of lower respiratory tract infection in infants and the elderly. Despite its substantial global health burden, RSV-targeted treatments remain unavailable for the majority of individuals. While vaccine development is underway, a detailed understanding of the host response to RSV infection and identification of required human host factors for RSV may provide insight into combatting this pathogen. Here, we utilized single-cell RNA sequencing and functional genomics to understand the host response in both RSV-infected and bystander cells, identify what host factors mediate infection, and contextualize these findings relative to dozens of previously reported screens across 17 additional viruses.},
}
@article {pmid40767522,
year = {2025},
author = {Guo, G and Zhang, M and Xu, Z and Xi, P and Zhu, H and Evers, A and Lebbink, RJ and Lang, Y and He, Q and Huang, Y-W and Li, T and Bosch, BJ and Li, W},
title = {Genome-wide CRISPR screen reveals key role of sialic acids in PEDV and porcine coronavirus infections.},
journal = {mBio},
volume = {16},
number = {9},
pages = {e0162825},
doi = {10.1128/mbio.01628-25},
pmid = {40767522},
issn = {2150-7511},
mesh = {*Porcine epidemic diarrhea virus/physiology/pathogenicity/genetics ; Animals ; *Coronavirus Infections/virology/veterinary/metabolism ; Swine ; Humans ; *CRISPR-Cas Systems ; *Sialic Acids/metabolism ; *Swine Diseases/virology/metabolism ; Virus Internalization ; Sialyltransferases/genetics/metabolism ; *N-Acetylneuraminic Acid/metabolism ; Clustered Regularly Interspaced Short Palindromic Repeats ; Host-Pathogen Interactions ; Cell Line ; },
abstract = {Porcine epidemic diarrhea virus (PEDV) is a globally distributed alphacoronavirus with economic importance that can cause severe watery diarrhea and even death in piglets. To identify host factors essential for PEDV infection, we performed a genome-wide CRISPR/Cas9 screen in human hepatocellular carcinoma cells (Huh7) using the highly virulent PEDV GIIb strain GDU. Several genes involved in the sialic acid and heparan sulfate biosynthesis pathway and cholesterol metabolism were highly enriched following PEDV selection. We validated that the host factor ST3 beta-galactoside alpha-2,3-sialyltransferase 4 (ST3GAL4), which catalyzes the transfer of sialic acid to sugar chains via α2,3-linked linkages, is important for PEDV infection. To systematically investigate the role of sialic acid in PEDV infection, we knocked out genes related to sialic acid synthesis. This led to a reduced abundance of sialic acid on the cell surface, which in turn inhibited PEDV adsorption and internalization. Furthermore, we found that both α2,3-linked and α2,6-linked sialic acids can serve as cellular attachment factors for PEDV. We conducted a glycan microarray screen to determine which sialoglycans are preferred by the PEDV spike protein. The results revealed that PEDV favors binding to α2,3-sialoglycans. Additionally, we found that not only current circulating PEDV strains but also other porcine coronaviruses rely on sialic acid for efficient infection. Collectively, our findings provide insights into critical host factors involved in PEDV infection and demonstrate that disrupting genes involved in sialic acid biosynthesis negatively affects the infectivity of multiple porcine enteric coronaviruses.IMPORTANCEA wide range of viruses utilize sialic acid as receptors. Sialic acid binding may serve as a key determinant of viral host range. Different viruses exhibit distinct preferences for specific types of sialic acid linkages. However, it remains unclear which specific subtypes of sialic acid are utilized during PEDV infection. In this study, we performed CRISPR-based genome-wide knockout screening and identified ST3GAL4 as a key host factor for PEDV infection. Furthermore, we found that both α2,3-linked and α2,6-linked sialic acids can function as attachment factors for PEDV infection. A glycan microarray screen revealed that PEDV S1 shows the strongest binding preference for α2,3-linked and α2,8-linked sialosides. Sialic acids were also implicated in infections by other porcine enteric coronaviruses. Overall, our findings advance our understanding of viral entry mechanisms of PEDV and other swine coronaviruses and may provide avenues for designing antiviral strategies.},
}
@article {pmid40744238,
year = {2025},
author = {Wen, Z and Yang, D and Yang, Y and Hu, J and Parviainen, A and Chen, X and Li, Q and VanDeusen, E and Ma, J and Tay, F},
title = {The path to biotechnological singularity: Current breakthroughs and outlook.},
journal = {Biotechnology advances},
volume = {84},
number = {},
pages = {108667},
doi = {10.1016/j.biotechadv.2025.108667},
pmid = {40744238},
issn = {1873-1899},
mesh = {Humans ; *Biotechnology/trends ; Gene Editing ; Artificial Intelligence ; Synthetic Biology ; Regenerative Medicine ; CRISPR-Cas Systems ; Brain-Computer Interfaces ; },
abstract = {Fueled by rapid advances in gene editing, synthetic biology, artificial intelligence, regenerative medicine, and brain-computer interfaces, biotechnology is approaching a transformative era often referred to as biotechnological singularity. CRISPR-based gene editing has revolutionized genetic engineering, enabling precise modifications for treating hereditary diseases and cancer. Synthetic biology facilitates sustainable biomaterial production and innovative therapeutic applications. Artificial intelligence accelerates drug discovery, enhances diagnostic accuracy, and personalizes treatment through deep learning models. Driven by stem cell research, regenerative medicine offers promising avenues for reversing aging and treating degenerative diseases. Brain-computer interfaces merge human cognition with technology, enabling direct neural control of prosthetics and expanding human-machine interactions. These breakthroughs, however, raise ethical, regulatory, and societal concerns, including equitable access, biosecurity risks, and the implications of human enhancement. The convergence of biological and computational technologies challenges traditional boundaries, necessitating comprehensive governance frameworks. By embracing responsible innovation, society can harness these advancements for transformative health interventions, environmental sustainability, and extended longevity. The realization of biotechnological singularity depends on interdisciplinary collaboration among scientists, policymakers, and the public to ensure that progress aligns with the well-being of humanity and ethical considerations.},
}
@article {pmid40552689,
year = {2025},
author = {Yao, B and Yang, Q and Snijders Blok, CJB and Daniels, MA and Doevendans, PA and Schiffelers, R and Sluijter, JPG and Lei, Z},
title = {Insights into pegRNA design from editing of the cardiomyopathy-associated phospholamban R14del mutation.},
journal = {FEBS letters},
volume = {599},
number = {17},
pages = {2543-2554},
doi = {10.1002/1873-3468.70097},
pmid = {40552689},
issn = {1873-3468},
support = {10250022110004//The Netherlands Organisation for Health Research and Development (ZonMw) Psider-Heart/ ; 202006170055//China Scholarship Council/ ; #725229//European Research Council (ERC), EVICARE project under H2020/ ; 2021/TTW/01038252//Netherlands Organization for Scientific Research (NWO) - Applied and Engineering Sciences (TTW)/ ; },
mesh = {*Gene Editing/methods ; Humans ; *Calcium-Binding Proteins/genetics ; *Cardiomyopathies/genetics/pathology ; CRISPR-Cas Systems ; *RNA, Guide, CRISPR-Cas Systems/genetics ; Mutation ; HEK293 Cells ; },
abstract = {Prime editing (PE) represents a transformative genome-editing technology and enables precise insertions, deletions, and base substitutions without introducing double-strand breaks, thereby reducing undesired indels and off-target effects. Despite advancements in enhanced prime editors and optimized prime editing guide RNAs (pegRNAs), designing effective pegRNAs remains a major challenge. The phospholamban (PLN) R14del mutation is associated with cardiomyopathies, making it a crucial target for precise gene-editing strategies. In this study, we explored pegRNA features that contribute to high editing efficiency using the FluoPEER.PLN R14del reporter cell line. Through systematic screening, we identified three pegRNAs with significantly enhanced editing efficiency. Our findings underscore the importance of pegRNA secondary structure and stability in optimizing prime editing, providing valuable insights into precise gene correction strategies.},
}
@article {pmid40920775,
year = {2025},
author = {Tan, Y and Kumagai-Takei, N and Shimizu, Y and Yamasaki, A and Hara-Yamamoto, M and Mitani, S and Ito, T},
title = {Targeting the Exon2 splice cis-element in PD-1 and its effects on lymphocyte function.},
journal = {PloS one},
volume = {20},
number = {9},
pages = {e0331468},
pmid = {40920775},
issn = {1932-6203},
mesh = {*Programmed Cell Death 1 Receptor/genetics/metabolism ; Humans ; *Exons/genetics ; CRISPR-Cas Systems ; *CD8-Positive T-Lymphocytes/metabolism/immunology ; *RNA Splicing ; Cytokines/metabolism ; RNA Precursors/genetics ; Lymphocyte Activation ; Cell Proliferation ; },
abstract = {T-cell therapies have proven to be a promising treatment option for cancer patients in recent years, especially in the case of chimeric antigen receptor (CAR)-T cell therapy. However, the therapy is associated with insufficient activation of T cells or poor persistence in the patient's body, which leads to incomplete elimination of cancer cells, recurrence, and genotoxicity. By extracting the splice element of PD-1 pre-mRNA using biology based on CRISPR/dCas13 in this study, our ultimate goal is to overcome the above-mentioned challenges in the future. PD-1 plays an important role in controlling T cell responses and is expressed at the cell surface of T cells following activation. The receptor PD-1 interferes with T cell receptor (TCR) signaling following interaction with PD-L1. The outcome of stimulation via PD-1 leads to decreases in cytokine secretion and cell proliferation. We extracted the RNA region of PD-1 pre-mRNA using CD8+T cell lines and examined the effect of targeting the Exon2 splice cis-element on the production of cytokines in the present study. In particular, the production of IFN-γ, TNF-α, GM-CSF was lower in RNA-targeted cells than in non-targeted cells, but the cytokine secretion capacity and cell proliferation were maintained in RNA-targeted cells. These results suggested that the use of the RNA editing technology, CRISPR/dCas13 strategy offers a novel approach to mitigate genotoxicity in lymphocytes with cytokine production and cell proliferation.},
}
@article {pmid40919784,
year = {2025},
author = {Leite, VLM and Faria, AR and Guerra, CF and Souza, SdSR and Freitas, AdAR and Morais, JM and Merquior, VLC and Planet, PJ and Teixeira, LM},
title = {Hidden diversity in Enterococcus faecalis revealed by CRISPR2 screening: eco-evolutionary insights into a novel subspecies.},
journal = {Microbiology spectrum},
volume = {},
number = {},
pages = {e0142825},
doi = {10.1128/spectrum.01428-25},
pmid = {40919784},
issn = {2165-0497},
abstract = {Enterococcus faecalis is a commensal bacterium that colonizes the gut of humans and animals and is a major opportunistic pathogen, known for causing multidrug-resistant healthcare-associated infections (HAIs). Its ability to thrive in diverse environments and disseminate antimicrobial resistance genes (ARGs) across ecological niches highlights the importance of understanding its ecological, evolutionary, and epidemiological dynamics. The CRISPR2 locus has been used as a valuable marker for assessing clonality and phylogenetic relationships in E. faecalis. In this study, we identified a group of E. faecalis strains lacking CRISPR2, forming a distinct, well-supported clade. We demonstrate that this clade meets the genomic criteria for classification as a novel subspecies, here referred to as "subspecies B." Through a comprehensive pangenome analysis and comparative genomics, we explored the adaptive ecological traits underlying this diversification process, identifying clade-specific features and their predicted functional roles. Our findings suggest that the frequent isolation of subspecies B from meat products and processing facilities may reflect dissemination routes involving environmental contamination (e.g., water, plants, soil) from avian species. The absence of key virulence traits required for pathogenicity in mammals, particularly humans, and the lack of clinically relevant resistance determinants indicate that subspecies B currently poses minimal threat to public health compared with the broadly disseminated "subspecies A." Nevertheless, the unclear potential for genetic exchange between these subspecies and the frequent association of subspecies B with food sources calls for continued genomic surveillance of E. faecalis from a One Health perspective to detect and mitigate the emergence of high-risk variants in advance.IMPORTANCEExploring intraspecific genetic variability in generalist bacteria with pathogenic potential, such as Enterococcus faecalis, is a key to uncovering stable evolutionary trends. By screening the CRISPR2 locus across a representative set of genomes from diverse sources, this study reveals a previously unrecognized lineage within the population structure of E. faecalis, associated with underexplored nonhuman and nonhospital reservoirs. These findings broaden our knowledge of the species' genetic landscape and shed light on its adaptive strategies and patterns of ecological dissemination. By bridging phylogenetic patterns with variation in genetic defense systems and accessory traits, the study generates testable hypotheses about the genomic determinants and corresponding selective pressures that shape the species' behavior and long-term dissemination. This work offers new perspectives on the eco-evolutionary dynamics of E. faecalis and highlights the value of genomic surveillance beyond clinical settings, in alignment with One Health principles.},
}
@article {pmid40845666,
year = {2026},
author = {Wang, M and Zhou, W and Wang, M and Zhang, K},
title = {A highly sensitive ECL biosensor for NF-κB p50 detection based on entropy-driven amplification and CRISPR/Cas12a signal enhancement.},
journal = {Bioelectrochemistry (Amsterdam, Netherlands)},
volume = {167},
number = {},
pages = {109081},
doi = {10.1016/j.bioelechem.2025.109081},
pmid = {40845666},
issn = {1878-562X},
mesh = {*Biosensing Techniques/methods ; *CRISPR-Cas Systems ; *NF-kappa B p50 Subunit/analysis ; Humans ; *Entropy ; *Luminescent Measurements/methods ; Electrochemical Techniques/methods ; Limit of Detection ; Exodeoxyribonucleases/metabolism ; Nucleic Acid Amplification Techniques/methods ; *CRISPR-Associated Proteins/metabolism ; Bacterial Proteins ; Endodeoxyribonucleases ; },
abstract = {Transcription factors, particularly NF-κB p50, play crucial roles in regulating gene expression and are involved in several diseases such as cancer, autoimmune disorders, and chronic inflammation. The sensitive detection of NF-κB p50 is essential for clinical diagnostics and therapeutic monitoring. In this study, we present an electrochemiluminescence (ECL) biosensor designed for the highly sensitive and specific detection of NF-κB p50. The biosensor integrates entropy-driven amplification and CRISPR/Cas12a-based signal enhancement to detect trace amounts of NF-κB p50. Upon detection of NF-κB p50, a ternary complex forms with a double-stranded DNA (dsDNA) probe, which prevents subsequent cleavage by exonuclease III (Exo III) and inhibits the CRISPR/Cas12a system. In the absence of NF-κB p50, Exo III digestion triggers entropy-driven amplification, which activates CRISPR/Cas12a, leading to enhanced electrochemical signals. The ECL biosensor demonstrated a detection limit of 0.56 pM, high selectivity, and excellent reproducibility. Furthermore, the biosensor successfully detected NF-κB p50 in complex biological samples, such as HeLa cell lysates, showcasing its potential for clinical applications in disease diagnostics.},
}
@article {pmid40845664,
year = {2026},
author = {Zhou, Y and Lv, Z and Geng, Y and Liu, R},
title = {An electrochemical RNA aptasensor based on sheet-like α-Fe2O3/Fe3O4 magnetic nanocomposites and CRISPR/Cas13a system for supersensitive detection of osteopontin.},
journal = {Bioelectrochemistry (Amsterdam, Netherlands)},
volume = {167},
number = {},
pages = {109085},
doi = {10.1016/j.bioelechem.2025.109085},
pmid = {40845664},
issn = {1878-562X},
mesh = {*CRISPR-Cas Systems ; *Aptamers, Nucleotide/chemistry/genetics ; *Biosensing Techniques/methods ; *Electrochemical Techniques/methods ; *Nanocomposites/chemistry ; *Osteopontin/analysis/blood ; Limit of Detection ; Humans ; *Ferric Compounds/chemistry ; },
abstract = {Osteopontin (OPN) exhibits markedly elevated expression in malignant tumor tissues, rendering it a crucial tumor marker for cancer prevention and monitoring-underscoring the significance of its detection. This work proposed an electrochemical RNA aptasensor based on a novel sheet-like α-Fe2O3/Fe3O4 magnetic nanocomposites (MNCs) and CRISPR/Cas13a system to effectively detect OPN. The proposed aptasensor used the sheet-like α-Fe2O3/Fe3O4 MNCs as the conduction matrix and applied their magnetic property to accomplish self-assembly of the sensing element onto the electrode. The current change of this aptasensor depended on the activation degree of the CRISPR/Cas13a system, which correlated with the amount of OPN expression. Double-stranded RNA (Apt/Activator) was compelled disassembly due to the OPN's strong affinity of Apt, while single-stranded RNA (Activator) could be guided by crRNA to combine with Cas13a/crRNA and activated the enzymatic activity of Cas13a. Cas13a enzyme affected the current and electrical resistance by shearing the nucleic acid strands (Reporter) on the electrode, ultimately enabling the quantitative detection of OPN. The aptasensor demonstrated excellent selectivity, reproducibility, and stability, with the detection limit (LOD) of 0.33 pg·mL[-1] and the wide linear detection range of 1 pg·mL[-1] - 10 ng·mL[-1]. These results offer a novel idea for advancing tumor marker electrochemical biosensors.},
}
@article {pmid40757822,
year = {2025},
author = {Echavarria Galindo, M and Lai, Y},
title = {CRISPR-based genetic tools for the study of host-microbe interactions.},
journal = {Infection and immunity},
volume = {93},
number = {9},
pages = {e0051024},
doi = {10.1128/iai.00510-24},
pmid = {40757822},
issn = {1098-5522},
support = {26100423//Research Grants Council, University Grants Committee/ ; PF22-69790//Hong Kong PHD Fellowship Scheme/ ; R9829//Hong Kong University of Science and Technology (HKUST)/ ; },
mesh = {Humans ; Animals ; *CRISPR-Cas Systems ; *Host Microbial Interactions/genetics ; Gene Editing/methods ; *Clustered Regularly Interspaced Short Palindromic Repeats ; *Microbiota/genetics ; *Host-Pathogen Interactions/genetics ; },
abstract = {CRISPR-based genetic tools have revolutionized our ability to interrogate and manipulate genes. These tools can be applied to both host and microbial cells, and their use can enhance our understanding of the dynamic nature of host-microbe interactions by uncovering their genetic underpinnings. As reviewed here, CRISPR-based tools are being used to explore the microbiome in an efficient, accurate, and high-throughput manner. By employing CRISPR screens, targeted genome editing, and recording systems to the study of host cells and microorganisms, we can gain critical insights into host defense mechanisms, potential vulnerabilities, and microbial pathogenesis, as well as essential or condition-specific genes involved in host-microbe interactions. Additionally, CRISPR-based genetic tools are being used in animal models to study host-microbe interactions in vivo. Recent advancements in CRISPR-derived technology can be combined with emerging techniques, such as single-cell RNA sequencing, to examine the complex interactions between hosts and microbes, shedding light on the role of the microbiome in health and disease. This review aims to provide a comprehensive overview of how these cutting-edge genetic tools are being used to investigate host-microbial systems, as well as their current limitations. Current research is likely to yield even more advanced genetic toolkits than those presently available, and these can serve researchers in identifying and exploring new therapeutic targets for diseases related to host-microbe interactions.},
}
@article {pmid40749504,
year = {2026},
author = {Ji, Z and Cheng, S and Li, W and Xing, Y and Tang, Z and Zhu, X and Wang, D and Hao, C and Wang, B and Shi, M},
title = {Ultrasensitive detection of miR-31 using a signal-on electrochemiluminescence biosensor based on CRISPR/Cas12a and MXene nanocomposites.},
journal = {Bioelectrochemistry (Amsterdam, Netherlands)},
volume = {167},
number = {},
pages = {109059},
doi = {10.1016/j.bioelechem.2025.109059},
pmid = {40749504},
issn = {1878-562X},
mesh = {*MicroRNAs/analysis/blood/genetics ; *Biosensing Techniques/methods ; Humans ; *Nanocomposites/chemistry ; *CRISPR-Cas Systems ; *Electrochemical Techniques/methods ; Limit of Detection ; *Luminescent Measurements/methods ; *Endodeoxyribonucleases/metabolism ; *CRISPR-Associated Proteins/metabolism ; Gold/chemistry ; Electrodes ; Metallocenes ; Bacterial Proteins ; Nitrites ; Transition Elements ; },
abstract = {MicroRNAs (miRNAs) have emerged as critical biomarkers for early cancer diagnosis due to their high specificity and stability in biological fluids. In this study, we report a highly sensitive and specific electrochemiluminescence (ECL) biosensor for the detection of miR-31, a miRNA closely associated with non-small cell lung cancer. The sensing platform integrates a PEI-Ru@Ti3C2@AuNPs-modified electrode with a ferrocene-labeled DNA probe (DNA1-Fc) to construct a target-responsive signal-on system. Upon recognition of miR-31, an isothermal strand displacement amplification (ISDA) reaction is initiated, producing abundant double-stranded DNA (dsDNA) that activates the CRISPR/Cas12a complex. The trans-cleavage activity of Cas12a then cleaves the DNA1-Fc probes on the electrode surface, removing the quenching ferrocene moiety and restoring the Ru-based ECL signal. Under optimized conditions, the biosensor exhibited a wide dynamic range from 10 aM to 100 pM and a remarkably low detection limit of 1.67 aM. The system also showed excellent specificity against homologous miRNAs, and its applicability was successfully validated in spiked human serum samples, achieving high recovery and reproducibility. The synergistic combination of nanomaterial-enhanced ECL emission, isothermal nucleic acid amplification, and CRISPR-based enzymatic cleavage provides a powerful strategy for ultrasensitive nucleic acid detection. This work offers a promising approach for early cancer diagnosis and has great potential for clinical translation and point-of-care testing.},
}
@article {pmid40719649,
year = {2025},
author = {Chen, M and Choi, HK and Goldston, LL and Hou, Y and Jiang, C and Lee, KB},
title = {Advanced Cancer Liquid Biopsy Platform for miRNA Detection in Extracellular Vesicles Using CRISPR/Cas13a and Gold Nanoarrays.},
journal = {ACS nano},
volume = {19},
number = {35},
pages = {31438-31456},
doi = {10.1021/acsnano.5c06940},
pmid = {40719649},
issn = {1936-086X},
mesh = {*MicroRNAs/genetics/analysis/blood ; Humans ; *Extracellular Vesicles/chemistry/metabolism/genetics ; *Gold/chemistry ; *CRISPR-Cas Systems ; Liquid Biopsy/methods ; *Colorectal Neoplasms/diagnosis/genetics ; Biomarkers, Tumor/genetics/blood ; Epithelial Cell Adhesion Molecule/metabolism ; Cell Line, Tumor ; Metal Nanoparticles/chemistry ; },
abstract = {Liquid biopsy is a transformative, noninvasive tool for cancer diagnosis and monitoring, with the potential to revolutionize personalized medicine. In this study, we introduce an advanced liquid biopsy platform for highly sensitive and selective detection of extracellular vesicle (EV) microRNAs (miRNA-21 and miRNA-23a) as biomarkers for colorectal cancer. The platform combines two innovations: (1) gold nanoarrays with epithelial cell adhesion molecule (EpCAM)-specific aptamers to selectively isolate tumor-derived EVs from plasma and (2) CRISPR/Cas13a-encapsulated liposomes that fuse with EVs to form nanoscale reactors. Upon fusion, the CRISPR/Cas13a complex is activated by target miRNA, triggering trans-cleavage of RNA reporters and generating an amplified fluorescence signal for enhanced detection sensitivity. The assay achieves a linear detection range of 10 to 10[6] EVs/μL and a detection limit of 2.5 × 10[1] EVs/μL on the gold nanoarray. Its performance relies on three strategies: (i) EpCAM-mediated tumor EV enrichment, (ii) CRISPR/Cas13a-based collateral activity for ultrasensitive miRNA detection, and (iii) fluorescence signal enhancement via localized nanoreactors. Validation with a 2D SW480 cell model, a 3D vascularized tumor spheroid (VTS) model, and clinical plasma samples confirms diagnostic accuracy, with miRNA quantification comparable to RT-qPCR but without the need for labor-intensive RNA extraction and amplification. By integrating nanotechnology with CRISPR-based diagnostics, this platform bridges research and clinical translation, improving diagnostic precision and streamlining workflows. Future development will focus on multiplexed biomarker detection and single-EV analysis to reveal insights into EV heterogeneity and function in cancer. This technology supports the application in precision oncology, offering a tool for early detection, treatment monitoring, and therapeutic decision-making.},
}
@article {pmid40653285,
year = {2025},
author = {Monteiro, JP and Vacante, F and De Pace, AL and Bennett, M and Rodor, J and O'Carroll, D and Wu, JC and Quertermous, T and Baker, AH},
title = {Targeting Cis-regulatory elements for CRISPR-mediated transcriptional activation of the human MIR503HG locus.},
journal = {Vascular pharmacology},
volume = {160},
number = {},
pages = {107521},
doi = {10.1016/j.vph.2025.107521},
pmid = {40653285},
issn = {1879-3649},
mesh = {Humans ; *MicroRNAs/genetics/metabolism ; *Transcriptional Activation ; *CRISPR-Cas Systems ; *RNA, Long Noncoding/genetics/metabolism ; Lentivirus/genetics ; HEK293 Cells ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Genetic Vectors ; *Gene Editing/methods ; *Regulatory Elements, Transcriptional ; Human Umbilical Vein Endothelial Cells/metabolism ; },
abstract = {Advances in genome annotation have revealed a striking increase in the number and complexity of non-coding RNA (ncRNA) genes, particularly multi-transcript loci that harbor long non-coding RNAs (lncRNAs) and microRNAs (miRNAs) within the same genomic region. These loci can often function as coordinated regulatory units, with their transcription modulated by shared cis-acting regulatory elements (CREs). Traditional gene manipulation approaches, which typically target individual transcripts, are insufficient to capture the full regulatory and therapeutic potential of these complex loci. Here, we present "cis-ON" a single-vector lentiviral delivery system based on CRISPR activation (CRISPRa), designed to simultaneously upregulate multiple noncoding RNA transcripts by targeting a single CRE. We focused on the evolutionarily conserved MIR503HG locus, which encodes seven lncRNA isoforms and hosts the miR-424/503 cluster, both implicated in various cellular processes, including proliferation, angiogenesis, and endothelial-to-mesenchymal transition. Using integrative analysis of histone marks (H3K27Ac, H3K4Me3), DNase hypersensitivity, and CAGE-seq data, we identified the primary promoter of the MIR503HG locus. A dual fluorescent reporter screen selected optimal single guide RNAs (sgRNAs) for targeting this region. We then engineered cis-ON, a novel lentiviral system combining dCas9-VPR and sgRNA to enable a streamlined single-vector delivery approach. Transduction of primary human endothelial cells with this system robustly activated the entire locus including the MIR503HG isoforms and co-embedded miRNAs miR-424 and miR-503, demonstrating coordinated transcriptional regulation. Additionally, the neighboring LINC00629 lncRNA locus remained unaffected, highlighting regulatory specificity. This approach demonstrates the feasibility of modulating complex ncRNA loci across a ∼ 10 kb genomic region by targeting a single CRE, bypassing limitations of transcriptspecific strategies. By enabling simultaneous upregulation of lncRNAs and miRNAs, the cis-ON platform provides a streamlined strategy for restoring regulatory networks disrupted in disease.},
}
@article {pmid39992014,
year = {2025},
author = {Chen, RH and Bai, Y and Shi, LD and Liu, XY and Li, DL and Di, M and Duan, JX and Zhang, ZH and Xu, JL and He, ZQ and Li, K},
title = {Functional exploration of the Sex combs reduced gene in Gryllus bimaculatus.},
journal = {Insect molecular biology},
volume = {34},
number = {5},
pages = {608-618},
doi = {10.1111/imb.12987},
pmid = {39992014},
issn = {1365-2583},
mesh = {Animals ; *Insect Proteins/genetics/metabolism ; Female ; CRISPR-Cas Systems ; Male ; Gene Knockout Techniques ; *Homeodomain Proteins/genetics/metabolism ; },
abstract = {The Hox gene Sex combs reduced (Scr) is recognized as a key factor in the development of the head and thorax in insects. However, its function in the growth, development and morphogenesis of Gryllus bimaculatus remains poorly understood. This study aimed to explore the function of the Scr gene in G. bimaculatus by using CRISPR/Cas9 technology to generate an Scr gene knock-out strain. Intercrossing the G0 generation knock-out individuals with wild-type individuals yielded the G1 generation to screen the mutant strain. It was found that the knock-out of the Scr gene had a severe impact on the growth and development of G. bimaculatus, resulting in high mortality and making it difficult to obtain Scr[-/-] mutants. Therefore, heterozygous individuals (Scr[+/-]) with 1 bp deleted were obtained for investigation. The results showed that the Scr deletion led to ectopic segment formation in the G0 generation. In the G2 generation, it was observed that stable Scr[-/-] strains displayed abnormal embryonic development, characterized by enlarged, blackened and lethal eggs during embryogenesis. During the post-embryonic stage, Scr[-/-] mutants exhibited abnormalities in body segmentation, particularly in the head-thorax region, resulting in a dorsal ridge structure. Furthermore, some Scr[+/-] individuals exhibited a dorsal ridge during the nymphal stage. Notably, this characteristic did not persist into the adult stage. Our findings highlight the distinct but crucial roles of the Scr gene in both embryonic and post-embryonic growth and development of G. bimaculatus.},
}
@article {pmid40918426,
year = {2025},
author = {Senbadejo, TY and Ntiamoah Osei, S and Isawumi, A},
title = {Snapshot of Defense Systems in Multidrug Resistant Klebsiella pneumoniae.},
journal = {microPublication biology},
volume = {2025},
number = {},
pages = {},
pmid = {40918426},
issn = {2578-9430},
abstract = {Bacterial defense mechanisms protect pathogens from host immunity, bacteriophages, and harsh environments. This study investigates defense systems in multidrug-resistant Klebsiella pneumoniae from Ghanaian hospital ICUs, focusing on CRISPR-Cas, restriction-modification (R-M), and toxin-antitoxin (TA) systems. Genomes of environmental (NS2) and clinical (PS4) strains were sequenced and analyzed using PADLOC, defensefinder, and TADB3.0. NS2 carries 12 defense systems, including CRISPR-Cas, while PS4 has five. Both possess diverse RM and TA systems. These strains, resistant to six antibiotic classes, encode clinically significant defense systems, suggesting microbial exchange between fomites and humans, potentially increasing infection risks in ICU environments requiring targeted surveillance.},
}
@article {pmid40918304,
year = {2025},
author = {Dash, B and Bhuyan, SS and Sahoo, RK and Swain, N and Jeughale, KP and Sarkar, S and Verma, RL and Parameswaran, C and Devanna, BN and Samantaray, S},
title = {CRISPR/Cas-mediated genome editing: playing a versatile role in mitigating the challenges of sustainable rice improvement.},
journal = {3 Biotech},
volume = {15},
number = {10},
pages = {327},
pmid = {40918304},
issn = {2190-572X},
abstract = {Just as Gregor Mendel's laws of inheritance laid the foundation for modern genetics, the emergence of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas systems has catalyzed a new era in precision genome engineering. CRISPR/Cas has revolutionized rice (Oryza sativa L.) breeding by enabling precise, transgene-free edits to improve yield, nutrition, and stress tolerance. Advanced tools like base and prime editing further refine these capabilities, offering powerful solutions for climate-resilient agriculture and global food security. The review synthesizes the CRISPR-mediated strategies for improving resistance against major biotic (bacterial blight, blast, sheath blight) and abiotic (drought, salinity, submergence, nutrient deficiency) stresses. Additionally, we explore the critical prerequisites for efficient genome editing in rice, ranging from target site design, PAM specificity, delivery systems (like Agrobacterium, RNPs, and nanoparticle-mediated delivery), to screening and validation of mutants. This review also highlights recent breakthroughs in multiplex genome editing for complex traits, including the development of haploid inducer lines and clonal seed technology. Haploid inducers accelerate breeding by producing homozygous lines without tissue culture, while engineered apomixis enables clonal propagation of elite hybrids. Beyond technical dimensions, this review underscores the broader socio-economic and regulatory implications of genome-edited rice, addressing the emerging ethical concerns, intellectual property issues, farmer access, and equitable technology dissemination in resource-limited agricultural regions. As the global policy landscape transitions to accommodate CRISPR-edited crops, transparent regulatory frameworks, stakeholder engagement, and public perception will play pivotal roles in ensuring sustainable, safe, and inclusive adoption of genome editing in agriculture.},
}
@article {pmid40918251,
year = {2025},
author = {Zu, H and Sun, R and Li, J and Guo, X and Wang, M and Guo, W and Wang, X},
title = {Integrated CRISPR-Cas12a and RAA one-pot visual strategy for the rapid identification of Streptococcus equi subspecies equi.},
journal = {Frontiers in cellular and infection microbiology},
volume = {15},
number = {},
pages = {1526516},
pmid = {40918251},
issn = {2235-2988},
mesh = {*Streptococcus equi/genetics/isolation &amp; purification/classification ; Horses ; *CRISPR-Cas Systems ; Animals ; *Streptococcal Infections/veterinary/diagnosis/microbiology ; *Horse Diseases/diagnosis/microbiology ; Sensitivity and Specificity ; *Nucleic Acid Amplification Techniques/methods ; *Recombinases/metabolism/genetics ; *Molecular Diagnostic Techniques/methods ; Iceland ; Bacterial Proteins/genetics ; Endodeoxyribonucleases ; Streptococcus ; CRISPR-Associated Proteins ; },
abstract = {Strangles, a highly contagious disease caused by Streptococcus equi subspecies equi (S.equi), significantly impacts horse populations worldwide, with Iceland as the only exception. This disease poses serious threats to equine health and results in considerable economic losses. Consequently, the accurate, sensitive, and rapid detection of S.equi from clinical samples is essential for early warning and effective disease management. This study introduces a novel detection method that integrates recombinase-aided amplification (RAA) with CRISPR/Cas12a technologies. We specifically designed RAA primers and CRISPR RNA to target the eqbE gene of S.equi, and we have carefully optimized the reaction systems for this purpose. The newly established visual diagnostic method has shown to be highly effective, demonstrating 97.14% specificity and 100% sensitivity, with the capability to detect as few as 5.6×10[0] copies of the target. This is the first study to propose the combined application of RAA and CRISPR/Cas12a for the on-site rapid detection of S.equi. This is the first study to propose the combined application of RAA and CRISPR/Cas12a for the on-site rapid detection of S.equi, which enables visual point-of-care diagnosis of Strangles.},
}
@article {pmid40918142,
year = {2025},
author = {Golinelli, G and Scholler, J and Roussel-Gervais, A and Šakić, A and Ilmjärv, S and Song, D and Gabunia, K and Ji, M and Fan, TJ and Gupta, A and Deshmukh, M and Berjis, A and Cuoghi Costantini, R and Apodaca, K and Sheppard, NC and Kili, S and Dominici, M and Alessandrini, M and June, CH and Levine, BL},
title = {Multiplex engineering using microRNA-mediated gene silencing in CAR T cells.},
journal = {Frontiers in immunology},
volume = {16},
number = {},
pages = {1647433},
pmid = {40918142},
issn = {1664-3224},
mesh = {*MicroRNAs/genetics ; Animals ; Humans ; *Receptors, Chimeric Antigen/genetics/immunology ; *Immunotherapy, Adoptive/methods ; Mesothelin ; *Gene Silencing ; Mice ; *T-Lymphocytes/immunology/metabolism ; Cell Line, Tumor ; Gene Editing ; CRISPR-Cas Systems ; Receptors, Antigen, T-Cell/genetics ; *Pancreatic Neoplasms/therapy/immunology/genetics ; Xenograft Model Antitumor Assays ; GPI-Linked Proteins/genetics/immunology ; },
abstract = {BACKGROUND: Multiplex gene-edited chimeric antigen receptor (CAR) T-cell therapies face significant challenges, including potential oncogenic risks associated with double-strand DNA breaks. Targeted microRNAs (miRNAs) may provide a safer, functional, and tunable alternative for gene silencing without the need for DNA editing.<br><br>METHODS: As a proof of concept for multiplex gene silencing, we employed an optimized miRNA backbone and gene architecture to silence T-cell receptor (TCR) and major histocompatibility complex class I (MHC-I) in mesothelin-directed CAR (M5CAR) T cells. The efficacy of this approach was compared to CD3&#x3b6; and &#x3b2;2-microglobulin (&#x3b2;2M) CRISPR/Cas9 knockout (KO) cells. miRNA-expressing cassettes were incorporated into M5CAR lentiviral vectors, enabling combined gene silencing and CAR expression. Antitumor activity was evaluated using in vitro assays and in vivo pancreatic ductal adenocarcinoma models.<br><br>RESULTS: Silenced (S) M5CAR T cells retained antitumor functionality comparable to, and in some cases exceeding, that of KO cells. In vivo, S M5CAR T cells achieved tumor control with higher persistence and superior metastasis prevention. In vitro assays demonstrated enhanced resistance to alloreactive natural killer (NK) cells and peripheral blood mononuclear cells (PBMCs).<br><br>CONCLUSIONS: Titratable multiplex gene silencing via targeted miRNAs offers an alternative to gene editing for CAR T cells, with potential advantages in potency, persistence, metastasis prevention, and immune evasion for allogeneic products. This strategy may overcome tumor-induced immunosuppression while avoiding the risks associated with DNA double-strand breaks.},
}
@article {pmid40916896,
year = {2025},
author = {Courtier-Orgogozo, V},
title = {Risks associated with CRISPR homing gene drive.},
journal = {Comptes rendus biologies},
volume = {348},
number = {},
pages = {211-227},
doi = {10.5802/crbiol.182},
pmid = {40916896},
issn = {1768-3238},
mesh = {*Gene Drive Technology/adverse effects ; Humans ; Animals ; *CRISPR-Cas Systems/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Ecosystem ; Gene Editing ; },
abstract = {CRISPR homing gene drive is a disruptive biotechnology developed over the past decade with potential applications in public health, agriculture, and conservation biology. This technology relies on an autonomous selfish genetic element able to spread in natural populations through the release of gene drive individuals. However, it has not yet been deployed in the wild. In this review, we examine the key risks associated with CRISPR homing gene drives. First, we explore technical limitations, where gene drives might not be as efficient as intended, and cases where mitigation strategies may not be able to block a gene drive. Then, we present four main categories of adverse effects: (a) ecological risks, corresponding to unintended consequences on ecosystems and non-target populations; (b) sociological risks, i.e. concerns over public perception, governance, and societal acceptance; (c) risks associated with research activities; and (d) risks associated with malevolent usage. Regulatory aspects are not addressed here. This article provides a foundation for evaluating gene drive risks to ensure responsible and informed decision-making.},
}
@article {pmid40916704,
year = {2025},
author = {Ficoseco, CMA and Chieffi, D and Montemurro, M and Bavaro, A and Rizzello, CG and Nader-Macias, MEF and Fadda, S and Fanelli, F and Fusco, V and Vignolo, GM},
title = {Genomic Characterisation of Limosilactobacillus fermentum CRL2085 Unveiling Probiotic Traits for Application in Cattle Feed.},
journal = {Environmental microbiology reports},
volume = {17},
number = {5},
pages = {e70176},
doi = {10.1111/1758-2229.70176},
pmid = {40916704},
issn = {1758-2229},
support = {//This work was financially supported by the Joint Bilateral Agreement CNR/CONICET (ITALY-Argentina) "Lactic Acid Bacteria as bioprotective agents against zoonotic pathogens in the meat chain" Biennial Programme 2023-2024/ ; },
mesh = {Animals ; *Probiotics ; Cattle ; *Limosilactobacillus fermentum/genetics/isolation &amp; purification/metabolism ; *Animal Feed/microbiology ; *Genome, Bacterial ; Genomics ; Genomic Islands ; Bacterial Adhesion ; },
abstract = {Limosilactobacillus fermentum CRL2085, isolated from feedlot cattle rations, displayed high efficiency as a probiotic when administered to animals. A comprehensive genomic analysis was performed to elucidate the genetic basis underlying its probiotic potential. Fifteen genomic islands and CRISPR-Cas elements were identified in its genome. Pan-genomic analysis highlighted the dynamic evolution of this species, and clustering based on the nucleotide genomic similarity only partially correlated with the source of isolation or the geographic origin of the strains. Several genes known to confer probiotic properties were identified, including those related to adhesion, resistance to acidic pH and bile salts, tolerance to oxidative stress, metabolism/transport of sugars and other compounds, and genes for exopolysaccharide biosynthesis. In silico analysis of antimicrobial resistance genes and virulence determinants confirmed the safety of this strain. Moreover, genes related to B-group vitamins biosynthesis and feruloyl esterase hydrolase were also found, showing the nutritional contribution of the strain, which also showed moderate adhesion capability, exopolysaccharide production when grown with sucrose, and the capacity to metabolise 42 out of 95 carbon substrates tested. This data provides the genetic basis for deciphering the mechanisms beyond the benefits demonstrated by its use during cattle intensive raising and confirms its promising role as a probiotic.},
}
@article {pmid40914328,
year = {2025},
author = {Rathored, J and Budhbaware, T},
title = {Integrative Strategies Against Multidrug-Resistant Bacteria: Synthesizing Novel Antimicrobial Frontiers for Global Health.},
journal = {Microbial pathogenesis},
volume = {},
number = {},
pages = {108018},
doi = {10.1016/j.micpath.2025.108018},
pmid = {40914328},
issn = {1096-1208},
abstract = {Concerningly, multidrug-resistant bacteria have emerged as a prime worldwide trouble, obstructing the treatment of infectious diseases and causing doubts about the therapeutic accidentalness of presently existing drugs. Novel antimicrobial interventions deserve development as conventional antibiotics are incapable of keeping pace with bacteria evolution. Various promising approaches to combat MDR infections are discussed in this review. Antimicrobial peptides are examined for their broad-spectrum efficacy and reduced ability to develop resistance, while phage therapy may be used under extreme situations when antibiotics fail. In addition, the possibility of CRISPR-Cas systems for specifically targeting and eradicating resistance genes from bacterial populations will be explored. Nanotechnology has opened up the route to improve the delivery system of the drug itself, increasing the efficacy and specificity of antimicrobial action while protecting its host. Discovering potential antimicrobial agents is an exciting prospect through developments in synthetic biology and the rediscovery of natural product-based medicines. Moreover, host-directed therapies are now becoming popular as an adjunct to the main strategies of therapeutics without specifically targeting pathogens. Although these developments appear impressive, questions about production scaling, regulatory approvals, safety, and efficacy for clinical employment still loom large. Thus, tackling the MDR burden requires a multi-pronged plan, integrating newer treatment modalities with existing antibiotic regimens, enforcing robust stewardship initiatives, and effecting policy changes at the global level. The international health community can gird itself against the growing menace of antibiotic resistance if collaboration between interdisciplinary bodies and sustained research endeavours is encouraged. In this study, we evaluate the synergistic potential of combining various medicines in addition to summarizing recent advancements. To rethink antimicrobial stewardship in the future, we provide a multi-tiered paradigm that combines pathogen-focused and host-directed strategies.},
}
@article {pmid40912804,
year = {2025},
author = {Qiu, Y and Li, X and Fan, M and Tang, H and Zhang, S and Huang, W and Han, Z and Wang, S and Peng, H and Xiao, Y and Ye, X and Wang, K},
title = {Modification of starch traits in commercial wheat through TaWaxy gene editing.},
journal = {Carbohydrate polymers},
volume = {368},
number = {Pt 1},
pages = {124105},
doi = {10.1016/j.carbpol.2025.124105},
pmid = {40912804},
issn = {1879-1344},
mesh = {*Triticum/genetics/metabolism/chemistry ; *Gene Editing/methods ; Amylose/genetics ; *Starch/genetics/metabolism/chemistry ; *Plant Proteins/genetics/metabolism ; Mutation ; CRISPR-Cas Systems ; Plants, Genetically Modified ; },
abstract = {Amylose content (AC) is a key determinant of wheat quality, and the TaWaxy gene determined amylose synthesis with a dose-dependent effect on AC. In this study, the TaWOX5 gene, which significantly enhances wheat transformation efficiency, was combined with CRISPR/SpCas9 system to generate TaWaxy mutants in a commercial winter wheat Jimai 22. Seven transgene-free mutant types were produced, compared to only three transgene-free mutants in the spring wheat variety Ningchun 4. The TaWaxy mutants from the two varieties showed decreased ACs ranging from 0 to 19.05 %. Results demonstrated that the Waxy-B1 protein has the most significant effect on amylose synthesis. The mutants with TaWaxy-abd, TaWaxy-ab, and TaWaxy-bd alleles showed waxy wheat trait. Interestingly, the TaWaxy-b mutant from Jimai 22 exhibited a waxy trait, unlike the TaWaxy-b mutant from Ningchun 4. Transmission electron microscope and scanning electron microscopy showed increased B-type starch granules in mutant grains. The mutants displayed varying effects on bread, cake, cookie, and noodle quality. All mutants showed decreased quality in bread and cake production, while TaWaxy-ad-JM and TaWaxy-b-NC mutants showed improved noodle and cookie quality. The generated mutants provide optimized optimized amylose content, enhancing noodle and biscuit quality as a practical alternative to blending.},
}
@article {pmid40912763,
year = {2025},
author = {Joshi, R and Paliwal, T and Sharma, S and Kapoor, DU and Prajapati, BG},
title = {Chitosan polyplexes for targeted gene delivery: From mechanisms to clinical applications.},
journal = {Carbohydrate polymers},
volume = {368},
number = {Pt 1},
pages = {124080},
doi = {10.1016/j.carbpol.2025.124080},
pmid = {40912763},
issn = {1879-1344},
mesh = {*Chitosan/chemistry ; Humans ; *Gene Transfer Techniques ; Animals ; Genetic Therapy/methods ; DNA/chemistry ; },
abstract = {As a diverse natural polymer called Chitosan, it created ground-breaking advancements in nucleic acid therapeutic delivery techniques for handling essential DNA and RNA delivery hurdles. The article investigates how nucleic acids form stable polyplexes with chitosan through electrostatic bonds, as well as explores their chemical and biological properties. The review explores how molecular weight, combined with the degree of deacetylation, combined with advanced functionalization strategies, help enhance delivery results. Cellular uptake and specific targeting of polyplexes are improved through research into different targeting methods, which include ligand attachment, charge manipulation, and cell-penetrating peptides. Endosomal release of polyplexes is enabled by pH-sensitive and redox-sensitive polyplex architectures, while simultaneous control strategies are achieved through these delivery systems. The field of nucleic acid therapy now includes applications with CRISPR-Cas delivery and mRNA vaccines, and gene silencing therapy. The safety assessment encompasses cytotoxicity testing alongside immunogenicity evaluation and biodegradability assessment, which is conducted alongside direct comparison with alternative polymeric delivery vehicles. The potential for clinical use and targeted delivery, and combined smart transport systems establishes chitosan polyplexes as an innovative solution for nucleic acid drug delivery according to recent research findings.},
}
@article {pmid40911134,
year = {2025},
author = {Jiang, C and Li, Y and Yu, P and Fang, M and Huang, D and Fang, X and Xu, Z},
title = {A rapid and ultrasensitive CRISPR/Cas12a-based assay for the accurate identification of T-even type phages.},
journal = {Biotechnology letters},
volume = {47},
number = {5},
pages = {95},
pmid = {40911134},
issn = {1573-6776},
support = {2021YFC2103200//the National Key Research and Development Program of China/ ; 22404149//the National Natural Science Foundation of China/ ; 2023M742994//the China Postdoctoral Science Foundation/ ; GZC20232319//the Postdoctoral Fellowship Program of CPSF/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; *Nucleic Acid Amplification Techniques/methods ; *T-Phages/genetics/isolation &amp; purification/classification ; Limit of Detection ; Wastewater/virology ; },
abstract = {Phage contamination poses a significant threat to industrial fermentation, leading to substantial economic losses. Virulent T-even type phages (T2/T4/T6) represent particularly concerning biological hazards in fermentation systems. This paper developed a novel CRISPR/Cas12a-based system integrated with recombinase polymerase amplification (RPA), enabling ultrasensitive identification of T-even type phages. This method targeted the TerL gene of T-even type phages as a detection marker. The optimized RPA-CRISPR assay demonstrated exceptional sensitivity with a limit of detection (LOD) reaching 1 aM for synthetic targets. Besides, this system achieved detection thresholds of 1 and 10 PFU/μL for T2 and T4 phages, respectively. Comparative validation with quantitative PCR (qPCR) confirmed the method's reliability through strong correlation in the detection for both spiked and wastewater samples. The detection platform exhibited remarkable potential for rapid, sensitive monitoring of T-even type phages contamination in fermentation processes, offering promising application prospects for quality control in biochemical industries.},
}
@article {pmid40904109,
year = {2025},
author = {Clabby, T and Tesson, F and Gaborieau, B and Bernheim, A},
title = {Why do bacteria accumulate antiphage defence systems?.},
journal = {Philosophical transactions of the Royal Society of London. Series B, Biological sciences},
volume = {380},
number = {1934},
pages = {20240082},
pmid = {40904109},
issn = {1471-2970},
support = {//MSDAVENIR/ ; //Pasteur Institute/ ; /ERC_/European Research Council/International ; },
mesh = {*Bacteria/classification/genetics/immunology/virology ; *Bacteriophages/genetics/physiology ; Ecology ; CRISPR-Cas Systems ; Gene Transfer, Horizontal ; Interspersed Repetitive Sequences ; *Phage Therapy ; },
abstract = {While it is well established that bacterial genomes encode multiple and diverse antiphage systems, the reasons for their co-occurrence and their heterogeneous distribution remain debated. This review examines why bacteria accumulate antiphage systems and how this influences phage-bacteria interactions, particularly in the context of phage therapy. Two main hypotheses may explain this phenomenon: (i) the pan-immunity hypothesis, which suggests that defence system accumulation provides protection against phage predation at the community level, and (ii) mobile genetic element (MGE) competition, where defence systems primarily protect intra-bacterial MGEs against other ones rather than the bacterial host itself. The ecological context also influences the distribution of antiphage systems, with defencee accumulation shaping phage-bacteria interactions in diverse communities but playing a lesser role at the species level, potentially explaining why multiple defences do not strongly limit phage host range in therapeutic settings. Finally, we address the challenges in understanding the drivers shaping the distribution of defence systems across bacterial genomes (expressions, costs, etc.) and their implications for elucidating the ecological role of defence systems and optimizing phage therapy strategies.This article is part of the discussion meeting issue 'The ecology and evolution of bacterial immune systems'.},
}
@article {pmid40889469,
year = {2025},
author = {Li, Y and Dou, Y and Lu, Z and Wang, Y and Zhou, H and Li, T},
title = {CRISPR/Cas12a-functionalized silicon nanowires field-effect transistor sensor for ultra-sensitive detection of pathogen nucleic acids.},
journal = {Biosensors &amp; bioelectronics},
volume = {289},
number = {},
pages = {117936},
doi = {10.1016/j.bios.2025.117936},
pmid = {40889469},
issn = {1873-4235},
mesh = {*Biosensing Techniques/instrumentation ; *Nanowires/chemistry/ultrastructure ; *Silicon/chemistry ; *CRISPR-Cas Systems/genetics ; Transistors, Electronic ; *Bacillus anthracis/genetics/isolation &amp; purification/pathogenicity ; Limit of Detection ; *DNA, Bacterial/genetics/isolation &amp; purification/analysis ; *Endodeoxyribonucleases/chemistry ; Bacterial Proteins/chemistry ; CRISPR-Associated Proteins ; },
abstract = {Rapid, sensitive, and accurate detection of pathogen nucleic acids is critical for ensuring public safety and health. Nevertheless, current methods still encounter significant challenges. Field-effect transistor (FET) biosensors are renowned for high sensitivity, rapid response, and label-free detection. However, when employed for the direct detection of long-chain DNA extracted from pathogens, these sensors exhibit low recognition efficiency, poor accuracy, and prolonged reaction times. To address these limitations, we propose a novel silicon nanowires FET sensing strategy functionalized with the CRISPR/Cas12a system. The Cas12a/crRNA complex rapidly scans and precisely cleaves target sequences within long double-stranded DNA (dsDNA). This mechanism effectively mitigates detection performance degradation caused by nucleic acid folding and entanglement, thereby significantly enhancing both sensitivity and accuracy. Additionally, Cas12a/crRNA cleaves long dsDNA into specific-length fragments, thereby ensuring their distribution within the Debye length and enhancing signal consistency. Using this approach, we successfully achieved quantitative detection of Bacillus anthracis dsDNA within 10 min, with a detection limit at the attomolar (aM) level. Furthermore, the correlation coefficient between detection results of real whole-genome samples and digital PCR reached 0.912, validating the reliability of this strategy. In summary, this strategy provides a highly valuable reference for the direct detection of pathogen nucleic acids.},
}
@article {pmid40886437,
year = {2025},
author = {Xu, Q and Wang, X and Gu, Z and Duan, N and Jiang, S and Wu, S and Yuan, W},
title = {An electrochemical/colorimetric sensor for 5-methyltetrahydrofolate based on a screened specific aptamer and DNA walker-driven CRISPR/Cas12a cascade amplification.},
journal = {Biosensors &amp; bioelectronics},
volume = {289},
number = {},
pages = {117931},
doi = {10.1016/j.bios.2025.117931},
pmid = {40886437},
issn = {1873-4235},
mesh = {*Biosensing Techniques/methods ; *Colorimetry/methods ; *Aptamers, Nucleotide/chemistry ; Electrochemical Techniques/methods ; Humans ; Limit of Detection ; CRISPR-Cas Systems/genetics ; *Tetrahydrofolates/analysis/isolation &amp; purification/chemistry ; Gold/chemistry ; DNA, Single-Stranded/chemistry ; Endodeoxyribonucleases/chemistry/genetics ; Bacterial Proteins ; CRISPR-Associated Proteins ; },
abstract = {5-Methyltetrahydrofolate (5-MTHF), the primary bioactive form of folate (vitamin B9), played a vital role in human metabolism. In this work, an electrochemical/colorimetric dual-mode aptasensor for 5-MTHF was constructed by combining a DNA Walker-driven CRISPR-Cas12a trans-cleavage system. A 5-MTHF aptamer D1a was obtained through Capture-SELEX with subsequent trimming of non-binding regions, which exhibiting high affinity and specificity. CuMOF@CuO@RuO2@IrO2, serving as a multifunctional indicator with high conductivity and peroxidase-like activity, was immobilized on a gold electrode (AuE) via ssDNA. The DNA Walker and nicking enzyme (Nt.BbvCI) continuously released Cas12a activators, triggering Cas12a-mediated cleavage of ssDNA on the AuE, leading to reduced electrochemical signals. The released materials were collected for colorimetric detection. The detection limits were determined to be 0.044 ng/mL (electrochemical) and 0.114 ng/mL (colorimetric). This dual-mode aptasensor demonstrated high selectivity, sensitivity, and stability for 5-MTHF detection, establishing a novel sensing platform for 5-MTHF analysis in food samples. The proposed aptasensor exhibited excellent sensitivity, selectivity, and reproducibility. This strategy offered a promising platform for 5-MTHF analysis in nutritional evaluation and dietary supplementation guidance.},
}
@article {pmid40886435,
year = {2025},
author = {He, X and Liang, J and Zhang, J and Fang, W and Liu, J and Zhang, M and Wang, L and Song, C},
title = {CRISPR/Cas13a triggered-DNA walker amplified SERS sensor for ultrasensitive detection of cancer-related exosomal miRNA.},
journal = {Biosensors &amp; bioelectronics},
volume = {289},
number = {},
pages = {117924},
doi = {10.1016/j.bios.2025.117924},
pmid = {40886435},
issn = {1873-4235},
mesh = {Humans ; *MicroRNAs/isolation &amp; purification/genetics/blood ; *Biosensing Techniques/methods ; *Exosomes/chemistry/genetics ; Limit of Detection ; *Spectrum Analysis, Raman/methods ; CRISPR-Cas Systems/genetics ; *Neoplasms/genetics/diagnosis/blood ; DNA/chemistry ; DNA, Catalytic/chemistry ; },
abstract = {Accurate quantification of cancer-related miRNA in exosomes offers a promising approach for early and effective cancer diagnosis. However, reliably detecting extremely low-abundance exosomal miRNAs in complex bodily fluids remains a significant challenge. Herein, a CRISPR/Cas13a triggered-DNA walker amplified SERS sensor has been proposed for detection of cancer cell-derived exosomal miRNA-106a. The sensor comprises three main components: SERS Tags, SERS capture substrates and the CRISPR/Cas13a system. In the presence of miRNA-106a, the CRISPR/Cas13a trans-cleavage is activated, and the cleavage product further enables the DNAzymes to 'walk' on the SERS Tags, leading to the linking of the SERS Tags to a SERS capture substrate, ultimately generating significantly amplified SERS signal. The proposed SERS sensor exhibits good detection capability for miRNA-106a, i.e., rapid detection time within 80 min, wide linear response ranging from 100 aM to 1 nM, limit of detection (LOD) as low as 53.16 aM, good specificity, excellent reproducibility, and satisfactory recovery rates in human serum. Furthermore, the exosomal miRNA-106a derived from gastric cancer cells were detected and a LOD as low as 6.1 × 10[3] particles/mL of exosome was achieved, and the sensing results of the clinical serum samples underscore the potential of accurately differentiate between cancer patients and healthy individuals via analyzing cancer-related exosomal miRNAs, which indicates that the proposed SERS sensor can be a powerful tool for exosomal miRNA detection and holds good potential for precise liquid biopsy of tumor-derived exosomes.},
}
@article {pmid40882515,
year = {2025},
author = {Li, QN and Huang, HR and Li, RY and Hou, XY and Yang, QF and Jiang, HX and Cai, QL and Kong, DM},
title = {Position-independent single-nucleotide polymorphism discrimination by CRISPR/Cas12a via rational activator strand engineering.},
journal = {Biosensors &amp; bioelectronics},
volume = {289},
number = {},
pages = {117929},
doi = {10.1016/j.bios.2025.117929},
pmid = {40882515},
issn = {1873-4235},
mesh = {*Polymorphism, Single Nucleotide/genetics ; *CRISPR-Cas Systems/genetics ; *Biosensing Techniques/methods ; Humans ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {Single-nucleotide polymorphisms (SNPs) are critical biomarkers for disease diagnosis and genetic research, yet their sensitive and specific detection remains challenging. Here, we report a rational activator strand design strategy that significantly enhances the SNP discrimination capability of CRISPR/Cas12a-based biosensing systems. By systematically optimizing the length of the crRNA-complementary region and the architecture of the 3'-terminal random extension sequence, we developed an engineered CRISPR/Cas12a platform capable of discrimination SNPs with single-nucleotide resolution, regardless of mutation position. Our optimized activator strand (ssAS13+3-X) leverages the "RESET" effect (random extending sequences enhance trans-cleavage activity) enables simple one-pot detection of low-abundance mutations (0.1 %) without target pre-amplification, offering significant advantages over conventional SNP detection methods in clinical settings. The single-stranded flexibility and length tolerance of the 3'-terminal extension further ensure broad applicability across diverse genomic contexts. This work not only deepens our fundamental understanding of CRISPR/Cas12a regulation, but also provides a versatile and streamlined platform for applications in molecular diagnostics, pathogen surveillance, and precision medicine.},
}
@article {pmid40865357,
year = {2025},
author = {Wang, H and Wang, Z and Wu, M and Chen, M and He, X and Deng, L and Huang, J and Lin, R and Mao, X and Li, Q and Sheng, Y},
title = {CrispHunter-enabled systematic crRNA design and structure-optimized hairpin probes for enhanced CRISPR-Cas12a detection of Burkholderia pseudomallei.},
journal = {Biosensors &amp; bioelectronics},
volume = {289},
number = {},
pages = {117912},
doi = {10.1016/j.bios.2025.117912},
pmid = {40865357},
issn = {1873-4235},
mesh = {*Burkholderia pseudomallei/genetics/isolation &amp; purification/pathogenicity ; *CRISPR-Cas Systems/genetics ; *Biosensing Techniques/methods ; Humans ; Limit of Detection ; *Melioidosis/diagnosis/microbiology ; *RNA, Bacterial/genetics ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {Effective control of genetically diverse pathogens necessitates rapid and accurate diagnostics, as their genomic variability undermines assay reliability and complicates public health interventions. Herein, we introduce a CRISPR-Cas12a-based molecular diagnostic platform integrating robust in silico crRNA design via our CrispHunter pipeline with a high-performance detection module featuring structure-optimized hairpin probes. We validated this approach using Burkholderia pseudomallei, whose high pathogenicity, genetic diversity, and diagnostic evasion exemplify the challenges of detecting heterogeneous pathogens. Applying CrispHunter to 3245 publicly available B. pseudomallei genomes, we systematically identified 12 highly conserved crRNA candidates and optimized a penta-crRNA strategy that reduced the limit of detection (LOD) from the previously reported 100 pM to 7.5 pM, thereby minimizing the risk of false negatives due to pathogen genomic diversity. Engineered hairpin-structured molecular beacons with iteratively optimized loop and stem lengths further enhanced assay performance, lowering the LOD to 2.1 pM without pre-amplification while improving signal-to-noise ratio by 42-fold, response sensitivity by 47-fold, and reducing time-to-positive from 56 to 3 min with excellent specificity. Employing recombinase polymerase amplification further enhanced sensitivity, lowering the LOD to 14.82 copies/μL, which is significantly below the concentrations typically found in clinical specimens. The modular architecture of CrispHunter, together with the optimized CRISPR-Cas12a detection platform, provides a versatile framework for rapid, sensitive, and specific molecular diagnostics. This strategy enhances detection of B. pseudomallei and can be readily adapted for the diagnosis of other genetically diverse pathogens, supporting broader applications in infectious disease management and public health.},
}
@article {pmid40848338,
year = {2025},
author = {Hiniduma, K and De Silva, PIT and Canete, R and Vora, P and Gunathillaka, H and Clement, O and Shawky, SM and Rouge, JL and Mosa, IM and Steffens, DC and Manning, K and Breno, D and Rusling, JF},
title = {ECL-CRISPR array for multiplexed detection of miRNAs.},
journal = {Biosensors &amp; bioelectronics},
volume = {289},
number = {},
pages = {117855},
doi = {10.1016/j.bios.2025.117855},
pmid = {40848338},
issn = {1873-4235},
mesh = {*MicroRNAs/genetics/isolation &amp; purification/blood ; Humans ; *Biosensing Techniques/methods ; *Electrochemical Techniques/methods ; Limit of Detection ; *Luminescent Measurements/methods/instrumentation ; *CRISPR-Cas Systems/genetics ; *Alzheimer Disease/genetics/blood/diagnosis ; Equipment Design ; },
abstract = {We describe here an electrochemiluminescent (ECL) array for individually detecting 3 miRNAs utilizing CRISPR/Cas13a. Detection involves binding a target miRNA to Cas 13a protein that includes the RNA complement to the target, This activated Cas13a then cleaves a poly-RNA rich in r-Guanosine to produce electrochemiluminescent (ECL) activators that increases ECL output proportional to target miRNA concentration. Specifically, poly-r-guanosine (poly-r-G) is cleaved by the collateral RNase activity of Cas13a to generate small poly-r-G fragments that are efficient in activating ECL of (bis-2,2'-bipyridyl) ruthenium polyvinylpyridine ([Ru(bpy)2PVP10] (ClO4)2) (RuPVP) films on sensor electrodes at +1.1 V vs. Ag/AgCl. The 3D-printed array was used to detect three Alzheimer's disease (ALZ) miRNA biomarkers (30e-5p, 34c-3p and 200c-5p). ECL is generated in the 3D-printed array designed with reference, counter and four separate RuPVP sensor electrodes. Detection limits for miRNAs were 7.4 fg/mL to 7 pg/mL with high sensitivities in linear dynamic ranges from 70 pg/mL to 70 μg/mL. Limits of detection (LOD) were 42 pg/mL, 0.074 fg/mL, and 0.15 fg/mL for miR30e-5p, miR34c-5p, and miR200c-3p, respectively. Spike recovery studies and patient plasma analyses after RNA extraction gave high accuracy and specificity, and excellent correlation with a referee CRISPR fluorescence method.},
}
@article {pmid40834995,
year = {2025},
author = {De Marco, F and Sebastian, IR and Napoleone, A and Molin, A and Riedl, M and Bydlinski, N and Motheramgari, K and Hussein, MK and Kramer, L and Kelly, T and Jostock, T and Borth, N},
title = {A genome-scale CRISPR deletion screen in Chinese hamster ovary cells reveals essential regions of the coding and non-coding genome.},
journal = {Metabolic engineering},
volume = {92},
number = {},
pages = {194-207},
doi = {10.1016/j.ymben.2025.08.007},
pmid = {40834995},
issn = {1096-7184},
mesh = {Animals ; CHO Cells ; Cricetulus ; *Genome ; *CRISPR-Cas Systems ; Cricetinae ; *Gene Deletion ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; *Sequence Deletion ; },
abstract = {The biopharmaceutical sector relies on CHO cells to investigate biological processes and as the preferred host for production of biotherapeutics. Simultaneously, advancements in CHO cell genome assembly have provided insights for developing sophisticated genetic engineering strategies. While the majority of these efforts have focused on coding genes, with some interest in transcribed non-coding RNAs (e.g., microRNAs and lncRNAs), there remains a lack of genome-wide systematic studies that precisely examine the remaining 90 % of the genome and its impact on cellular phenotypes. This unannotated "dark matter" includes regulatory elements and other poorly characterized genomic features that may be potentially critical for cell behaviour. In this study, we deployed a genome-scale CRISPR screening platform with 112,272 paired guide RNAs targeting 14,034 genomic regions for complete deletion of 150 kb long sections. This platform enabled the execution of a negative screen that selectively identified dying cells to determine regions essential for cell survival. By using paired gRNAs, we overcame the intrinsic limitations of traditional frameshift strategies, which will likely have little or no effect on the non-coding genome. This study revealed 427 regions essential for CHO cell survival, many of which currently lack gene annotation or known functions. For these regions, we present their annotation status, transcriptional activity and annotated chromatin states. Selected regions, particularly those lacking all of the above, were individually deleted to confirm their essentiality. This work sheds a novel light on a substantial portion of the mammalian genome that has been traditionally difficult to investigate and therefore neglected. Notably, the fact that the deletion of some of these regions is lethal to cells suggests they encode critical regulatory functions. A better genome-wide understanding of these functions could open new avenues for engineering cells with improved bioprocess relevant properties.},
}
@article {pmid40803011,
year = {2025},
author = {Jiang, Y and Wu, J and Xiang, X and Wei, J and Cheng, K and Cui, L and Xu, H and Li, Z},
title = {A cleavage-gated terminal exposure-driven CRISPR-RCA self-amplifying system for ultra-fast DNA detection.},
journal = {Biosensors &amp; bioelectronics},
volume = {289},
number = {},
pages = {117857},
doi = {10.1016/j.bios.2025.117857},
pmid = {40803011},
issn = {1873-4235},
mesh = {*Biosensing Techniques/methods ; *DNA, Viral/genetics/isolation &amp; purification/analysis ; *Nucleic Acid Amplification Techniques/methods ; *CRISPR-Cas Systems/genetics ; Limit of Detection ; Humans ; CRISPR-Associated Proteins/chemistry ; Endodeoxyribonucleases/chemistry ; Bacterial Proteins ; },
abstract = {The one-pot detection technology based on nucleic acid isothermal amplification combined with CRISPR has a significant advantage in on-site detection of infectious diseases. It is superior to quantitative polymerase chain reaction (qPCR) due to its lack of temperature variation and significantly faster reaction speed. Nevertheless, Cas proteins compete with amplification enzymes for reaction substrates, which results in the signal amplification effect being less than expected. To overcome this limitation, we have developed a single-tube self-amplifying system driven by cleavage-gated terminal exposure based on CRISPR and rolling circle amplification (AURORA), enabling ultra-fast and sensitive monkeypox virus (MPXV) detection. This method innovatively designs a dual-function probe (DF probe). In the presence of the target, the trans-cleavage activity of Cas12a is activated, only cleaving the single-stranded DNA to expose the 3' terminal of the DF probe. The cleaved DF probe hybridizes with the circular DNA template and is bound by phi29 DNA polymerase to initiate RCA. Here, we utilize the characteristics of Cas12a and phi29 DNA polymerase acting on substrates in different strand states to avoid substrate competition between the two enzymes in a single-tube reaction. This assay can achieve ultra-fast signal amplification of MPXV DNA within 8 min, with a limit of detection (LOD) of 88 aM (53 copies/μL). Combining the viral nucleic acid thermal lysis method, it is possible to achieve results from sample input to output in 10 min. The AURORA detection strategy was further used to detect MPXV in clinical samples (36 MPXV samples), and the results were completely consistent with qPCR. The AURORA system features ultra-fast and precise detection, providing a more efficient tool for the prevention and control of severe infectious diseases.},
}
@article {pmid40752104,
year = {2025},
author = {Pavlikova, L and Krepop, J and Sulova, Z and Breier, A and Seres, M},
title = {Differential UVC radiation sensitivity in multidrug-resistant l1210 cells: Insights into p53 and Bcl-XL expression/function.},
journal = {Journal of photochemistry and photobiology. B, Biology},
volume = {271},
number = {},
pages = {113229},
doi = {10.1016/j.jphotobiol.2025.113229},
pmid = {40752104},
issn = {1873-2682},
mesh = {*Ultraviolet Rays ; *Tumor Suppressor Protein p53/genetics/metabolism ; *bcl-X Protein/metabolism/genetics ; Animals ; Mice ; Cell Line, Tumor ; *Drug Resistance, Multiple/radiation effects ; Drug Resistance, Neoplasm/radiation effects ; Doxorubicin/pharmacology ; DNA Damage/radiation effects ; Vincristine/pharmacology ; Apoptosis/radiation effects/drug effects ; CRISPR-Cas Systems ; Radiation Tolerance ; },
abstract = {Multidrug-resistant (MDR) variants of L1210 cells, selected for resistance to vincristine (R) or doxorubicin (D), exhibit elevated ABCB1 (P-glycoprotein) expression but differ in UVC sensitivity. D cells resemble parental L1210 (S) cells, whereas R cells are significantly more UVC vulnerable. To investigate this, we analyzed the expression of genes involved in DNA damage response, Trp53 family members, cyclin-dependent kinase inhibitors (p15, p21), Bcl-2 family genes, and DNA repair genes in S, R, and D cells before and after UVC irradiation. The most striking difference was the absence of Trp53 expression in R cells at both mRNA and protein levels, while S and D cells expressed this gene. Instead, R cells uniquely expressed Trp63. CRISPR/Cas9-generated p53-null mutants of S and D cells showed increased UVC-induced cell death, but their sensitivity did not reach that of R cells. Bcl-XL, a protein linked to resistance against UVC-induced apoptosis, was also reduced at both transcript and protein levels in R cells. Pharmacological inhibition of Bcl-XL in S and D cells with A-1155463 and A-1331852 enhanced UVC-induced cell death but did not replicate the high sensitivity observed in R cells. Our findings suggest that the heightened UVC sensitivity of R cells results from a combined deficiency of p53 and Bcl-XL, impairing DNA damage response and apoptosis. These results reveal distinct molecular adaptations in MDR variants and provide insight into the mechanisms underlying differential UVC sensitivity.},
}
@article {pmid40835108,
year = {2025},
author = {Shan, X and Zhang, X and Tao, B and Song, Y and Zhu, Z and Hu, W and Chen, J},
title = {CRISPR/Cas9-mediated editing of ptprfb (protein tyrosine phosphatase receptor type fb) reveals its regulatory role in zebrafish spermatogenesis.},
journal = {Comparative biochemistry and physiology. Part B, Biochemistry &amp; molecular biology},
volume = {280},
number = {},
pages = {111146},
doi = {10.1016/j.cbpb.2025.111146},
pmid = {40835108},
issn = {1879-1107},
mesh = {Animals ; *Zebrafish/genetics/physiology ; Male ; *CRISPR-Cas Systems ; *Spermatogenesis/genetics ; *Gene Editing ; *Zebrafish Proteins/genetics/metabolism ; Spermatozoa/metabolism ; },
abstract = {Gonadal development and gamete maturation are essential for fish reproduction. The protein tyrosine phosphatase receptor type Fb (Ptprfb) is a member of the tyrosine phosphatase family. In the present study, we used CRISPR/Cas9 to mutate ptprfb in zebrafish. A significantly reduced natural fertilization rate of sperm from mutant fish was observed. The mutant fish produced fewer sperm with shorter flagella, and a smaller proportion of sperm could be activated. RNA-seq analysis revealed abnormal expression of some genes in testicular cells, such as hemoglobin subunit βA1 (hbba1), myosin heavy chain 11b (myh11b), and transgelin (tagln), as well as some genes involved in focal adhesion formation. These findings demonstrate that ptprfb contributes to spermatogenesis in zebrafish, and its dysfunction can adversely affect both the quantity and quality of sperm.},
}
@article {pmid40190036,
year = {2025},
author = {Wang, J and Xu, K and Liu, T and Zhao, H and Jamal, MA and Chen, G and Huo, X and Yang, C and Jiao, D and Wei, T and Huang, H and Zhao, H and Guo, J and Wang, F and Zhang, X and Liu, K and Qu, S and Wang, G and Guo, H and Chen, G and Zhao, HY and Zeng, Z and Dou, K and Wei, HJ},
title = {Production and Functional Verification of 8-Gene (GGTA1, CMAH, β4GalNT2, hCD46, hCD55, hCD59, hTBM, hCD39)-Edited Donor Pigs for Xenotransplantation.},
journal = {Cell proliferation},
volume = {58},
number = {9},
pages = {e70028},
doi = {10.1111/cpr.70028},
pmid = {40190036},
issn = {1365-2184},
support = {202102AA310047//Major Science and Technology Project of Yunnan Province/ ; 2019YFA0110700//National Key Research and Development Program of China/ ; 2023-JCJQ-ZD-118-00//173 Basic Strengthening Program/ ; },
mesh = {Animals ; *Transplantation, Heterologous/methods ; *Gene Editing/methods ; Swine ; Humans ; CRISPR-Cas Systems/genetics ; Galactosyltransferases/genetics ; Animals, Genetically Modified ; CD59 Antigens/genetics ; N-Acetylgalactosaminyltransferases/genetics ; Membrane Cofactor Protein/genetics ; Mixed Function Oxygenases ; },
abstract = {Gene-edited (GE) pig-to-human xenotransplantation continues to make breakthroughs, but which kind of gene combination is suitable for organ-specific transplantation remains unclear. In this study, we utilised CRISPR/Cas9 gene editing technology, PiggyBac transposon system, and serial somatic cell cloning technology to develop GTKO/CMAHKO/β4GalNT2KO/hCD46/hCD55/hCD59/hCD39/hTBM 8 gene-edited cloned (GEC) donor pigs and performed pig-to-non-human primate (NHP) transplantation to evaluate the effectiveness of these GEC pigs. The 8-GEC pigs were obtained by recloning with a 33-day-old 8-GEC fetus with O blood type, which was generated after cell transfection, screening of cell colonies, and somatic cell cloning. Molecular identification at DNA, mRNA, and protein levels confirmed successful 8-gene editing. Three copies of transgenes were identified by droplet digital polymerase chain reaction and whole genome sequencing, which were inserted into the introns of pig RFTN1 and MYO10 genes, as well as the intergenic region between PRLR and LOC110257300 genes of these 8-GEC pigs. The 8-GEC pigs also exhibited the ability of germline transmission when mated with our previously generated 4-GEC male pigs. Moreover, antigen-antibody binding assay and complement-dependent cytotoxicity assay demonstrated that 8-gene editing effectively reduced the immune incompatibility and kidney xenograft from 8-GEC pigs survived for 15 and 17 days in two NHPs, respectively. Postoperatively, the recipient serum antibodies IgA, IgG and IgM, complements C3 and C4, coagulation indicators PT, APTT, TT and FIB, as well as most electrolytes and liver function indicators remained relatively stable. Serum creatinine was normal within 10 days post operation. However, the kidney xenograft developed active antibody-mediated rejection at necropsy, characterised by the deposition of antibodies IgG and IgM, as well as complements C4d, C3c and C5b-C9, infiltration of CD68[+] macrophages, and micro-thrombotic embolism of glomerular capillaries, etc. In conclusion, we successfully developed fertile 8-GEC pigs, which effectively alleviated immune rejection and exerted life-supporting kidney function in the recipients.},
}
@article {pmid40909725,
year = {2025},
author = {Hong, A and Liu, M and Truta, A and Talaie, A and Smith, GR and Bondy-Denomy, J},
title = {Gabija restricts phages that antagonize a conserved host DNA repair complex.},
journal = {bioRxiv : the preprint server for biology},
volume = {},
number = {},
pages = {},
doi = {10.1101/2025.08.30.673261},
pmid = {40909725},
issn = {2692-8205},
abstract = {Anti-bacteriophage systems like restriction-modification and CRISPR-Cas have DNA substrate specificity mechanisms that enable identification of invaders. How Gabija, a highly prevalent nuclease-helicase anti-phage system, executes self- vs. non-self-discrimination remains unknown. Here, we propose that phage-encoded DNA end-binding proteins that antagonize host RecBCD sensitize phages to Gabija. When targeting temperate phage D3 in Pseudomonas aeruginosa, Gabija functions early by preventing phage genome circularization in a non-abortive manner. Phage and plasmid DNA-end sensitivity to Gabija is licensed by a phage exonuclease and ssDNA-annealing protein. Unrelated F8 and JBD30 phages are sensitized to Gabija by Gam_Mu, a distinct DNA end-binding protein that antagonizes loading of the host repair complex RecBCD. Escape phages lacking these end-binding proteins become protected from Gabija by RecBCD activities, which also prevent Gabija from targeting self-DNA. Therefore, we propose that Gabija antagonizes circularization of linear DNA devoid of RecBCD as a mechanism to identify foreign invaders.},
}
@article {pmid40543483,
year = {2026},
author = {Wang, H and Yan, Y and Codjia, CR and Dan, Y and Li, L and He, S and Yang, X and Chen, L and Liu, H and Wang, X},
title = {CRISPR-Cas13a coupled with recombinase aided amplification: Development of an ultrasensitive point - of - care detection method for visual diagnosis of egg drop syndrome virus.},
journal = {Talanta},
volume = {296},
number = {},
pages = {128470},
doi = {10.1016/j.talanta.2025.128470},
pmid = {40543483},
issn = {1873-3573},
mesh = {Animals ; *CRISPR-Cas Systems/genetics ; *Recombinases/metabolism ; *Nucleic Acid Amplification Techniques/methods ; *Atadenovirus/isolation &amp; purification/genetics ; *Poultry Diseases/diagnosis/virology ; Chickens/virology ; *Point-of-Care Systems ; Limit of Detection ; },
abstract = {Egg Drop Syndrome Virus (EDSV) infection in poultry causes a significant drop in egg - laying rate, harming poultry farms' economic benefits. A sensitive and specific EDSV detection method is urgently needed in clinical practice. The CRISPR-Cas13a system can detect various targets specifically, and recombinase-aided amplification (RAA) can rapidly amplify nucleic acids. Therefore, in this experiment, the RAA was integrated with the CRISPR-Cas13a system to develop a novel, visual, ultrasensitive and point - of - care detection method for EDSV. In this experiment, the reaction system was optimized, and its sensitivity, specificity, repeatability, and clinical sample validation were conducted and evaluated. The results showed that the optimal concentration of the obtained Cas13a protein was 2.4 mg/mL, and that of crRNA 1 was 100 μg/μL. This method not only showed rapid detection (30-50 min), very high sensitivity, with the detection limit reaching 1 copy/μL, but also showed good specificity with no cross - reaction to Marek's Disease Virus (MDV), Infectious Laryngotracheitis Virus (ILTV), Avian Leukosis Virus (ALV), Chicken Anemia Virus (CAV), Astrovirus (AstV), H9N2 subtype of Avian Influenza Virus (H9N2 AIV), Fowl Adenovirus serotype 4 (FAdV-4), Fowl Adenovirus serotype 8 (FAdV-8) and Fowl Adenovirus serotype 11 (FAdV-11). Furthermore, the method displayed excellent repeatability, with the coefficient of variation of both intra - group and inter - group no more than 4 %. Evaluation of this method through 210 clinical samples found that compared with the traditional polymerase chain reaction (PCR) which is the industry standard, its positive coincidence rate was 100 %, the negative coincidence rate was 98.35 %, the overall coincidence rate was 98.57 %, and the kappa value was 0.94. This assay provides a potential point - of - care testing approach for the clinical detection, virology, and epidemiological studies of EDSV.},
}
@article {pmid40499384,
year = {2026},
author = {Yang, Y and Ji, X and Zhou, F and He, Z},
title = {A one-pot assay based on PAM-free recombinase polymerase amplification and CRISPR/Cas12a for rapid detection of SARS-CoV-2 N gene.},
journal = {Talanta},
volume = {296},
number = {},
pages = {128448},
doi = {10.1016/j.talanta.2025.128448},
pmid = {40499384},
issn = {1873-3573},
mesh = {*SARS-CoV-2/genetics/isolation &amp; purification ; *CRISPR-Cas Systems/genetics ; *Nucleic Acid Amplification Techniques/methods ; Humans ; *Recombinases/metabolism/genetics ; *COVID-19/diagnosis/virology ; CRISPR-Associated Proteins/metabolism ; *Phosphoproteins/genetics ; Endodeoxyribonucleases/metabolism ; Coronavirus Nucleocapsid Proteins ; Bacterial Proteins ; },
abstract = {SARS-CoV-2 is a pathogenic virus, which exhibits high contagiousness. Therefore, a rapid and sensitive SARS-CoV-2 detection strategy is imperative. Herein, a one-pot assay by the combination of protospacer adjacent motif (PAM)-free recombinase polymerase amplification with CRISPR/Cas12a for detecting SARS-CoV-2 N gene was reported. To avoid the constraint of the PAM site for double-stranded DNA (dsDNA) in CRISPR/Cas12a system, we designed two individual crRNAs to hybridize with two different regions of the target sequence. The presence of N gene DNA was able to initiate the amplification of RPA, exposing the recognition site of crRNA and activating the Cas12a. Whereafter, the Cas12a activation resulted in the digestion of nontarget DNA reporters to induce significant fluorescence signal. The assay completed the detection of N gene DNA within 30 min. And a high sensitivity of 100 aM was obtained because of RPA amplification and Cas12a trans cleavage activity. Meanwhile, the proposed assay showed excellent specificity due to the site-specific recognition ability of CRISPR/Cas12a. More importantly, analysis of spiked samples verified the excellent practical application of the proposed method. Thus, the assay earned promising potential in molecular diagnostics.},
}
@article {pmid40909132,
year = {2025},
author = {Biswas, I},
title = {Ethical dimensions and societal implications: ensuring the social responsibility of CRISPR technology.},
journal = {Frontiers in genome editing},
volume = {7},
number = {},
pages = {1593172},
pmid = {40909132},
issn = {2673-3439},
abstract = {CRISPR-Cas9 is a breakthrough genome-editing platform that can cut chosen DNA sequences with unprecedented speed, accuracy, and affordability. By reprogramming a single guide RNA, researchers now alter gene function, correct pathogenic variants, or introduce novel traits. Earlier tools such as zinc-finger nucleases and TALENs performed similar tasks but were significantly more complex and costly. Yet CRISPR's very power raises urgent ethical concerns: Who controls its use, and how can society prevent germ-line enhancement, eugenic selection, or unequal access that favors wealthy nations and patients? A well-publicized case of embryo editing already showed how premature, unregulated experiments can erode public trust. This perspective therefore frames CRISPR's scientific promise alongside its social responsibilities, arguing that proactive, globally coordinated governance is essential to unlock benefits while preventing new forms of genetic inequality.},
}
@article {pmid40907709,
year = {2025},
author = {Chugh, P and Soni, S and Ghanghas, N and Kumar, S and Mohan, H},
title = {Comprehensive insights into Japanese encephalitis virus: From molecular characterization to advanced detection and vaccine strategies.},
journal = {Antiviral research},
volume = {},
number = {},
pages = {106268},
doi = {10.1016/j.antiviral.2025.106268},
pmid = {40907709},
issn = {1872-9096},
abstract = {The Japanese encephalitis virus (JEV) remains a major cause of viral encephalitis in Asia, with significant morbidity and mortality. This review offers a comprehensive overview of the current landscape of JEV research, focusing on its genomic structure, protein composition, and global epidemiology. We highlight the complexity of JEV transmission and pathogenesis, examining the interplay of demographic factors and geographic spread. In particular, we assess the evolution of diagnostic methodologies from traditional molecular and serological techniques to emerging biosensor-based approaches, emphasizing advancements in sensitivity and rapidity. The application of CRISPR/Cas systems for JEV detection marks a promising frontier in molecular diagnostics. Additionally, we review the current status of JEV vaccines, discussing recent innovations in vaccine development aimed at enhancing immunogenicity and accessibility. Beyond prevention, a spectrum of antiviral strategies-including direct-acting antivirals, entry inhibitors, host-directed modulators, neuroprotective agents, and steroidal/synthetic compounds-has demonstrated potent in vitro and in vivo efficacy, targeting viral enzymes, structural proteins, and host pathways. This review underscores the critical role of advanced detection strategies and vaccines in controlling JEV, offering insights into ongoing efforts to mitigate its impact in endemic regions.},
}
@article {pmid40906807,
year = {2025},
author = {Han, Z and Huang, C and Luo, T and Mirkin, CA},
title = {A general genome editing strategy using CRISPR lipid nanoparticle spherical nucleic acids.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {122},
number = {36},
pages = {e2426094122},
doi = {10.1073/pnas.2426094122},
pmid = {40906807},
issn = {1091-6490},
support = {FA9550-22-1-0300//DOD | AF | AMC | AFRL | Air Force Office of Scientific Research (AFOSR)/ ; DMR-2428112//National Science Foundation (NSF)/ ; DMR-2308691//National Science Foundation (NSF)/ ; NSF ECCS-2025633//National Science Foundation (NSF)/ ; },
mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; *Nanoparticles/chemistry ; Humans ; *Lipids/chemistry ; RNA, Guide, CRISPR-Cas Systems/genetics ; *Nucleic Acids/chemistry/genetics ; HEK293 Cells ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Transfection ; Liposomes ; },
abstract = {Genome editing with CRISPR-Cas systems hold promise for treating a wide range of genetic disorders and cancers. However, efficient delivery of genome editors remains challenging due to the requirement for the simultaneous delivery or intracellular generation of Cas proteins, guide RNAs, and, in some applications, donor DNAs. Furthermore, the immunogenicity and toxicity of delivery vehicles can limit the safety and efficacy of genetic medicines. Here, we combine two nucleic acid delivery approaches to create CRISPR lipid nanoparticle-spherical nucleic acids (LNP-SNAs) that are both efficient and biocompatible. Compared to lipid nanoparticles (LNPs) lacking a surface-bound DNA shell, CRISPR LNP-SNAs exhibit two- to three-fold higher cellular uptake, reduced cytotoxicity, and improved gene transfection efficiency. Across multiple cell lines and genomic loci, CRISPR LNP-SNAs induce insertion-deletion mutations at average frequencies two- to three-fold higher than those observed with LNPs. When codelivered with donor templates, CRISPR LNP-SNAs enable homology-directed repair at an average efficiency of 21 ± 7%, a 2.5-fold improvement over LNPs (8 ± 4%). The ease of synthesis and biocompatibility of CRISPR LNP-SNAs highlight their potential as a versatile delivery platform for CRISPR-Cas and other gene therapies.},
}
@article {pmid40906401,
year = {2025},
author = {Luo, J and Lu, W and Liu, R and Zhang, S and Cao, J and Ma, C},
title = {From Panels to Pathogen Networks: The Expanding Role of Targeted Sequencing in Veterinary Medicine.},
journal = {Biology},
volume = {14},
number = {8},
pages = {},
pmid = {40906401},
issn = {2079-7737},
abstract = {Targeted sequencing, a pivotal branch of next-generation sequencing (NGS), enables the selective enrichment of specific genomic regions and has demonstrated significant advantages in the detection of animal pathogens. This review systematically explores the underlying principles of targeted sequencing, various enrichment strategies-including PCR amplification, probe hybridization, and CRISPR-Cas systems-and their key applications in veterinary pathogen diagnostics. Due to its high throughput, sensitivity, and cost-effectiveness, targeted sequencing has been successfully applied in the multiplex detection of pathogens in economically significant livestock, such as cattle, as well as in the surveillance of antimicrobial resistance (AMR) genes, pathogen typing, and source tracing. It is particularly effective in identifying mixed infections and low-abundance pathogens. Nonetheless, wide application is restricted by some factors, like incomprehensive reference databases, cost-effectiveness, and limited application in primary-level laboratories. Further development directions are AI-based panel design, multimodal diagnostic platform integration, standard workflow construction, and introduction of a multi-omics method. Such progress focuses on enhancing the targeted sequencing scalability and precision consistent with the "One Health" initiative objective.},
}
@article {pmid40905590,
year = {2025},
author = {Cheng, L},
title = {Topology-Engineered Guide RNAs for Programmable Control of CRISPR/Cas Activity.},
journal = {Angewandte Chemie (International ed. in English)},
volume = {},
number = {},
pages = {e202511756},
doi = {10.1002/anie.202511756},
pmid = {40905590},
issn = {1521-3773},
support = {2025YFA0920900//National Key R&amp;D Program of China/ ; XDB0960103//Strategic Priority Research Program of the Chinese Academy of Sciences/ ; BNLMS-CXTD-202401//Beijing National Laboratory for Molecular Sciences/ ; 22537005//National Natural Science Foundation of China/ ; },
abstract = {CRISPR/Cas systems have transformed genome editing, yet achieving precise temporal and conditional control remains challenging. Traditional strategies involving linear guide RNAs (gRNAs) modified with multiple chemical groups throughout their strands often face limitations such as heterogeneous reaction outcomes, irreversibility, and variable editing efficiencies. To overcome these issues, topology-engineered guide RNAs (TE-gRNAs) have emerged, featuring defined structural architectures including polymeric, circular, and dendrimer-like topologies that enable precise spatial control, reversibility, and programmable activation of CRISPR activity. By selectively incorporating physical or chemically responsive linkers and stimuli-sensitive groups at specific sites, TE-gRNAs facilitate dynamic and conditional genome editing that can be activated or deactivated with external triggers such as light or chemical signals. These engineered RNA structures significantly improve synthesis feasibility, stability, reduce off-target effects, and provide unprecedented control over gene editing processes. Recent advancements in TE-gRNAs demonstrate their broad applicability in synthetic biology, functional genomics, and therapeutic interventions, highlighting their potential to achieve precise spatiotemporal modulation of CRISPR systems. This review summarizes the current strategies, benefits, and challenges associated with TE-gRNAs, and discusses future directions for enhancing their performance and utility in complex genome editing applications.},
}
@article {pmid40848498,
year = {2026},
author = {Chen, J and Shi, K},
title = {A fluorescent aptasensor for accurate and sensitive detection of glyphosate based on asymmetrically competitive CRISPR/Cas12a with phosphorothioate-modified G-quadruplex.},
journal = {Spectrochimica acta. Part A, Molecular and biomolecular spectroscopy},
volume = {345},
number = {},
pages = {126840},
doi = {10.1016/j.saa.2025.126840},
pmid = {40848498},
issn = {1873-3557},
mesh = {Glyphosate ; *Glycine/analogs &amp; derivatives/analysis ; *G-Quadruplexes ; *Aptamers, Nucleotide/chemistry/genetics ; *CRISPR-Cas Systems ; *Biosensing Techniques/methods ; Limit of Detection ; Spectrometry, Fluorescence/methods ; *Fluorescent Dyes/chemistry ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {Accurate detection of glyphosate (GLY) residues is essential for protecting public health, ensuring food safety, and preventing environmental pollution caused by excessive or improper herbicide application. Based on the asymmetric CRISPR/Cas12a system as a signal amplification technique, with phosphorothioate-modified hairpin G-quadruplex (psHG4) acting as a signal probe, a platform named ACC-GLY is developed for the sensitive and accurate detection of GLY. In the designed ACC-GLY platform, the target GLY specifically binds to the aptamer, and the cascading signal amplification strategy, driven by the DNase activity of a single Cas12a, is initiated by two competitive guide RNAs. Under the influence of Cas12a's DNase activity, the psHG4 probe is cleaved, releasing the psG4 sequence. The released psG4 sequence then binds to Thioflavin T (ThT), forming a complex that generates a fluorescence signal. Under optimal conditions, the detection platform can specifically detect GLY at a concentration as low as 0.3 pM. The detection platform demonstrates significant capability in detecting GLY in tap water and corn samples, highlighting its vast potential for applications in environmental monitoring and food safety. To the best of our knowledge, this is the first study to utilize the CRISPR/Cas12a system for GLY detection.},
}
@article {pmid40795705,
year = {2026},
author = {Li, H and Tang, X and Li, F and Yang, M and Jing, H and Li, L and Liu, J and Cao, Z and Yang, X and Wang, J},
title = {Neutrophil-inspired CRISPR/dCas9 nanomedicine to program self-destructing and bystander killing of tumor cell for selective cancer therapy.},
journal = {Biomaterials},
volume = {325},
number = {},
pages = {123619},
doi = {10.1016/j.biomaterials.2025.123619},
pmid = {40795705},
issn = {1878-5905},
mesh = {*Neutrophils/metabolism ; Humans ; Animals ; *CRISPR-Cas Systems/genetics ; *Bystander Effect ; *Nanomedicine/methods ; *Neoplasms/therapy/pathology ; Cell Line, Tumor ; Leukocyte Elastase/metabolism/genetics ; Mice ; Female ; },
abstract = {Achieving high efficiency, selectivity, and durability remains a major challenge in the development of innovative cancer treatments, as current clinical therapies often fall short. Inspired by neutrophil-mediated tumor clearance, we engineered a nanocarrier-mediated CRISPR/dCas9 system to activate endogenous expression of neutrophil elastase (ELANE) for precise cancer therapy. Although ELANE was upregulated in both normal and tumor cells, selective killing occurred only in tumor cells through a histone H1.0-dependent self-destructing mechanism. Additionally, secreted ELANE further efficiently eliminated neighboring tumor cells via bystander killing. ELANE-mediated tumor cell death also induced immunogenic responses, potently enhancing antitumor immunity and synergizing with anti-PD-L1 therapy to inhibit tumor metastasis. This study presents a novel CRISPRa-based therapeutic strategy that mimic neutrophil function to achieve potent, selective, and durable tumor eradication through combined mechanisms of self-destruction, bystander killing and immune activation.},
}
@article {pmid40904932,
year = {2025},
author = {Wang, HM and Xu, SJ and Cai, BY and Qiu, WY and Lu, H and Tang, YD},
title = {Highly efficient gene editing of Feline herpesvirus 1 using CRISPR/Cas9 combined with FACS.},
journal = {Frontiers in cellular and infection microbiology},
volume = {15},
number = {},
pages = {1660446},
pmid = {40904932},
issn = {2235-2988},
mesh = {*CRISPR-Cas Systems ; *Gene Editing/methods ; Animals ; *Varicellovirus/genetics ; Cats ; *Flow Cytometry/methods ; Thymidine Kinase/genetics ; Genes, Reporter ; Herpesviridae Infections/virology/veterinary ; Green Fluorescent Proteins/genetics ; },
abstract = {Feline herpesvirus 1 (FHV-1) is a major causative agent of feline viral rhinotracheitis and ocular lesions. Due to its large DNA genome, the construction of recombinant FHV-1 viruses presents considerable challenges for conventional methodologies. In this study, we implemented an integrated strategy combining CRISPR/Cas9-mediated gene editing with fluorescence-activated cell sorting (FACS) to enable the rapid and efficient generation of recombinant FHV-1 viruses. Specifically, the thymidine kinase (tk) gene was disrupted by inserting a monomeric Cherry (mCherry) reporter gene, and the glycoprotein E (gE) gene was similarly interrupted through the incorporation of a green fluorescent protein (GFP) reporter. The CRISPR/Cas9 system enables precise, site-specific genomic modifications, while FACS allows for effective enrichment and isolation of the desired recombinant viral populations. This combined approach significantly reduces the time required for recombinant virus generation from weeks to days, thereby offering substantial potential to expedite vaccine development and advance functional genomics research.},
}
@article {pmid40904117,
year = {2025},
author = {Koonce, KC and Mauritzen, JJ and Hitz, IF and Vangsgaard, EF and Putz, EHM and Wajn, AS and Leth, FH and Høyland-Kroghsbo, NM},
title = {The H-NS homologues MvaT and MvaU repress CRISPR-Cas in Pseudomonas aeruginosa.},
journal = {Philosophical transactions of the Royal Society of London. Series B, Biological sciences},
volume = {380},
number = {1934},
pages = {20240073},
pmid = {40904117},
issn = {1471-2970},
support = {//Danmarks Frie Forskningsfond/ ; //Lundbeck Foundation/ ; },
mesh = {*Pseudomonas aeruginosa/genetics/metabolism ; *CRISPR-Cas Systems/genetics ; *Bacterial Proteins/genetics/metabolism ; *DNA-Binding Proteins/genetics/metabolism ; Trans-Activators ; },
abstract = {CRISPR-Cas is an adaptive immune system of bacteria and archaea that protects against foreign genetic elements. In Escherichia coli and Salmonella, CRISPR-Cas is inhibited by the conserved global repressor the histone-like nucleoid structuring protein (H-NS), which blocks the expression of AT-rich horizontally acquired genes. While the opportunistic pathogen Pseudomonas aeruginosa harbours two partially redundant H-NS homologues, MvaT and MvaU, their role in CRISPR-Cas regulation in this bacterium remains unexplored. Here, we demonstrate that in the absence of both MvaT and MvaU, CRISPR-Cas activity increases more than tenfold, as measured by a reduction in the transformation efficiency of a CRISPR-targeted plasmid. Importantly, we find that in the absence of MvaT and MvaU, Cas proteins are already produced at low cell density prior to the onset of quorum sensing-mediated activation of CRISPR-Cas, which occurs at high cell density. Moreover, the ∆mvaT ∆mvaU mutant has a significantly reduced growth rate, known to independently increase CRISPR-Cas activity. In addition to regulating CRISPR-Cas, the absence of MvaT and MvaU affects phage-host interactions, including enhancing the adsorption of the LPS-binding phage JBD44, highlighting their broader role in coordinating bacterial defence mechanisms.This article is part of the discussion meeting issue 'The ecology and evolution of bacterial immune systems'.},
}
@article {pmid40904116,
year = {2025},
author = {Hoikkala, V and Chi, H and Grüschow, S and Graham, S and White, MF},
title = {Diversity and abundance of ring nucleases in type III CRISPR-Cas loci.},
journal = {Philosophical transactions of the Royal Society of London. Series B, Biological sciences},
volume = {380},
number = {1934},
pages = {20240084},
pmid = {40904116},
issn = {1471-2970},
support = {/ERC_/European Research Council/International ; },
mesh = {*CRISPR-Cas Systems/genetics ; *Bacteria/genetics/enzymology ; *Archaea/genetics/enzymology ; *Endonucleases/genetics/metabolism ; *Bacterial Proteins/genetics/metabolism ; },
abstract = {Most type III CRISPR-Cas systems facilitate immune responses against invading mobile genetic elements such as phages by generating cyclic oligoadenylates (cOAs). Downstream effectors activated by cOAs are typically non-specific proteins that induce damage to essential cellular components, thereby preventing phage epidemics. Owing to these toxic effects, it is crucial that the production and concentration of cOAs remain under tight regulatory control during infection-free periods or when deactivating the immune response after clearing an infection. Type III CRISPR loci often encode enzymes known as ring nucleases (RNs) that bind and degrade specific cOAs, while some effectors are auto-deactivating. Despite the discovery of several classes of RNs, a comprehensive bioinformatic analysis of type III CRISPR-Cas loci in this context is lacking. Here, we examined 38 742 prokaryotic genomes to provide a global overview of type III CRISPR loci, focusing on the known and predicted RNs. The candidate RNs Csx16 and Csx20 are confirmed as active enzymes, joining Crn1-3. Distributions and patterns of co-occurrence of RNs and associated effectors are explored, allowing the conclusion that a sizeable majority of type III CRISPR systems regulate cOA levels by degrading the signalling molecules, which has implications for cell fate following viral infection.This article is part of the discussion meeting issue 'The ecology and evolution of bacterial immune systems'.},
}
@article {pmid40904105,
year = {2025},
author = {Elliott, JFK and Cozens, K and Cai, Y and Waugh, G and Watson, BN and Westra, E and Taylor, TB},
title = {Phage susceptibility to a minimal, modular synthetic CRISPR-Cas system in Pseudomonas aeruginosa is nutrient dependent.},
journal = {Philosophical transactions of the Royal Society of London. Series B, Biological sciences},
volume = {380},
number = {1934},
pages = {20240473},
pmid = {40904105},
issn = {1471-2970},
support = {//UK Government's Horizon Europe funding guarantee/ ; //Royal Society/ ; //Philip Leverhulme Prize/ ; /BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; },
mesh = {*Pseudomonas aeruginosa/virology/genetics ; *CRISPR-Cas Systems ; *Pseudomonas Phages/physiology/genetics ; *Nutrients/metabolism ; *Bacteriophages/physiology ; },
abstract = {CRISPR-Cas systems can provide adaptive, heritable immunity to their prokaryotic hosts against invading genetic material such as phages. It is clear that the importance of acquiring CRISPR-Cas immunity to anti-phage defence varies across environments, but it is less clear if and how this varies across different phages. To explore this, we created a synthetic, modular version of the type I-F CRISPR-Cas system of Pseudomonas aeruginosa. We used this synthetic system to test CRISPR-Cas interference against a panel of 13 diverse phages using engineered phage-targeting spacers. We observed complete protection against eight of these phages, both lytic and lysogenic and with a range of infectivity profiles. However, for two phages, CRISPR-Cas interference was only partially protective in high-nutrient conditions, yet completely protective in low-nutrient conditions. This work demonstrates that nutrient conditions modulate the strength of CRISPR-Cas immunity and highlights the importance of environmental conditions when screening defence systems for their efficacy against various phages.This article is part of the discussion meeting issue 'The ecology and evolution of bacterial immune systems'.},
}
@article {pmid40904104,
year = {2025},
author = {David, E and Plantady, C and Poissonnier, S and Elliott, JFK and Kenck, E and Le Boulch, J and Gutierrez, A and Chevallereau, A},
title = {Systematic functional assessment of anti-phage systems in their native host.},
journal = {Philosophical transactions of the Royal Society of London. Series B, Biological sciences},
volume = {380},
number = {1934},
pages = {20240067},
pmid = {40904104},
issn = {1471-2970},
support = {//ATIP-Avenir/ ; //Agence Nationale de la Recherche/ ; //Idex Paris Cit&#xe9;/ ; //Emergence ville de Paris/ ; },
mesh = {*Escherichia coli/virology/genetics ; *Coliphages/physiology ; *Bacteriophages/physiology ; DNA Restriction-Modification Enzymes/genetics ; },
abstract = {Bacterial resistance to bacteriophages (phages) relies on two primary strategies: preventing phage attachment and blocking post-attachment steps. These post-attachment mechanisms are mediated by diverse defence systems, including DNA-degrading systems such as restriction-modification and CRISPR-Cas, along with abortive infection systems that induce cell death or dormancy. Computational analyses suggest that bacterial genomes encode multiple defence systems, which may act synergistically to enhance phage resistance. However, the regulation, interactions and ecological roles of these systems in native hosts remain poorly understood. This study explored the role of eight predicted defence systems in the clinical isolate NILS69 of Escherichia coli by testing its susceptibility to 93 phages. Infectivity and adsorption assays using mutants defective in these systems revealed that only PD-T4-3 and restriction-modification systems restricted phages that were able to adsorb. The restriction-modification system acted via a predicted type IV endonuclease and was also able to limit plasmid conjugation if the plasmid was transferred from a donor strain lacking a methylase, which is the hallmark of type I, II or III restriction-modification systems. Other defence systems showed no detectable activity, likely owing to phage specificity, environmental regulation or cofactor requirements. These findings underscore the need for further studies to investigate the regulation and ecological roles of bacterial defence systems in their native host contexts.This article is part of the discussion meeting issue 'The ecology and evolution of bacterial immune systems'.},
}
@article {pmid40904102,
year = {2025},
author = {Pons, BJ and Łapińska, U and Lopes-Domingues, I and Chisnall, MAW and Westra, ER and Pagliara, S and van Houte, S},
title = {Phage provoke growth delays and SOS response induction despite CRISPR-Cas protection.},
journal = {Philosophical transactions of the Royal Society of London. Series B, Biological sciences},
volume = {380},
number = {1934},
pages = {20240474},
pmid = {40904102},
issn = {1471-2970},
support = {/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; /MRC_/Medical Research Council/United Kingdom ; //UK Government's Horizon Europe funding guarantee/ ; //Leverhulme Trust/ ; },
mesh = {*Pseudomonas aeruginosa/virology/growth &amp; development/genetics ; *CRISPR-Cas Systems ; *Pseudomonas Phages/physiology ; *SOS Response, Genetics ; *Bacteriophages/physiology ; },
abstract = {Bacteria evolve resistance against their phage foes with a wide range of resistance strategies whose costs and benefits depend on the level of protection they confer and on the costs for maintainance. Pseudomonas aeruginosa can evolve resistance against its phage DMS3vir either by surface mutations that prevent phage binding or through CRISPR-Cas immunity. CRISPR immunity carries an inducible cost whose exact origin is still unknown, and previous work suggested it stems from the inability of the CRISPR-Cas system to completely prevent phage DNA injection and subsequent gene expression before clearing the phage infection. However, the bacterial processes involved are still unknown, and we hypothesize that CRISPR-immunity-associated costs could come from increased mortality rate or reduced growth ability compared with surface-resistant bacteria. To tease apart these two mechanisms with divergent ecological consequences, we use a novel microfluidics-based single-cell approach combined with flow cytometry methods to monitor the effects of phage exposure on the survival and growth of its host. We observed that while CRISPR immunity protects from phage-induced lysis, it cannot prevent phage-induced division lag, filamentation and SOS response activation in a subpopulation of the host bacteria. These results suggest that the costs associated with CRISPR immunity at the population level are caused by heterogeneity in phage-induced growth defects.This article is part of the discussion meeting issue 'The ecology and evolution of bacterial immune systems'.},
}
@article {pmid40903118,
year = {2025},
author = {Zhou, J and Shi, X and He, C and Zheng, X and Yuan, R and Yang, X},
title = {SERS biosensor based on the Cas13a assisted entropy-driven system and lychee-like Fe-TiO2 with excellent exciton capture and separation.},
journal = {Analytica chimica acta},
volume = {1372},
number = {},
pages = {344442},
doi = {10.1016/j.aca.2025.344442},
pmid = {40903118},
issn = {1873-4324},
mesh = {*Titanium/chemistry ; *Spectrum Analysis, Raman/methods ; *Entropy ; *Biosensing Techniques/methods ; *Iron/chemistry ; CRISPR-Cas Systems ; *MicroRNAs/analysis ; Limit of Detection ; },
abstract = {BACKGROUND: Entropy-Driven Circuits (EDC), distinguished by their spontaneous operation and absence of enzymatic reactions, represent a superior strategy for integration with CRISPR/Cas systems, as they obviate the potential for interference among various enzymes during the process of DNA amplification and CRISPR/Cas system integration. Due to the wide band gap of TiO2, its response to visible light is limited, and owing to its high crystallinity and exceptionally stable crystal lattice, the charge transfer (CT) process in TiO2 is suboptimal.<br><br>RESULTS: In this study, lychee-like Fe-TiO2 was firstly prepared to serve as Raman enhanced substrate, facilitating exciton capture and separation to exhibit an excellent Surface-enhanced Raman spectroscopy (SERS) performance. It is proven that the incorporation of Fe results in a significantly narrower band gap for TiO2, facilitating exciton resonance. The amount of Fe in TiO2 was optimized to fabricate a SERS biosensor for detection of miRNA-21 based on the Cas13a assisted entropy-driven system. The detection limit of miRNA was 43.88&#xa0;fmol/L.<br><br>SIGNIFICANCE: This work proposes a sensing strategy that integrates the CRISPR/Cas system with EDC, leveraging a semiconductor substrate exhibiting superior SERS performance to provide a stable Raman signal, thereby enabling highly sensitive detection of miRNA-21, which has a potential application in disease early warning and treatment.},
}
@article {pmid40902823,
year = {2025},
author = {Zhou, X and Diao, R and Li, X and Ziegler, CA and Gramelspacher, MJ and Freddolino, L and Hou, Z and Zhang, Y},
title = {Cas9 senses CRISPR RNA abundance to regulate CRISPR spacer acquisition.},
journal = {Nature},
volume = {},
number = {},
pages = {},
doi = {10.1038/s41586-025-09577-9},
pmid = {40902823},
issn = {1476-4687},
abstract = {Prokaryotes create adaptive immune memories by acquiring foreign DNA snippets, known as spacers, into the CRISPR array[1]. In type II CRISPR-Cas systems, the RNA-guided effector Cas9 also assists the acquisition machinery by selecting spacers from protospacer adjacent motif (PAM)-flanked DNA[2,3]. Here, we uncover the first biological role for Cas9 that is independent of its dual RNA partners. Following depletion of crRNA and/or tracrRNA, Neisseria apoCas9 stimulates spacer acquisition efficiency. Physiologically, Cas9 senses low levels of crRNA in cells with short CRISPR arrays - such as those undergoing array neogenesis or natural array contractions - and dynamically upregulates acquisition to quickly expand the small immune memory banks. As the CRISPR array expands, rising crRNA abundance in turn reduces apoCas9 availability, thereby dampening acquisition to mitigate autoimmunity risks associate with elevated acquisition. While apoCas9's nuclease lobe alone suffices for stimulating acquisition, only full-length Cas9 responses to crRNA levels to boost acquisition in cells with low immunity depth. Finally, we show that this activity is evolutionarily conserved across multiple type II-C Cas9 orthologs. Altogether, we establish an auto-replenishing feedback mechanism in which apoCas9 safeguards CRISPR immunity depth by acting as both a crRNA sensor and a regulator of spacer acquisition.},
}
@article {pmid40902040,
year = {2025},
author = {Li, L and Dai, H and Sun, R and Zhang, Z and Zhang, B},
title = {MicroRNAs as Biotechnological Targets for Future Food Security and Agricultural Sustainability.},
journal = {Journal of agricultural and food chemistry},
volume = {},
number = {},
pages = {},
doi = {10.1021/acs.jafc.5c07100},
pmid = {40902040},
issn = {1520-5118},
abstract = {MicroRNAs (miRNAs) are small noncoding RNAs that post-transcriptionally regulate gene expression, playing key roles in plant growth, development, and stress responses. Their regulatory functions make miRNAs ideal targets for enhancing crop yield, quality, and stress tolerance using biotechnologies, such as transgenic overexpression and CRISPR/Cas genome editing. By targeting multiple genes, miRNAs address complex agricultural challenges effectively. This review focuses on the diverse roles of miRNAs in enhancing crop productivity and resilience; miRNAs are an important biotechnological target for ensuring food security and agricultural sustainability. We also highlight transgenic and CRISPR/Cas genome editing approaches to demonstrate miRNA-driven trait improvements, such as drought/salinity tolerance, pest resistance, and nutrient use efficiency. Due to the quick development of advanced biotechnology tools, both upregulated and downregulated miRNAs can be manipulated for optimizing agronomic traits. Challenges including off-target effects, regulatory barriers, and environmental concerns are analyzed with strategies proposed to overcome them. By leveraging miRNA technologies, this perspective emphasizes their transformative potentials in achieving sustainable agriculture and global food security.},
}
@article {pmid40901825,
year = {2025},
author = {Thiam, R and Ceballos, MS and Beneke, T and Kuk, N and Pasquier, G and Crobu, L and Jeffares, DC and Vergnes, B and Barckmann, B and Sterkers, Y},
title = {A novel Leishmania infantum reference strain for gene editing and the study of visceral leishmaniasis.},
journal = {PloS one},
volume = {20},
number = {9},
pages = {e0327390},
pmid = {40901825},
issn = {1932-6203},
mesh = {*Leishmania infantum/genetics/growth &amp; development/pathogenicity ; *Leishmaniasis, Visceral/parasitology ; *Gene Editing/methods ; Animals ; Humans ; CRISPR-Cas Systems ; Mice ; Macrophages/parasitology ; Genome, Protozoan ; },
abstract = {Parasites of the Leishmania donovani complex are responsible for visceral leishmaniasis, a vector-borne disease transmitted through the bite of female phlebotomine sand flies. As well as the human hosts, these parasites infect many mammals which can serve as reservoirs. Dogs are particularly important reservoirs. Transmission is widespread across Asia, Africa, the Americas, and the Mediterranean basin, including South of France. Visceral leishmaniasis poses a fatal threat if left untreated. Research into the pathophysiology of this neglected disease is of prime importance, as is the development of new drugs. In this study, we evaluated the growth, differentiation, and macrophage infectivity of four L. donovani complex strains and identified L. infantum S9F1 (MHOM/MA/67/ITMAP263, clone S9F1) as a well-adapted strain for genetic engineering studies. We present here the genome sequence and annotation of L infantum S9F1 T7 Cas9, providing the scientific community with easy access to its genomic information. The data has been integrated into the LeishGEdit online resource to support primer design for CRISPR-Cas9 experiments. We now aim to make this strain widely available to foster studies of visceral leishmaniasis.},
}
@article {pmid40788276,
year = {2025},
author = {Gao, H and Zhang, Y and Wang, Y and Wu, Y and Zheng, Z and Quan, F and Han, Q and Li, Y and Zhang, K},
title = {Integration of CRISPR/Cas12a and Cas13a in one pot for ratiometric calibration of single-nucleotide variations.},
journal = {Chemical communications (Cambridge, England)},
volume = {61},
number = {72},
pages = {13675-13678},
doi = {10.1039/d5cc03931b},
pmid = {40788276},
issn = {1364-548X},
mesh = {*Polymorphism, Single Nucleotide ; *CRISPR-Cas Systems/genetics ; *CRISPR-Associated Proteins/genetics/metabolism ; Calibration ; *Endodeoxyribonucleases/genetics/metabolism ; *Bacterial Proteins/genetics/metabolism ; Humans ; },
abstract = {Accurate detection of single-nucleotide variations (SNVs) plays a pivotal role in medical diagnosis. Herein, by integrating Cas12a and Cas13a to simultaneously detect wild-type and mutated sites on a single RNA strand, we developed an innovative one-pot SNV analysis method, namely iCasdrop, which is capable of reducing non-specific signals induced by the wild type sequence and achieving ratiometric calibration of SNVs.},
}
@article {pmid40599075,
year = {2025},
author = {Zhang, T and Cai, L and Chu, Z and She, A and Yang, J and Su, X},
title = {Rapid and Sensitive Detection of miRNA by Single-Molecule Fluorescence Dequenching Assay with Target Recycled CRISPR/Cas12a Amplification System.},
journal = {Small (Weinheim an der Bergstrasse, Germany)},
volume = {21},
number = {35},
pages = {e2412228},
doi = {10.1002/smll.202412228},
pmid = {40599075},
issn = {1613-6829},
support = {32271521//National Natural Science Foundation of China/ ; 31971361//National Natural Science Foundation of China/ ; 2022YFC2603902//State Key Research Development Program of China/ ; PT2406//Fundamental Research Funds for the Central Universities/ ; 2023-NHLHCRF-YXHZ-ZRMS-05//National High Level Hospital Clinical Research Funding/ ; },
mesh = {*MicroRNAs/genetics/metabolism/analysis ; Humans ; *CRISPR-Cas Systems/genetics ; Fluorescence ; Carcinoma, Non-Small-Cell Lung/genetics ; Lung Neoplasms/genetics ; Female ; Cell Line, Tumor ; },
abstract = {Dysregulated miRNAs play a critical role in the development of cancers. A rapid and sensitive single-molecule fluorescence dequenching assay combined with a CRISPR/Cas12a-based target recycling amplification system for miRNA detection is developed. This single-molecule assay detects miRNAs down to ≈10 fM within 10 min. An automated single-molecule fluorescent puncta analysis procedure is also created, improving the signal-to-noise ratio by 3.76-fold compared to traditional hidden Markov model (HMM)based methods. The clinical applicability of this technique is demonstrated. Two key miRNA targets associated with non-small cell lung cancer (NSCLC) and ovarian cancer (OC) from 2867 datasets of the TCGA database are screened. Validation is initially conducted at the cell line level, followed by testing with tissue and blood samples from 10 patients with NSCLC and OC. The assay demonstrated high diagnostic accuracy, with receiver operating characteristic curves (area under the curve (AUC) > 0.93) and significant statistical differentiation (p < 0.001) between cancer and healthy samples. This method's exceptional sensitivity and speed highlight its potential for early cancer diagnostics and personalized medicine.},
}
@article {pmid40536333,
year = {2025},
author = {Zhao, G and Li, Z and Zhao, MJ and Li, SY and Xia, Q and Xu, S and Zhang, Y and Wang, Y and Li, F and Liu, YL and Guo, YH and Xu, RX and Zhou, H and Zhou, H and Ding, WW and Wang, YC and Miao, Y and Wang, Z},
title = {A High-Fidelity RNA-Targeting Cas13X Downregulates Connexin43 in Macroglia: A Novel Neuroprotective Strategy for Glaucoma.},
journal = {Advanced science (Weinheim, Baden-Wurttemberg, Germany)},
volume = {12},
number = {33},
pages = {e15856},
doi = {10.1002/advs.202415856},
pmid = {40536333},
issn = {2198-3844},
support = {82301215//National Natural Science Foundation of China/ ; 32271043//National Natural Science Foundation of China/ ; 32471057//National Natural Science Foundation of China/ ; 82171047//National Natural Science Foundation of China/ ; 2018SHZDZX01//Shanghai Municipal Science and Technology Major Project/ ; },
mesh = {*Connexin 43/genetics/metabolism ; *Glaucoma/genetics/metabolism/therapy ; Animals ; Mice ; Retinal Ganglion Cells/metabolism ; Disease Models, Animal ; CRISPR-Cas Systems/genetics ; Down-Regulation ; Intraocular Pressure ; Mice, Inbred C57BL ; Humans ; Optic Nerve/metabolism ; },
abstract = {Glaucoma is a neurodegenerative disease characterized by the progressive degeneration of retinal ganglion cells (RGCs) and their axons, ultimately leading to irreversible vision loss. Elevated intraocular pressure (IOP) is one of the significant risk factors in glaucoma; however, neurodegeneration continues even after effective IOP management, underscoring the need for neuroprotective therapies. This study investigates the role of connexin43 (Cx43), which is extensively expressed in retinal macroglia, in regulating microglial activation and optic nerve degeneration in glaucoma. A high-fidelity CRISPR-Cas13 (hfCas13X) system is employed to selectively target and knock down Cx43 expression in macroglia. The findings reveal that Cx43-mediated ATP release through hemichannels exacerbates microglial activation and neuroinflammation, thereby contributing to RGC loss. Notably, in a mouse model of chronic ocular hypertension (COH) glaucoma, knocking down Cx43 in macroglia using the hfCas13X system significantly promoted the survival of RGCs and the integrity of the optic nerve, and improved visual function. The hfCas13X system, which offers high-fidelity RNA editing with minimal off-target effects, represents a novel and promising therapeutic strategy for glaucoma, highlighting the potential of gene editing technologies in the management of neurodegenerative diseases.},
}
@article {pmid40534129,
year = {2025},
author = {Nicosia, L and Harrison, PT},
title = {CRISPR for cystic fibrosis: Advances and insights from a systematic review.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {33},
number = {9},
pages = {4091-4112},
doi = {10.1016/j.ymthe.2025.06.021},
pmid = {40534129},
issn = {1525-0024},
mesh = {*Cystic Fibrosis/genetics/therapy ; Humans ; *Gene Editing/methods ; *Cystic Fibrosis Transmembrane Conductance Regulator/genetics ; *CRISPR-Cas Systems ; *Genetic Therapy/methods ; Mutation ; Animals ; *Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {Cystic fibrosis (CF) is a severe genetic disorder caused by loss-of-function mutations in the CFTR gene. Gene-editing approaches have the potential to correct such mutations. This systematic review outlines the mechanisms of the main CRISPR-based technologies, and, through cross-study comparisons, analyzes 27 research articles that applied them to target CF-causing variants. We report and discuss the strategy design, target cell selection, editing efficiency, prevalence of editing byproducts, and levels of CFTR functional restoration achieved in each work, with the aim of providing technical insights for further exploration of CRISPR-based gene-editing approaches. Our findings show that the F508del and W1282X mutations were the most extensively studied CF-causing variants, though over fifteen mutations were targeted overall. The majority of works under review explored the use of homology-directed repair or base editing, with a growing number of studies reporting efficient prime editing. Some studies tackled multiple individual mutations, compared different editors, or tested strategies across various models, while others focused on approaches that rescue CFTR function without directly correcting a mutation. Several works also proposed strategies that could address multiple variants with a single approach, while others highlighted technical difficulties in editing certain regions of the CFTR gene. This cross-study comparison also emphasizes the need for standardized reporting of editing efficiency and functional recovery, and stresses the importance of further single-cell RNA sequencing and in vivo studies to reach clinically relevant conclusions. As gene-editing techniques continue to evolve, and with over 60 ongoing CRISPR-based clinical trials, there is growing optimism for meaningful advancements in CF gene-editing therapeutics.},
}
@article {pmid40518666,
year = {2025},
author = {Yang, Y and Fu, Z and Deng, S and Wu, G and Wang, C and Luo, X and Kang, R and Chen, Y and Peng, C and Zhang, P and Cui, K and Wan, F and Wang, J and Zhou, Q and Chen, W and Xiong, Y and Ma, W and Songyang, Z and Liang, P},
title = {RNA-DNA hybrid binding domain broadens the editing window of base editors.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {33},
number = {9},
pages = {4431-4446},
doi = {10.1016/j.ymthe.2025.06.024},
pmid = {40518666},
issn = {1525-0024},
mesh = {*Gene Editing/methods ; Humans ; Animals ; Mice ; *DNA/genetics/metabolism/chemistry ; CRISPR-Cas Systems ; Ribonuclease H/genetics/metabolism ; *RNA/genetics/metabolism/chemistry ; },
abstract = {Adenine base editors (ABEs) and cytosine base editors (CBEs) are prominent tools for precise genome editing but are hindered by limited editing activity at positions proximal to the protospacer adjacent motif (PAM). This study investigates the potential of enhancing base editors editing activity by fusing them with RNA-DNA hybrid binding domains (RHBDs). Specifically, fusing ABE8e with the RHBD of Homo sapiens RNaseH1 (RHBD1) significantly increased A-to-G editing efficiency in the PAM-proximal region (A9-A15) by up to 3.5-fold, while reducing off-target cytosine editing. Additionally, RHBD1 is compatible with ABEmax, BE4max, and dual base editor (eA&amp;C-BEmax), enhancing their editing activity at the PAM-proximal bases. Notably, RHBD1-fused BE4max led to a 3.1-fold improvement in C-to-T editing efficiency at PAM-proximal region (C9-C12). Furthermore, we demonstrated that RHBD1-fused ABE8e could effectively edit disease-related single nucleotide variations (SNVs) in human cells and validated its efficacy in adult mouse liver. These findings highlight the significance of the RHBD in expanding editing window and the applicability of base editors for gene therapy and disease modeling.},
}
@article {pmid40493884,
year = {2025},
author = {Loke, J and Kim, PG and Nguyen, TTP and Boileau, M and McConkey, M and Miller, A and Shin, W and Hergott, CB and Ericsson, M and Nordstrom, A and Llopis, PM and Armstrong, SA and Mancias, JD and Ebert, BL},
title = {An in vivo barcoded CRISPR-Cas9 screen identifies Ncoa4-mediated ferritinophagy as a dependence in Tet2-deficient hematopoiesis.},
journal = {Blood},
volume = {146},
number = {10},
pages = {1174-1186},
doi = {10.1182/blood.2024028033},
pmid = {40493884},
issn = {1528-0020},
mesh = {*CRISPR-Cas Systems ; *Hematopoiesis/genetics ; *Ferritins/metabolism/genetics ; *DNA-Binding Proteins/genetics/deficiency/metabolism ; Animals ; *Nuclear Receptor Coactivators/metabolism/genetics ; Dioxygenases ; Mice ; *Proto-Oncogene Proteins/genetics/deficiency ; Hematopoietic Stem Cells/metabolism ; Mice, Knockout ; *Autophagy ; Iron/metabolism ; Humans ; },
abstract = {TET2 is among the most commonly mutated genes in both clonal hematopoiesis and myeloid malignancies; thus, the ability to identify selective dependencies in TET2-deficient cells has broad translational significance. Here, we identify regulators of Tet2 knockout (KO) hematopoietic stem and progenitor cell (HSPC) expansion using an in vivo CRISPR-Cas9 KO screen, in which nucleotide barcoding enabled large-scale clonal tracing of Tet2-deficient HSPCs in a physiologic setting. Our screen identified candidate genes, including Ncoa4, that are selectively required for Tet2 KO clonal outgrowth compared with wild type. Ncoa4 targets ferritin for lysosomal degradation (ferritinophagy), maintaining intracellular iron homeostasis by releasing labile iron in response to cellular demands. In Tet2-deficient HSPCs, increased mitochondrial adenosine triphosphate production correlates with increased cellular iron requirements and, in turn, promotes Ncoa4-dependent ferritinophagy. Restricting iron availability reduces Tet2 KO stem cell numbers, revealing a dependency in TET2-mutated myeloid neoplasms.},
}
@article {pmid40492429,
year = {2025},
author = {Meng, R and Li, J and Wang, W and Liang, D and Li, Z and Mao, C and Li, Q and Zhang, Y and Chen, H and Tang, J and Hu, P and Niu, Q and Huang, X and Shen, B and Zhang, J},
title = {Engineered Cas12j-8 is a Versatile Platform for Multiplexed Genome Modulation in Mammalian Cells.},
journal = {Advanced science (Weinheim, Baden-Wurttemberg, Germany)},
volume = {12},
number = {33},
pages = {e02593},
doi = {10.1002/advs.202502593},
pmid = {40492429},
issn = {2198-3844},
support = {2022YFC2702705//National Key Research and Development Project of China/ ; 82221005//Creative Research Groups of China/ ; 32371546//National Natural Science Foundation of China/ ; 82071434//National Natural Science Foundation of China/ ; 81971398//National Natural Science Foundation of China/ ; },
mesh = {*Gene Editing/methods ; Humans ; *CRISPR-Cas Systems/genetics ; Animals ; HEK293 Cells ; *CRISPR-Associated Proteins/genetics ; },
abstract = {Cas12j-8 is a compact Cas nuclease discovered from the metagenome of giant bacteriophages, consisting of only 717 amino acids and recognizing the '5-TTN-3' protospacer adjacent motif (PAM) sequence. However, its low gene editing efficiency in mammalian cells limits its application in therapeutic gene editing. To address this limitation, structure-guided mutagenesis is employed to replace key negatively charged residues with arginine, strengthening DNA binding. The resulting quintuple mutant, engineered Cas12j-8 (enCas12j-8), demonstrates robust on-target editing efficiency comparable to LbCas12a while maintaining low off-target effects. Cytosine base editors (CBEs) and adenine base editors (ABEs) are developed using enCas12j-8, achieving up to 29.54-fold C-to-T and 36.57-fold A-to-G conversion efficiency compared with the wild-type at the dominated sites, respectively. Notably, enCas12j-8 enables multiplexed editing of three genomic loci simultaneously via a single crRNA array, achieving efficiencies comparable to single-guide approaches. Additionally, enCas12j-8-ABE facilitates the disruption of splice acceptor sites, effectively inducing exon skipping in the SOD1 gene. This strategy holds potential significance for therapeutic genome modulation. These findings establish enCas12j-8 as a versatile, high-precision tool for genome engineering, combining efficient delivery, multiplexing capability, and compatibility with diverse editing modalities.},
}
@article {pmid40901634,
year = {2025},
author = {Jiang, X and Wang, X and Shen, S and Hou, S and Yu, C},
title = {3D Genome Engineering: Current Advances and Therapeutic Opportunities in Human Diseases.},
journal = {Research (Washington, D.C.)},
volume = {8},
number = {},
pages = {0865},
doi = {10.34133/research.0865},
pmid = {40901634},
issn = {2639-5274},
abstract = {Dynamic chromatin 3-dimensional (3D) conformation is a key mechanism regulating gene expression and cellular function during development and disease. Elucidating the structure, functional dynamics, and spatiotemporal organization of the 3D genome requires integrating multiple experimental approaches, including chromatin conformation capture techniques, precise genome manipulation tools, and advanced imaging technologies. Notably, CRISPR/Cas systems have emerged as a revolutionary genome-editing platform, offering unprecedented opportunities for manipulating 3D genome organization and investigating disease mechanisms. This review systematically examines recent advances in CRISPR-based mammalian 3D genome engineering and explores the therapeutic potential of 3D genome engineering strategies in disease intervention.},
}
@article {pmid40900077,
year = {2025},
author = {Yu, Y and Zhang, Z and Zhai, Z and Sun, B and Yang, D and Wang, Z and Lin, Q and Zhou, X and Zhao, J},
title = {A Novel Mouse Model of Granular Corneal Dystrophy Type II Reveals Impaired Autophagy and Recapitulates Human Pathogenesis.},
journal = {Investigative ophthalmology &amp; visual science},
volume = {66},
number = {12},
pages = {7},
doi = {10.1167/iovs.66.12.7},
pmid = {40900077},
issn = {1552-5783},
mesh = {Animals ; *Corneal Dystrophies, Hereditary/genetics/pathology/metabolism ; *Autophagy/physiology ; *Disease Models, Animal ; Mice ; Microscopy, Electron, Transmission ; Tomography, Optical Coherence ; Transforming Growth Factor beta/genetics/metabolism ; Blotting, Western ; Extracellular Matrix Proteins/genetics/metabolism ; Humans ; *Mutation ; Mice, Inbred C57BL ; Slit Lamp Microscopy ; CRISPR-Cas Systems ; Phenotype ; *Cornea/pathology ; },
abstract = {PURPOSE: To develop and characterize a novel mouse model of granular corneal dystrophy type II (GCD2) using CRISPR/Cas9 technology and explore the underlying pathogenesis of transforming growth factor-beta-induced protein (TGFBIp) aggregation.<br><br>METHODS: CRISPR/Cas9 technology was employed to introduce the R124H mutation in the TGFBI gene of mice. Genomic sequencing and polymerase chain reaction confirmed the mutation. Phenotypic characteristics were evaluated through slit-lamp examination, optical coherence tomography, histological analysis, electron microscopy, and immunofluorescence, comparing wild-type (WT), heterozygous (HE), and homozygous (HO) mice. Transcriptome sequencing was conducted to identify the pathogenesis of GCD2. The findings were further validated through western blotting and transmission electron microscopy.<br><br>RESULTS: The R124H mutation in TGFBI was successfully introduced, with breadcrumb-like deposits observed in the corneas of mutant mice, with HO mice displaying more severe phenotypes than HE mice. TGFBIp levels were elevated in HE and HO mice (both P < 0.001). Histological and electron microscopy analyses revealed abnormal collagen arrangement and TGFBIp deposits in the corneal stroma of the HE and HO mice. Transcriptome analysis indicated that the TGFBI-R124H mutation was associated with impaired autophagy, endocytosis, and extracellular matrix signaling. Additional experiments confirmed autophagy-related markers LC3 and SQSTM1 were upregulated in the corneas of mutant mice, accompanied by increased autophagosome formation in corneal keratocytes, indicating impaired autophagy flux in HE and HO mice.<br><br>CONCLUSIONS: We established a GCD2 mouse model caused by the R124H mutation using CRISPR/Cas9, providing a reliable platform for understanding pathogenesis for GCD2.},
}
@article {pmid40897812,
year = {2025},
author = {Wolter, JM and James, LM and Boeshore, SL and Mao, H and McCoy, ES and Ryan, DF and Fragola, G and Taylor-Blake, B and Stein, JL and Zylka, MJ},
title = {AAV-dCas9 vector unsilences paternal Ube3a in neurons by impeding Ube3a-ATS transcription.},
journal = {Communications biology},
volume = {8},
number = {1},
pages = {1332},
pmid = {40897812},
issn = {2399-3642},
support = {631904//Simons Foundation/ ; 1R01NS109304//U.S. Department of Health &amp; Human Services | NIH | National Institute of Neurological Disorders and Stroke (NINDS)/ ; T32HD040127//U.S. Department of Health &amp; Human Services | NIH | Eunice Kennedy Shriver National Institute of Child Health and Human Development (NICHD)/ ; },
mesh = {Animals ; *Dependovirus/genetics ; Mice ; *Neurons/metabolism ; Humans ; *Ubiquitin-Protein Ligases/genetics/metabolism ; Genetic Vectors/genetics ; *Angelman Syndrome/genetics/therapy ; *Transcription, Genetic ; *CRISPR-Associated Protein 9/genetics/metabolism ; CRISPR-Cas Systems ; Male ; *RNA, Long Noncoding/genetics ; },
abstract = {Angelman syndrome (AS) is a debilitating neurodevelopmental disorder caused by loss of maternally-inherited UBE3A. In neurons, paternally-inherited UBE3A is silenced in cis by a long non-coding RNA called Ube3a-ATS. Here, we found that Neisseria meningitidis Cas9 with two mutations (D15A and H587A) in the nuclease domains (dNmCas9) can unsilence the dormant paternal Ube3a allele in mouse and human neurons when targeted to Snord115 snoRNA genes located in introns of Ube3a-ATS. Importantly, dNmCas9 disrupted Ube3a-ATS with a non-template bias and in the absence of a chromatin modifying domain, supporting a transcriptional interference mechanism. When packaged into an adeno-associated virus (AAV) vector, dNmCas9 exhibited dose-dependent Ube3a-ATS knock-down and paternal Ube3a unsilencing in vitro and in vivo. This vector also partially rescued the hind limb clasp phenotype when delivered to neonatal AS model mice. Collectively, our study underscores the potential of dCas9-based therapeutics without chromatin repression domains to mediate transcriptional downregulation.},
}
@article {pmid40897312,
year = {2025},
author = {Bircheneder, M and Parniske, M},
title = {Engineering and comparison of cas12a-based genome editing systems in plants.},
journal = {The Plant journal : for cell and molecular biology},
volume = {123},
number = {5},
pages = {e70410},
pmid = {40897312},
issn = {1365-313X},
support = {401867691//Deutsche Forschungsgemeinschaft/ ; //Bayerisches Staatsministerium f&#xfc;r Unterricht und Kultus/ ; },
mesh = {*Gene Editing/methods ; *Arabidopsis/genetics ; Nicotiana/genetics ; CRISPR-Cas Systems/genetics ; Genome, Plant/genetics ; *Lotus/genetics ; *CRISPR-Associated Proteins/genetics/metabolism ; *Endodeoxyribonucleases/genetics/metabolism ; *Bacterial Proteins/genetics/metabolism ; Plants, Genetically Modified ; },
abstract = {While Cas9 and Cas12a are both RNA-guided endonucleases used for genome editing, only Cas12a is able to process pre-crRNA via its additional ribonuclease activity. This feature reduces the complexity of Cas12a versus Cas9-based genome editing systems thus providing an attractive alternative for generating site-specific mutations in plants. Here we aimed to improve the efficiency of the cas12a-based generation of two double-strand breaks flanking the open reading frame of a target gene, leading to its full deletion. To this end, we compared the relative impact of different components on cas12a-based gene deletion efficiency in three different eudicotyledons, Arabidopsis thaliana, Lotus japonicus, and Nicotiana benthamiana. We detected the highest cas12a-based editing efficiency with a combination of suitable promoters for crRNA and cas12a expression, a tandem terminator to control cas12a expression, a re-coded cas12a, adapted to the codon usage of Arabidopsis and engineered to carry introns, and encoding a Cas12a flanked by a nuclear localization signal at both ends. Our work revealed the high potential for improving cas12a-based genome editing systems for plant genetic research.},
}
@article {pmid40896718,
year = {2025},
author = {Madsen, CK and Hanak, T and Aronsson, H and Brinch-Pedersen, H},
title = {Rapid one-step CRISPR-cas vector assembly by isothermal spacer removal linearization and sequence-ligation independent cloning (ISRL-SLIC).},
journal = {MethodsX},
volume = {15},
number = {},
pages = {103567},
pmid = {40896718},
issn = {2215-0161},
abstract = {CRISPR-Cas genome editing is a powerful tool in various fields, but current cloning methods can be time-consuming due to the frequent use of intermediate entry vectors and multiple steps involving restriction enzymes and ligases. These multiple steps can create a bottleneck in CRISPR-Cas experiments. In response to this challenge, we propose a highly efficient streamlined approach, which enables simultaneous linearization of the acceptor plasmid and protospacer cloning in a single isothermal reaction. This eliminates the need for entry vectors, pre-linearization of vectors, and in vitro ligation, thus significantly simplifying the cloning process. The method can be applied to clone short synthetic oligos for single protospacer constructs or multiple amplicons for multiplex genome editing designs. Either way, researchers can proceed directly to Escherichia coli transformation after a one-hour isothermal reaction and recover the final construct within two days. By combining the advantages of sequence-ligation independent cloning (SLIC) cloning with a streamlined workflow, our approach facilitates rapid and efficient construction of CRISPR-Cas vectors and holds the promise of accelerating research and development in genome editing and related fields. To expedite the cloning of constructs, we propose a rapid one-step CRISPR-Cas vector assembly method that combines isothermal spacer removal with a sequence-ligation-independent cloning reaction. We could show that Isothermal Spacer Removal Linearization and Sequence-Ligation Independent Cloning (ISRL-SLIC) can create single, double and triple protospacer constructs in one reaction with scalability. The ISRL-SLIC reaction delivers clones under a broad range of oligo concentration making it a robust and time saving alternative to other methods for constructing CRISPR-Cas vectors.},
}
@article {pmid40895203,
year = {2025},
author = {Kesarwani, P and Sundar, D},
title = {Conformational changes induced by K949A mutation in the CRISPR-Cas12a complex drives an effective target-binding mechanism.},
journal = {Current research in structural biology},
volume = {10},
number = {},
pages = {100173},
pmid = {40895203},
issn = {2665-928X},
abstract = {The CRISPR/Cas system is a potential tool for genome editing, yet it faces challenges due to off-target activity caused by mismatches at specific positions. However, Off-target activity can be minimized by optimal design of guide RNA (gRNA) but there remains a possibility of unintended cleavage, highlighting the role of the Cas nuclease in off-target recognition and binding the target site. This study focuses on comparing the conformational dynamics and stability of Wildtype, RR, RVR, RRm and RVRm variants of AsCas12a with gRNA-DNA bound complexes. It was found that the cross-correlation coefficient between His1167 of the NUC domain and Thr384 of the REC II domain significantly increased after the K949A mutation compared to other variants. The extensive spread of principal components also revealed flexibility in both Cas nuclease and gRNA-DNA hybrid of RVR variant and wildtype AsCas12a whereas the confined clusters in PCA plot suggests increased stability in both the variants after mutation. This study shows the role of K949A mutation in improving stability of PAM variants and predicted critical residues such as His1167, Thr384 and Ser959, in inducing stability in mutants of PAM variants.},
}
@article {pmid40893972,
year = {2025},
author = {},
title = {Correction to "High-efficiency genome editing of an extreme thermophile Thermus thermophilus using endogenous type I and type III CRISPR-Cas systems".},
journal = {mLife},
volume = {4},
number = {4},
pages = {470},
doi = {10.1002/mlf2.70010},
pmid = {40893972},
issn = {2770-100X},
abstract = {[This corrects the article DOI: 10.1002/mlf2.12045.].},
}
@article {pmid40891977,
year = {2025},
author = {Li, X and Zhong, Y and Jin, C and Chen, X and Cui, X and Xu, Y and Fan, Y and Song, F and Cen, P and Dong, L and Yu, K and He, Q and Wang, J and Hu, S and Zhang, XY and Li, C and Tian, M and Zhang, H},
title = {CRISPR/Cas9-Engineered Triple-Fusion Reporter Gene Imaging System for Monitoring Transplanted Neural Progenitor Cells in Ischemic Stroke.},
journal = {Radiology},
volume = {316},
number = {3},
pages = {e250305},
doi = {10.1148/radiol.250305},
pmid = {40891977},
issn = {1527-1315},
mesh = {Animals ; *Neural Stem Cells/transplantation ; Rats ; Male ; *Ischemic Stroke/diagnostic imaging/therapy ; *CRISPR-Cas Systems/genetics ; *Genes, Reporter/genetics ; Magnetic Resonance Imaging/methods ; Humans ; Disease Models, Animal ; Rats, Sprague-Dawley ; Positron Emission Tomography Computed Tomography/methods ; },
abstract = {Background Neural progenitor cell therapy holds great potential for repairing brain damage induced by ischemic stroke, and molecular imaging plays a crucial role in evaluating the therapeutic efficacy of neural progenitor cell transplantation. However, the presence of the blood-brain barrier significantly limits the effectiveness of such imaging methods. Purpose To enable long-term monitoring of transplanted human neural progenitor cells (hNPCs) in a rat model of ischemic stroke by combining a clustered regularly interspaced short palindromic repeats/CRISPR-associated nuclease 9 (CRISPR/Cas9)-engineered triple-fusion (TF) reporter gene system with a noninvasive adenosine agonistic micelle (AM)-based probe delivery strategy. Materials and Methods Between January 2021 and May 2025, 60 male rats that were 2 months old were included. Thirty-seven rats with stroke were administered either TF human NPCs (hNPCs) or vehicle (culture media) and underwent MRI, bioluminescence imaging, PET/CT, and neurologic assessments at weeks 1, 2, 4, and 8 after transplantation. Comparisons between groups were determined by t tests, one-way analysis of variance, linear regression, and linear mixed-effects model. Results TF-hNPCs proliferated within the ischemic rat brain (week 8 vs week 1, bioluminescence imaging and PET: P < .001 and P = .02, respectively) and exhibited progressive migration and maturation by 8 weeks after transplantation (proportion of microtubule-associated protein 2-positive TF-hNPCs at week 8 vs week 4: 94.08% ± 3.02 vs 85.47% ± 6.54, respectively [P = .04]; proportion of doublecortin-positive TF-hNPCs at week 4 vs week 2: 83.90% ± 2.84 vs 59.74% ± 0.55, respectively [P = .02]). Moreover, TF-hNPC transplantation increased glucose (fluorine 18 fluorodeoxyglucose) uptake in the ischemic brain (TF-hNPCs vs vehicle at week 4, 0.58 ± 0.04 vs 0.37 ± 0.05, respectively [P = .008]; TF-hNPCs vs vehicle at week 8, 0.52 ± 0.06 vs 0.29 ± 0.02, respectively [P = .01]) and attenuated neurologic deficits compared with the vehicle group (neurologic score, TF-hNPCs vs vehicle at week 8: 9.6 ± 0.25 vs 7.6 ± 0.3, respectively; P = .003). Conclusion A CRISPR/Cas9-engineered TF reporter gene imaging system combined with a noninvasive AM-based approach enabled in vivo monitoring of transplanted human NPCs in a rat model of ischemic stroke. © RSNA, 2025 Supplemental material is available for this article. See also the editorial by Chapelin in this issue.},
}
@article {pmid40857331,
year = {2025},
author = {Farr, GH and Reid, W and Hasegawa, EH and Azzam, A and Young, I and Li, ML and Olson, AK and Beier, DR and Maves, L},
title = {A systems genetics approach identifies roles for proteasome factors in heart development and congenital heart defects.},
journal = {PLoS genetics},
volume = {21},
number = {8},
pages = {e1011579},
pmid = {40857331},
issn = {1553-7404},
mesh = {Animals ; Zebrafish/genetics/embryology ; *Proteasome Endopeptidase Complex/genetics/metabolism ; *Heart Defects, Congenital/genetics/pathology ; *Heart/embryology/growth &amp; development ; Humans ; *Zebrafish Proteins/genetics/metabolism ; Protein Interaction Maps/genetics ; Gene Expression Regulation, Developmental ; Transcriptome/genetics ; Mutation ; CRISPR-Cas Systems ; },
abstract = {Congenital heart defects (CHDs) occur in about 1% of live births and are the leading cause of infant death due to birth defects. While there have been remarkable efforts to pursue large-scale whole-exome and genome sequencing studies on CHD patient cohorts, it is estimated that these approaches have thus far accounted for only about 50% of the genetic contribution to CHDs. We sought to take a new approach to identify genetic causes of CHDs. By combining analyses of genes that are under strong selective constraint along with published embryonic heart transcriptomes, we identified over 200 new candidate genes for CHDs. We utilized protein-protein interaction (PPI) network analysis to identify a functionally-related subnetwork consisting of known CHD genes as well as genes encoding proteasome factors, in particular POMP, PSMA6, PSMA7, PSMD3, and PSMD6. We used CRISPR targeting in zebrafish embryos to preliminarily identify roles for the PPI subnetwork genes in heart development. We then used CRISPR to create new mutant zebrafish strains for two of the proteasome genes in the subnetwork: pomp and psmd6. We show that loss of proteasome gene functions leads to defects in zebrafish heart development, including dysmorphic hearts, myocardial cell blebbing, and reduced outflow tracts. We also identified deficits in cardiac function in pomp and psmd6 mutants. These heart defects resemble those seen in zebrafish mutants for known CHD genes and other critical heart development genes. Our study provides a novel systems genetics approach to further our understanding of the genetic causes of human CHDs.},
}
@article {pmid40779935,
year = {2025},
author = {Wu, J and Li, X and Yin, X and Hu, J and Zhou, P and Zhong, X and Wu, M},
title = {Rapid detection of plasma exosomal LncRNA CASC9 for HCC using RT-RPA-CRISPR/Cas12a assay.},
journal = {Journal of pharmaceutical and biomedical analysis},
volume = {266},
number = {},
pages = {117085},
doi = {10.1016/j.jpba.2025.117085},
pmid = {40779935},
issn = {1873-264X},
mesh = {Humans ; *Carcinoma, Hepatocellular/blood/diagnosis/genetics ; *Liver Neoplasms/blood/diagnosis/genetics ; *Exosomes/genetics/metabolism ; *RNA, Long Noncoding/blood/genetics ; Biomarkers, Tumor/blood/genetics ; Male ; Female ; Middle Aged ; ROC Curve ; CRISPR-Cas Systems/genetics ; alpha-Fetoproteins ; Real-Time Polymerase Chain Reaction/methods ; Aged ; },
abstract = {Hepatocellular carcinoma (HCC) is a leading cause of cancer-related mortality worldwide. Early detection is essential for improving patient outcomes. Long non-coding RNAs (lncRNAs) in plasma exosomes have emerged as promising non-invasive biomarkers. However, sensitive detection methods remain limited. Plasma exosomes were isolated and validated using transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA), and Western blot (WB). RNA sequencing identified CASC9 as the most significantly upregulated exosomal lncRNA in HCC patients. Its diagnostic value was evaluated using real-time quantitative PCR (RT-qPCR) and a novel RT-RPA-CRISPR/Cas12a fluorescence assay. Diagnostic performance was assessed through receiver operating characteristic (ROC) curve analysis and compared with alpha-fetoprotein (AFP). Exosomal CASC9 levels were significantly elevated in HCC patients and correlated with tumor size, stage, and number (P < 0.001). ROC analysis demonstrated that CASC9 had superior diagnostic accuracy (area under the curve [AUC] = 0.822) compared to AFP (AUC = 0.795), with further improvement when combined (AUC = 0.875). The RT-RPA-CRISPR/Cas12a assay achieved a detection limit of 0.1 copies/μL, outperforming RT-qPCR. When combined with RT-qPCR and AFP, the method achieved an AUC of 0.987 against normal controls and 0.975 against benign cases. Plasma exosomal CASC9 is a promising diagnostic biomarker for HCC. The RT-RPA-CRISPR/Cas12a assay offers a rapid, ultra-sensitive, and clinically feasible detection strategy.},
}
@article {pmid40764055,
year = {2025},
author = {Stewart-Ornstein, J and Irby, MJ and Lilieholm, MK and Laprise, D and Collier, MD and Aunins, T and Harjanto, D and Chang, AN and Reyon, D and Duffield, JS},
title = {3'-end ligation sequencing is a sensitive method to detect DNA nicks at potential sites of off-target activity induced by prime editors.},
journal = {Genome research},
volume = {35},
number = {9},
pages = {2064-2075},
pmid = {40764055},
issn = {1549-5469},
mesh = {Humans ; *Gene Editing/methods ; *Sequence Analysis, DNA/methods ; Genome, Human ; CRISPR-Cas Systems ; *DNA/genetics ; DNA Breaks, Double-Stranded ; },
abstract = {Gene editing makes precise changes in DNA to restore normal function or expression of genes; however, the advancement of gene editing to the clinic is limited by the potential genotoxicity of off-target editing. To comprehensively identify potential sites in the genome that may be recognized by gene editing agents, in vitro approaches, in which the editor is combined with human genomic DNA and sites where editing may occur are identified biochemically, are important tools. Existing biochemical approaches for off-target discovery recognize double-stranded breaks generated by nuclease-based gene editors such as SpCas9, but novel approaches are needed for new editing modalities, such as prime editing, that nick one strand of DNA. To fill this gap, we have developed 3'-end ligation sequencing (PEG-seq), which can identify prime editor-induced nicks throughout the genome on in vitro digested human genomic DNA to identify potential off-target sites. Here we show that PEG-seq is an important addition to the off-target detection toolkit, enabling off-target discovery for DNA nicking gene editors such as prime editors.},
}
@article {pmid40757665,
year = {2025},
author = {Carlsson, PO and Hu, X and Scholz, H and Ingvast, S and Lundgren, T and Scholz, T and Eriksson, O and Liss, P and Yu, D and Deuse, T and Korsgren, O and Schrepfer, S},
title = {Survival of Transplanted Allogeneic Beta Cells with No Immunosuppression.},
journal = {The New England journal of medicine},
volume = {393},
number = {9},
pages = {887-894},
doi = {10.1056/NEJMoa2503822},
pmid = {40757665},
issn = {1533-4406},
mesh = {Humans ; Male ; *Diabetes Mellitus, Type 1/therapy/surgery ; *Islets of Langerhans Transplantation/methods ; Transplantation, Homologous ; Gene Editing ; Adult ; *Graft Survival ; CRISPR-Cas Systems ; Insulin/metabolism ; *Insulin-Secreting Cells/transplantation ; },
abstract = {The need to suppress a patient's immune system after the transplantation of allogeneic cells is associated with wide-ranging side effects. We report the outcomes of transplantation of genetically modified allogeneic donor islet cells into a man with long-standing type 1 diabetes. We used clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 12b (Cas12b) editing and lentiviral transduction to genetically edit the cells to avoid rejection; the cells were then transplanted into the participant's forearm muscle. He did not receive any immunosuppressive drugs and, at 12 weeks after transplantation, showed no immune response against the gene-edited cells. C-peptide measurements showed stable and glucose-responsive insulin secretion. A total of four adverse events occurred, none of which were serious or related to the study drug. (Funded by the Leona M. and Harry B. Helmsley Charitable Trust; EudraCT number, 2023-507988-19-00; ClinicalTrials.gov number, NCT06239636.).},
}
@article {pmid40744866,
year = {2025},
author = {Schmal, M and Kramer, LTS and Mach, RL and Mach-Aigner, AR and Zimmermann, C},
title = {Providing a toolbox for genomic engineering of Trichoderma aggressivum.},
journal = {Microbiology spectrum},
volume = {13},
number = {9},
pages = {e0096625},
pmid = {40744866},
issn = {2165-0497},
support = {P 34036//Austrian Science Fund/ ; },
mesh = {*Gene Editing/methods ; CRISPR-Cas Systems ; Genome, Fungal ; Transformation, Genetic ; *Trichoderma/genetics ; *Genetic Engineering/methods ; Hypocreales/genetics ; Agaricus ; Plasmids/genetics ; Fungal Proteins/genetics ; Plant Diseases/microbiology ; Genomics/methods ; },
abstract = {Fungi belonging to the genus of Trichoderma have a long history of infecting crops of edible fungi and reducing the yield. Trichoderma aggressivum is the main causal agent of green mold disease in Agaricus bisporus. Despite its infamous role as a persistent and aggressive contamination in A. bisporus crops, T. aggressivum can also be used as a biocontrol agent or as a promoter of plant growth. In order to work efficiently with T. aggressivum on a molecular biology level, a transformation protocol is required. This study provides a detailed protocol on how to perform a transformation in T. aggressivum using plasmid DNA for ectopic integration. In addition, a Cas9-RNP-based approach has been established for genome editing. We performed two transformations to confirm the usability of the hph gene as well as the pyr4 gene from Trichoderma reesei as selection markers. First, we integrated the hph gene ectopically and determined the overall transformation efficiency. In the second transformation experiment, we disrupted the ornithine-5' phosphate decarboxylase gene of T. aggressivum by using the CRISPR-Cas9 approach. In total, four candidates showed the expected uridine auxotrophy and resistance to 5-FOA. Additionally, the genomic locus around the CRISPR target sites was sequenced to determine the exact circumstances around the gene disruption. To complete the genetic toolbox for T. aggressivum, the pyr4 gene of T. reesei was tested as a suitable selection marker in one of the generated uridine auxotrophic strains.IMPORTANCEResearchers need an efficient tool for genomic manipulation to investigate the fundamental biology of mycoparasitism of T. aggressivum and its correlation to secondary metabolites. We provide a protocol for transformation of T. aggressivum and successfully demonstrated transformation of T. aggressivum using a plasmid and genome editing applying a Cas9-RNP-based strategy. Simultaneously, we established two selection markers, the hph gene and pyr4 gene from T. reesei. By applying these methods, we give researchers the tools needed to investigate T. aggressivum on a deeper level. Possible applications include activation of biosynthetic gene clusters of secondary metabolites to determine the biosynthetic pathway and biotechnological applications of these compounds.},
}
@article {pmid40646716,
year = {2025},
author = {Coirry, C and Manessier, J and Clot, C and Mortier, M and Fauvarque, MO and Taillebourg, E},
title = {The deubiquitinase USP36 funtions through catalytic-dependent and catalytic-independent mechanisms in Drosophila.},
journal = {Genetics},
volume = {231},
number = {1},
pages = {},
pmid = {40646716},
issn = {1943-2631},
support = {ANR-17-EURE-0003//French National Research Agency/ ; //IDEX Universit&#xe9; Grenoble Alpes/ ; //GRAL PhD Operating Costs/ ; },
mesh = {Animals ; *Drosophila Proteins/genetics/metabolism ; Male ; *Drosophila melanogaster/genetics/growth &amp; development ; *Ubiquitin Thiolesterase/genetics/metabolism ; Spermatogenesis/genetics ; CRISPR-Cas Systems ; Female ; Proto-Oncogene Proteins c-myc/metabolism/genetics ; Deubiquitinating Enzymes/metabolism/genetics ; },
abstract = {Deubiquitinases (DUBs) form a specific class of proteases removing ubiquitin from target proteins. They are involved in the regulation of many cellular processes including cell growth and proliferation. Among them, USP36 is a key regulator of the oncogenic transcription factor c-Myc, preventing its degradation by the proteasome. These 2 proteins form an evolutionary conserved complex providing the opportunity to investigate USP36 mechanisms of action in vivo in a genetically tractable model such as Drosophila melanogaster. Null mutants of dUsp36 die early during larval development and exhibit severe growth defects. Strikingly, we report here that flies expressing a catalytically inactive version of dUSP36 produced by CRISPR/Cas9 gene editing survive to adulthood with only minor growth defects, yet males are infertile. This finding indicates that dUSP36 deubiquitinating activity is dispensable for cell growth but essential for spermatogenesis. Our results thus reveal that dUSP36 functions through both catalytic-dependent and catalytic-independent mechanisms, highlighting a dual mode of action with implications for the understanding of DUBs mechanism of action.},
}
@article {pmid40555854,
year = {2025},
author = {Basit, A and Liu, A and Zheng, W and Zhu, J},
title = {A review on the mechanism and potential diagnostic application of CRISPR/Cas13a system.},
journal = {Mammalian genome : official journal of the International Mammalian Genome Society},
volume = {36},
number = {3},
pages = {709-726},
pmid = {40555854},
issn = {1432-1777},
mesh = {*CRISPR-Cas Systems/genetics ; Humans ; Animals ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Bacteria/genetics ; CRISPR-Associated Proteins/genetics ; },
abstract = {Clustered regularly Interspaced short palindromic repeats (CRISPRs) and CRISPR-associated (Cas) proteins form a natural immune defense system in prokaryotic species, with approximately 90% of archaea and 40% of bacteria possessing these systems, highlighting their widespread role in microbial immunity. Among these, the CRISPR/Cas13a system, guided by a single-stranded RNA (crRNA), selectively targets RNA sequences and has shown immense potential in developing sensitive diagnostic tools. Recent advancements have combined Cas13a with amplification methods and lateral flow detection (CRISPR/Cas13a-LFD), improving its application for rapid and accurate RNA detection. In this review, we explore the history, structure, and functional mechanism of the CRISPR/Cas13a system, focusing on its diagnostic capabilities. We compare CRISPR/Cas13a to conventional diagnostic approaches, highlighting their advantages in sensitivity, specificity, speed, and flexibility for point-of-care application. Given the rapid development of CRISPR-based diagnostics in recent years, the Cas13a system shows great potential as a next-generation platform for accurate, portable, and cost-effective detection of viral and bacterial diseases. Furthermore, we address the existing challenges, including reliance upon amplification and off-target effects, and highlight the need for ongoing research to develop amplification-free systems suitable for clinical application.},
}
@article {pmid40541099,
year = {2025},
author = {Tian, J and Wang, Y and Zhou, Z and Li, Y and Chang, C and Meng, F and Shi, W and Li, H and Wang, W and Wei, Q and Liu, H and Yang, Z and Xiao, S},
title = {Genome-wide CRISPR/Cas9 screen identifies host factors for Newcastle disease virus replication.},
journal = {Poultry science},
volume = {104},
number = {9},
pages = {105421},
pmid = {40541099},
issn = {1525-3171},
mesh = {Animals ; *Newcastle disease virus/physiology ; *Virus Replication/genetics ; *CRISPR-Cas Systems ; Mice ; *Newcastle Disease/virology/genetics ; Cell Line ; },
abstract = {Viruses rely strictly on host factors to determine their susceptibility. Newcastle disease virus (NDV), an avian pathogen, can infect a wide range of avian, mammalian and human cell lines. However, NDV can only infect certain mammals and humans, and it does not cause disease in mammals or mice. A comprehensive identification of NDV-susceptible host genes that promote NDV infection is lacking. Here, we performed a mouse genome-wide CRISPR knockout (GeCKO) screening in murine fibroblast L929 cells infected with NDV. Fifty host genes were highly enriched in the screening. Using a viral minigenome assay and gene overexpression as well as single guide RNA (sgRNA) knockout, one of the genes was shown to predominantly promote viral replication. This gene is the solute carrier family 35 member A1 (SLC35A1), which is a cytidine monophosphate (CMP)-Sia transporter involved in the synthesis of sialic acid (SA). Knockout of SLC35A1 in L929 cells decreased the expression of the NDV receptors α2,3-SA and α2,6-SA on the cell surface, resulting in significant reductions in viral adsorption, internalization and replication. Furthermore, the knockout of a six-residue stretch, [82]LGSPKE[87], of SLC35A1 in cells specifically reduced the expression of the α2,6-SA receptor but not the α2,3-SA receptor, which decreased viral adsorption and replication. The reconstitution of SLC35A1 in the knockout cells completely recovered the α2,3-SA receptor, partially recovered the α2,6-SA receptor and almost completely recovered viral replication. In chicken fibroblast DF-1 cells, siRNA-mediated knockdown of chicken SLC35A1 reduced the expression levels of both the α2,6-SA and α2,3-SA receptors, decreasing viral replication. Our research indicated that SLC35A1 is a key host factor that promotes NDV replication. The CRISPR/Cas9 system can be used to identify essential host factors for the replication of intracellular pathogens. This study provides valuable insights into host susceptibility to NDV.},
}
@article {pmid40527130,
year = {2025},
author = {Jin, S and Wang, X and Liu, X and Xu, Y and Wang, W and Chen, W and Chang, H and Li, Z and Geng, Z},
title = {Effective IHH gene knockout by CRISPR/Cas9 system in chicken DF-1 cells.},
journal = {Poultry science},
volume = {104},
number = {9},
pages = {105433},
pmid = {40527130},
issn = {1525-3171},
mesh = {Animals ; *Chickens/genetics ; *CRISPR-Cas Systems ; *Hedgehog Proteins/genetics/metabolism ; *Gene Knockout Techniques/veterinary ; *Avian Proteins/genetics/metabolism ; Cell Line ; },
abstract = {Indian hedgehog (IHH) gene codes an important signal molecule mediating chondrogenesis and bone development in chickens, which are key factors that affect body weight and several other significant economic traits. The aim of this study was to construct an IHH knockout cell model using CRISPR-associated protein 9 (CRISPR/Cas9) technology to further analyze the function of IHH. TA cloning was used to screen the single-guide RNA (sgRNA1) [45 %] and sgRNA3 (30.8 %) with the highest targeting efficiency. Monoclonal cells were selected by flow cytometry for TA cloning sequencing to construct the IHH knockout cell model. Quantitative PCR (qPCR) was used to detect the changes in downstream gene expression levels after IHH knockout. TA cloning sequencing results showed that the IHH knockout cell model was successfully constructed, and two mutation types were generated with a 100 % mutation rate. In addition, qPCR results revealed that the expression of patched 1 (PTCH1), smoothened, frizzled class receptor (Smo), glioma-associated oncogene homolog 1 (Gli1), glioma-associated oncogene homolog 2 (Gli2), and osteopontin (OPN) was significantly lower in the IHH knockout group, while that of type II collagen (Col Ⅱ) was significantly higher. These results lay a theoretical foundation for the successful application of knockout technology in poultry functional genomics research and provide a stable knockout cell line model for further study of chicken IHH gene function.},
}
@article {pmid40891974,
year = {2025},
author = {Chapelin, F},
title = {CRISPR-based Triple-Modality Imaging Ushers a New Era for Stem Cell Tracking in Stroke.},
journal = {Radiology},
volume = {316},
number = {3},
pages = {e252546},
doi = {10.1148/radiol.252546},
pmid = {40891974},
issn = {1527-1315},
mesh = {Humans ; *Cell Tracking/methods ; *Stroke/diagnostic imaging/therapy ; *Multimodal Imaging/methods ; *Clustered Regularly Interspaced Short Palindromic Repeats ; *Stem Cells ; *CRISPR-Cas Systems ; },
abstract = {"Just Accepted" papers have undergone full peer review and have been accepted for publication in Radiology. This article will undergo copyediting, layout, and proof review before it is published in its final version. Please note that during production of the final copyedited article, errors may be discovered which could affect the content.},
}
@article {pmid40891659,
year = {2025},
author = {Takizawa, A and Foeckler, J and Knapp, E and Grzybowski, M and Geurts, AM and Carroll, J and Merriman, DK},
title = {Successful Generation of Germline Tyrosinase Gene Edited Thirteen-Lined Ground Squirrels (Ictidomys tridecemlineatus Mitchill 1821).},
journal = {Molecular reproduction and development},
volume = {92},
number = {9},
pages = {e70055},
doi = {10.1002/mrd.70055},
pmid = {40891659},
issn = {1098-2795},
support = {//This study was supported by the National Eye Institute of the National Institutes of Health (NIH) under award number T32EY014537, U24EY029891 and by the National Center for Advancing Translational Sciences, National Institutes of Health, Award Number UL1TR001436./ ; },
mesh = {Animals ; *Monophenol Monooxygenase/genetics/metabolism ; *Sciuridae/genetics ; *Gene Editing/methods ; CRISPR-Cas Systems ; Female ; Male ; *Germ-Line Mutation ; *Germ Cells/metabolism ; },
abstract = {Thirteen-lined ground squirrels (Ictidomys tridecemlineatus Mitchill 1821; 13-LGS) are useful diurnal rodent models of human cone-mediated vision due to their cone photoreceptor-dominant retinas. To develop the 13-LGS as a better model of inherited human visual disorders, we report a gene-editing protocol targeting the 13-LGS tyrosinase (Tyr) gene. CRISPR/Cas9 microinjection into donor embryos, followed by transfer to pseudo-pregnant recipients, yielded two Tyr-mutated founders. Mating these two to wild-type 13-LGS resulted in 22 offspring, of which five were genotyped with either a 17-bp deletion, 1-bp insertion, or 7-bp deletion Tyr mutation. These results demonstrated that this valuable mammalian model is amenable to germline gene editing by conventional methods.},
}
@article {pmid40891290,
year = {2025},
author = {Deng, Z and Sha, R and Qin, H and Shang, Y and Yuan, A and Xie, W and Peng, H},
title = {A CRISPR Cas protein coronated AuNP nanostructure for enhanced uptake efficiency into cells.},
journal = {Chemical communications (Cambridge, England)},
volume = {},
number = {},
pages = {},
doi = {10.1039/d5cc03658e},
pmid = {40891290},
issn = {1364-548X},
abstract = {The effectiveness of nanotechnologies is often limited by their non-specific aggregation in biological environments. We developed a protein coronated nanostructure by functionalizing AuNPs with nucleic acid scaffolds and CRISPR Cas proteins, significantly enhancing nanoparticle stability and cellular uptake efficiency, making it a promising tool for imaging and biomedical applications.},
}
@article {pmid40890415,
year = {2025},
author = {Laidoudi, Y and Davoust, B and Lepidi, H and Levasseur, A},
title = {Emergence of the zoonotic bacterium Necropsobacter rosorum in nutria Myocastor coypus with implications for wildlife and human health.},
journal = {Scientific reports},
volume = {15},
number = {1},
pages = {32252},
pmid = {40890415},
issn = {2045-2322},
mesh = {Animals ; Humans ; Phylogeny ; Genome, Bacterial ; France/epidemiology ; *Pasteurellaceae/genetics/isolation &amp; purification/classification/pathogenicity ; Animals, Wild/microbiology ; *Zoonoses/microbiology ; Whole Genome Sequencing ; *Rodentia/microbiology ; *Bacterial Zoonoses/microbiology/epidemiology ; },
abstract = {The nutria (Myocastor coypus), a semi-aquatic rodent native to South America, poses significant ecological and agricultural threats as an invasive species in France, where it continues to proliferate despite sustained control efforts. A fatal case of pneumonia in a nutria from Marseille (France) prompted a microbiological investigation that led to the isolation, taxonomic classification, genomic characterization, and phylogenetic analysis of Necropsobacter rosorum. Whole-genome sequencing of the N. rosorum strain RG01 revealed a genome size of 2,505,657 base pairs and 2303 predicted open reading frames, showing high similarity to other publicly available N. rosorum genomes. Comparative pan-genomic analysis indicated a high level of genomic conservation among N. rosorum strains. The presence of putative virulence factors and a CRISPR-Cas system suggests both pathogenic potential and adaptive defense mechanisms against bacteriophage predation. This study also explored the genetic epidemiology of members of the Pasteurellaceae family, highlighting a considerable overlap between species infecting animals and humans. Among the 408,387 sequence records retrieved from GenBank, 62.1% were deemed suitable for genomic epidemiological analysis. Notably, N. rosorum was underrepresented, with only 13 entries spanning nine countries and three host types, revealing critical gaps in current surveillance and research. Collectively, these findings contribute to a better understanding of the microbiology and epidemiology of N. rosorum and Pasteurellaceae-associated infections, and underscore the importance of integrated, genomics-informed approaches for the monitoring, control, and prevention of zoonotic diseases.},
}
@article {pmid40890128,
year = {2025},
author = {Álvarez-Pérez, JC and Sanjuán-Hidalgo, J and Arenas, AM and Hernández-Navas, I and Benitez-Cantos, MS and Andrades, A and Calabuig-Fariñas, S and Jantus-Lewintre, E and Paz-Ares, L and Ferrer, I and Medina, PP},
title = {High-fidelity Cas9-mediated targeting of KRAS driver mutations restrains lung cancer in preclinical models.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {7080},
pmid = {40890128},
issn = {2041-1723},
support = {LAB-AECC-2018//Fundaci&#xf3;n Cient&#xed;fica Asociaci&#xf3;n Espa&#xf1;ola Contra el C&#xe1;ncer (Scientific Foundation, Spanish Association Against Cancer)/ ; B-CTS-480-UGR20, C-EXP-051-UGR23, C-CTS-149-UGR23//Universidad de Granada (University of Granada)/ ; },
mesh = {*Lung Neoplasms/genetics/therapy/pathology ; Animals ; Humans ; *Proto-Oncogene Proteins p21(ras)/genetics/metabolism ; *CRISPR-Cas Systems/genetics ; *Carcinoma, Non-Small-Cell Lung/genetics/therapy/pathology ; Mice ; Xenograft Model Antitumor Assays ; Cell Line, Tumor ; Gene Editing/methods ; *Genetic Therapy/methods ; Mutation ; Female ; Adenoviridae/genetics ; },
abstract = {Missense mutations in the 12[th] codon of KRAS are key drivers of lung cancer, with glycine-to-cysteine (G12C) and glycine-to-aspartic acid (G12D) substitutions being among the most prevalent. These mutations are strongly associated with poor survival outcomes. Given the critical role of KRAS in lung cancer and other cancers, it remains as a major target for the development of new and complementary treatments. We have developed a CRISPR-High Fidelity (HiFi)-Cas9-based therapy strategy that can effectively and specifically target KRAS[G12C] and KRAS[G12D] mutants, avoiding KRAS[WT] off-targeting and affecting KRAS downstream pathways, thereby significantly reducing tumorgenicity. The delivery of HiFiCas9 components via ribonucleoprotein particles (RNPs) and adenovirus (AdV) effectively abrogates cell viability in KRAS-mutant Non-Small Cell Lung Cancer (NSCLC) preclinical models, including 2D and 3D cell cultures, cell-derived xenografts (CDX), and patient-derived xenograft organoids (PDXO). Our in vitro studies demonstrate that HiFiCas9-based therapy achieves superior KRAS inhibition compared to Sotorasib and effectively circumvents certain resistance mechanisms associated with Sotorasib treatment. Moreover, in vivo delivery using adenoviral particles significantly suppresses tumor growth in preclinical NSCLC models. Collectively, our findings establish HiFiCas9 as an effective therapeutic strategy with promising clinical applications, especially if in vivo delivery methods are further optimized.},
}
@article {pmid40825745,
year = {2025},
author = {Zhuang, S and Huang, X and Diao, M and Dong, R and Dong, Y and Liu, J and Liu, Y},
title = {CONVERT: Dynamic crRNA Reconfiguration for Universal One-Pot CRISPR/Cas12a-Based Nucleic Acid Detection.},
journal = {Analytical chemistry},
volume = {97},
number = {34},
pages = {18776-18785},
doi = {10.1021/acs.analchem.5c03577},
pmid = {40825745},
issn = {1520-6882},
mesh = {*CRISPR-Cas Systems ; *CRISPR-Associated Proteins/metabolism/genetics ; *Endodeoxyribonucleases/metabolism/genetics ; *RNA/genetics ; *Bacterial Proteins/metabolism/genetics ; },
abstract = {Current one-pot CRISPR diagnostics necessitate meticulous control of nucleic acid hybridization parameters or extensive modification of CRISPR components to achieve complete enzymatic silencing, a fundamental bottleneck limiting their robustness and generalizability. Here, we resolve this challenge through dynamic crRNA reconfiguration, a paradigm-shifting strategy that exploits the intrinsic structural duality of CRISPR RNA. We present CONVERT (CRISPR One-Pot Nucleic acid detection Via Engineered crRNA Reconfiguration Technology), a universal platform where nontarget intact crRNA acts as a universal suppressor, achieving complete Cas12a inhibition during RPA (recombinase polymerase amplification) by irreversible enzyme sequestration. Target detection is initiated through programmable conversion to split crRNA activators, bypassing sequence-specific design constraints entirely. This crRNA conformational switching mechanism is implemented via photocleavable linker cleavage and subsequent assembly with presupplied truncated crRNA (tcrRNA), activating trans-cleavage signal amplification. Three transformative advances emerge: First, the endogenous crRNA engineering eliminates dependency on exogenous nucleic acid blockers or chemical modifications, reducing optimization costs and time. Second, near-total enzymatic suppression ensures zero cross-talk between amplification and detection phases. Third, the unified workflow enables contamination-resistant operation through spatiotemporal control, requiring no physical partitioning. Validated for Enterocytozoon bieneusi detection, the system demonstrates attomolar sensitivity of 1 aM, 100% diagnostic concordance with qPCR, and absolute specificity against related pathogens. By effectively decoupling CRISPR regulation from sequence-context constraints through rational crRNA structural plasticity, this work establishes a generalized framework for next-generation molecular diagnostics.},
}
@article {pmid40742143,
year = {2025},
author = {Liu, G and Wu, J and Yang, B and Kong, J and Li, Y and Chen, C and Hu, J and Sun, Z and Hou, Y and Sun, R and Li, D},
title = {Ultrasensitive eccDNA Detection for Tumor Diagnostics by Using CRISPR/Cas12a-Coupled Nested PCR.},
journal = {Analytical chemistry},
volume = {97},
number = {34},
pages = {18527-18536},
doi = {10.1021/acs.analchem.5c02316},
pmid = {40742143},
issn = {1520-6882},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Polymerase Chain Reaction/methods ; *DNA, Circular/genetics/blood/analysis ; Limit of Detection ; *Biomarkers, Tumor/genetics/blood ; *Neoplasms/diagnosis/genetics ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {Extrachromosomal circular DNA (eccDNA) has emerged as a novel biomarker for cancer detection due to its tumor-specific amplification and stable structure in circulation. However, its clinical application is hindered by extremely low abundance in biofluids and the lack of robust detection techniques. To address this, we screened for tumor-associated eccDNA biomarkers and developed NPCC (Nested PCR-CRISPR/Cas12a), a novel method combining nested PCR for ultrasensitive amplification with CRISPR/Cas12a for sequence-specific detection. The assay employs two rounds of junction-specific PCR to enrich eccDNA, followed by CRISPR/Cas12a-mediated cleavage guided by target-specific crRNA. Validation using synthetic circular DNA standards demonstrated a limit of detection (LoD) of 10[-6] fM, representing a >100-fold improvement over conventional PCR, with no cross-reactivity to linear or genomic DNA fragments. In plasma samples from 88 cancer patients, NPCC successfully detected multiple tumor-specific eccDNAs, including the hepatocellular carcinoma marker eccDNA-HCC-1 (AUC = 0.8977). NPCC overcomes key technical barriers in liquid biopsy, offering a cost-effective, highly sensitive, and specific platform for noninvasive cancer diagnostics.},
}
@article {pmid40614014,
year = {2025},
author = {Shan, L and He, Q and Zhang, W and Zheng, X and Zhao, J and Yang, P and Mao, Q and Xia, H},
title = {CRISPR/Cas9-mediated promoterless gene targeting reduces lysosome storage in MPS VII mice.},
journal = {Science China. Life sciences},
volume = {68},
number = {9},
pages = {2697-2706},
pmid = {40614014},
issn = {1869-1889},
mesh = {Animals ; *CRISPR-Cas Systems/genetics ; Mice ; Glucuronidase/genetics/metabolism/blood ; *Mucopolysaccharidosis VII/therapy/genetics/metabolism ; *Gene Targeting/methods ; *Lysosomes/metabolism ; Disease Models, Animal ; Genetic Vectors/genetics ; Promoter Regions, Genetic ; Genetic Therapy/methods ; Actins/genetics ; Adenoviridae/genetics ; Liver/metabolism ; },
abstract = {Targeted gene integration mediated by CRISPR/Cas9 is a promising therapeutic strategy for monogenic autosomal recessive diseases. In this study, we established a novel all-in-one high-capacity adenovirus (HCAd) that can pack both CRISPR/Cas9 and donor DNA into the same vector and tested it on a mouse model of mucopolysaccharidosis type VII (MPS VII) caused by mutations in the β-glucuronidase (GUSB) gene. This system allowed targeted integration of promoterless GUSB in the mouse beta-actin gene (mActb) locus and the co-expression of GUSB with the self-cleaving peptide T2A (T2A) controlled by a strong endogenous mActb promoter. The in vivo results indicated that the serum GUSB level of MPS VII mice treated with a single intraperitoneal injection of the HCAd vector achieved 14% of that of wild-type mice, resulting in significant amelioration of lysosomal storage in the liver and spleen. Furthermore, the HCAd was injected intraventricularly in the brain of newborn MPS VII mice, leading to strongly positive GUSB enzyme staining in the choroid plexus and perivascular spaces of the periventricular regions and reduced lysosome storage. In summary, by using an all-in-one vector, the study provides a universal, one-for-all therapeutic for MPSVII, a disease caused by different mutations of the GUSB gene.},
}
@article {pmid40377870,
year = {2025},
author = {Amen, RA and Hassan, YM and Essmat, RA and Ahmed, RH and Azab, MM and Shehata, NR and Elgazzar, MM and El-Sayed, WM},
title = {Harnessing the Microbiome: CRISPR-Based Gene Editing and Antimicrobial Peptides in Combating Antibiotic Resistance and Cancer.},
journal = {Probiotics and antimicrobial proteins},
volume = {17},
number = {4},
pages = {1938-1968},
pmid = {40377870},
issn = {1867-1314},
mesh = {Humans ; *Gene Editing/methods ; *Neoplasms/microbiology/therapy/drug therapy ; *Microbiota ; *Antimicrobial Peptides/pharmacology ; CRISPR-Cas Systems ; Animals ; *Drug Resistance, Microbial ; Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {The growing crisis of antibiotic resistance and the increasing incidence of cancer have prompted the exploration of innovative approaches, such as gene editing and antimicrobial peptides (AMPs). The human microbiome is integral to various aspects of health, disease, and therapeutic development, influencing metabolic pathways, immune function, and pathogen resistance. Recent advances in gene editing technologies, particularly CRISPR (clustered regularly interspaced short palindromic repeats), have opened new avenues for leveraging the microbiome to address complex medical challenges, including combating multidrug-resistant pathogens and cancer. The microbiome plays a crucial role in combating antibiotic resistance by modulating microbial communities, influencing pathogen survival and susceptibility to treatments. This review explores the microbiome's dynamic role in metabolic regulation, its contribution to cancer management, and how AMPs help maintain homeostasis and exhibit emerging anticancer properties, supported by both preclinical findings and clinical evidence. Additionally, CRISPR-based microbiome engineering offers potential to enhance host-microbiome interactions, optimizing therapeutic outcomes. The integration of microbiome metagenomics and proteomics has led to the discovery of novel AMPs with targeted anticancer effects. Innovative strategies, such as engineered probiotics and CRISPR-based microbiome engineering, present exciting prospects for next-generation therapies. Despite these advances, the translation of microbiome-based therapies into clinical settings remains challenging due to ethical, regulatory, and ecological hurdles. This review underscores the transformative potential of microbiome-based interventions, emphasizing the role of personalized medicine in maximizing therapeutic efficacy. Furthermore, we also address critical research gaps, limitations, and future directions, including optimizing AMP stability, delivery, and bioavailability, as well as overcoming the regulatory and ethical challenges in clinical translation.},
}
@article {pmid40326293,
year = {2025},
author = {Lazarides, K and Engel, JL and Meseonznik, M and Feng, T and Choi, AH and Yu, Y and Liu, S and Meier, SR and Zhang, H and Shen, B and Tjin Tham Sjin, R and Whittington, DA and McMillan, BJ and Doyon, B and Pan, X and Wilker, E and Huang, A and Andersen, JN and Mallender, WD and Bandi, M},
title = {CRISPR Screens Identify POLB as a Synthetic Lethal Enhancer of PARP Inhibition Exclusively in BRCA-Mutated Tumors.},
journal = {Molecular cancer therapeutics},
volume = {24},
number = {9},
pages = {1466-1479},
doi = {10.1158/1535-7163.MCT-24-0822},
pmid = {40326293},
issn = {1538-8514},
mesh = {Humans ; *Poly(ADP-ribose) Polymerase Inhibitors/pharmacology ; Animals ; Mice ; *BRCA2 Protein/genetics ; *Synthetic Lethal Mutations ; Female ; Cell Line, Tumor ; *BRCA1 Protein/genetics ; Xenograft Model Antitumor Assays ; CRISPR-Cas Systems ; Mutation ; Apoptosis/drug effects ; },
abstract = {PARP inhibitors (PARPi) are an approved class of anticancer therapeutics that inhibit the activities of PARP1/2 and produce synthetic lethality in BRCA1/2-mutated cancers because of the absence of a functional homologous recombination-dependent DNA repair pathway. Although PARPis have led to successful clinical outcomes, two thirds of patients develop acquired resistance, limiting long-term utility as maintenance therapy. Motivated by this clinical need, we utilized a CRISPR target discovery screening platform to identify DNA polymerase beta (POLB) as a gene that acts selectively and synergistically with PARPis in BRCA1/2-mutated cancers and found that POLB knockout (KO) along with PARPi treatment enhanced loss of viability in BRCA1/2-mutant and BRCA2-null cells but not in isogenic BRCA1/2 wild-type cells. Overexpression of either POLB wild-type or catalytically inactive mutants confirmed that perturbation of both the polymerase and lyase catalytic activities of POLB are required for synergistic PARP-BRCA synthetic lethality. Mechanistically, POLB KO was associated with an increase in single- and double-strand DNA breaks, cell-cycle arrest, and apoptosis when in combination with PARP inhibition. The translational nature of this interaction was further examined using murine xenograft models of BRCA1-mutant and BRCA2-null cell lines, wherein the combination of POLB KO and niraparib led to profound tumor regression and prevented tumor regrowth even after cessation of treatment. Together, these results suggest that POLB is a synergistic enhancer of the synthetic lethal interaction between PARP and BRCA and support POLB as a promising therapeutic target for improving antitumor responses to PARPis in homologous recombination-deficient cancers.},
}
@article {pmid40889353,
year = {2025},
author = {Yu, Y and Yuan, Q and Liu, Z and Tong, B and Shi, S},
title = {Enhancement of Free Fatty Acids Production in Rhodotorula toruloides Using the CRISPR/Cas9-Based Base Editor.},
journal = {ACS synthetic biology},
volume = {},
number = {},
pages = {},
doi = {10.1021/acssynbio.5c00359},
pmid = {40889353},
issn = {2161-5063},
abstract = {Rhodotorula toruloides is a promising cell factory to produce various value-added chemicals, including fatty acid derivatives. However, their metabolic engineering development has been hindered by the limited availability of genetic tools. In this study, an accurate and specific gene-editing tool, CRISPR/Cas-based cytidine base editor (CBE) system, was developed for the first time in R. toruloides to broaden its genetic toolbox. The target gene was disrupted by introducing a premature stop codon via C to T mutation. This system achieved single-gene disruption efficiencies of up to 90% and successfully disrupted four genes in parallel with 5% efficiency, marking a breakthrough in multiplexed editing for this yeast. To enable iterative engineering, an inducible Cre-loxP system was integrated, achieving an over 70% selection marker recycling efficiency. Application of this system enabled the construction of uracil-auxotrophic strains. Furthermore, the CBE system was employed to disrupt four genes involved in lipid metabolism, resulting in an engineered strain capable of producing 512.3 mg/L of free fatty acids, thereby demonstrating the utility of the CBE system as an efficient genome editing tool in R. toruloides. The study provides valuable tools to expand the genetic toolbox of R. toruloides and paves the way for fully exploiting its metabolic engineering potential.},
}
@article {pmid40888633,
year = {2025},
author = {Kelleher, AM and Kim, HI and Bayammagari, GS and Davis, DJ and Spencer, TE},
title = {A Cxcl15 Cre Recombinase Mouse Model Useful to Study Gland Development in the Uterus.},
journal = {Genesis (New York, N.Y. : 2000)},
volume = {63},
number = {5},
pages = {e70026},
doi = {10.1002/dvg.70026},
pmid = {40888633},
issn = {1526-968X},
support = {HD042311/NH/NIH HHS/United States ; HD112315/NH/NIH HHS/United States ; },
mesh = {Animals ; Female ; *Integrases/genetics/metabolism ; Mice ; Hepatocyte Nuclear Factor 3-beta/genetics/metabolism ; *Uterus/metabolism/growth &amp; development ; *Endometrium/metabolism/growth &amp; development ; Pregnancy ; CRISPR-Cas Systems ; },
abstract = {The mammalian uterus contains glands in the endometrium that develop only or primarily after birth. In the mouse, endometrial glands govern post implantation pregnancy establishment via regulation of blastocyst implantation, stromal cell decidualization, and placental development. Here, we describe a new uterine glandular epithelium (GE) specific Cre recombinase mouse line that is useful to study endometrial gland development and function. Utilizing CRISPR-Cas9 genome editing, improved Cre recombinase (iCre) was inserted into the endogenous C-X-C motif chemokine ligand 15 (Cxcl15) gene. Cxcl15 mRNA, Cxcl15 protein, and Cxcl15-iCre recombinase activity were specific to the developing GE of the uterus. Cxcl15-iCre mice were crossed with floxed Foxa2 mice to conditionally delete Foxa2 specifically in the glands of the neonatal mouse uterus. This conditional deletion of Foxa2 in the developing neonatal uterus resulted in adult mice that lacked Foxa2 in the GE of the uterus, and the adult mice were infertile. The studies described here establish that Cxcl15-iCre mice are a valuable resource to elucidate and explore mechanisms regulating the development and function of glands in the uterus.},
}
@article {pmid40887498,
year = {2025},
author = {Gwon, LW and Badon, IW and Lee, Y and Kim, HJ and Lee, SH},
title = {Advances in large-scale DNA engineering with the CRISPR system.},
journal = {Experimental &amp; molecular medicine},
volume = {},
number = {},
pages = {},
pmid = {40887498},
issn = {2092-6413},
support = {the Chung-Ang University Research Grants in 2024//Chung-Ang University (CAU)/ ; },
abstract = {In recent years, DNA engineering technology has undergone significant advancements, with clustered regularly interspaced short palindromic repeats (CRISPR)-based target-specific DNA insertion emerging as one of the most rapidly expanding and widely studied approaches. Traditional DNA insertion technologies employing recombinases typically involve introducing foreign DNA into genes in vivo by either pre-engineering recognition sequences specific to the recombinase or through genetic crossing to incorporate the requisite recognition sequence into the target gene. However, CRISPR-based gene insertion technologies have advanced to streamline this engineering process by combining the CRISPR-Cas module with recombinase enzymes. This process enables accurate and efficient one-step insertion of foreign DNA into the target gene in vivo. Here we provide an overview of the latest developments in CRISPR-based gene insertion technologies and discusses their potential future applications.},
}
@article {pmid40885801,
year = {2025},
author = {Zirman, A and Abed El-Nabi, M and Samuel, E and Anavy, L and Yakhini, Z and Nachman, I and Rak, R},
title = {Pooled CRISPR screens identifies key regulators of bovine stem cell expansion for cultured meat.},
journal = {Communications biology},
volume = {8},
number = {1},
pages = {1313},
pmid = {40885801},
issn = {2399-3642},
support = {20-04-0001//Ministry of Agriculture and Rural Development (Israeli Ministry of Agriculture and Rural Development)/ ; },
mesh = {Animals ; Cattle ; Cell Proliferation/genetics ; *Mesenchymal Stem Cells/cytology/metabolism/physiology ; *CRISPR-Cas Systems ; Cell Differentiation/genetics ; Cells, Cultured ; *Meat ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Gene Knockout Techniques ; PTEN Phosphohydrolase/genetics ; Tumor Suppressor Protein p53/genetics ; In Vitro Meat ; },
abstract = {Cultured meat presents a sustainable alternative to traditional meat production but faces significant challenges in scalability and cost efficiency. A key limitation is the restricted proliferation capacity of bovine mesenchymal stem cells (bMSCs), a widely used cell source in the field. Using a pooled, lentiviral CRISPR knockout screen, we interrogated 3000 CRISPR guides targeting 600 genes involved in stem cell regulation or proliferation. Notably, knockouts of TP53 and PTEN significantly increased proliferation rates and delayed senescence. Validation with individual gene knockouts confirms these effects, showing enhanced growth but reduced differentiation potential. We also identified chondrogenic differentiation as a promising target whose repression may further promote MSC expansion. These findings demonstrate the utility of CRISPR screening for optimizing bovine stem cell traits and offer a path toward more scalable cultured meat production in the future.},
}
@article {pmid40669350,
year = {2025},
author = {Suvá, M and Bastón, JI and Wiedenmann, EA and Pose Ortiz de Rozas, MB and Jordán, R and Ghetti, A and Viale, DL and Moro, LN and Vichera, GD},
title = {Use of an exogenous DNA-free system to generate MSTN-KO calves by CRISPR/Cas9 and SCNT.},
journal = {Reproductive biology},
volume = {25},
number = {3},
pages = {101050},
doi = {10.1016/j.repbio.2025.101050},
pmid = {40669350},
issn = {2300-732X},
mesh = {Animals ; Cattle/genetics ; *Nuclear Transfer Techniques/veterinary ; *Myostatin/genetics ; *CRISPR-Cas Systems ; *Gene Editing/veterinary/methods ; *Gene Knockout Techniques/veterinary ; Animals, Genetically Modified ; Female ; Fibroblasts ; Male ; },
abstract = {This study aimed to obtain myostatin (MSTN)-knockout calves, while avoiding the risk of exogenous DNA integration during gene editing. To achieve this, we combined CRISPR/Cas9 ribonucleoprotein gene editing with somatic cell nuclear transfer (SCNT) technology. In the first experiment (E1), we compared the gene editing efficiency of four gRNAs targeting different coding regions of the MSTN gene using plasmid-based CRISPR/Cas9 in bovine fetal fibroblasts (BFF-E1 cells). The highest bioinformatically-predicted editing rate (BPE) was obtained with gRNA2 (96 %), which was subsequently used for further experiments. Next, embryos were produced by SCNT using BFF-E1-edited cells as nuclear donors. Sanger sequencing of the embryos showed biallelic MSTN editing. In the second experiment (E2), plasmid-based editing was replaced with CAS9 protein and trac:crRNA oligoribonucleotides. Editing efficiency was assessed on one edited bovine fetal fibroblast line (BFF-E2-male[ed]) and two edited bovine mesenchymal stem lines (MSC-E2-male[ed] and MSC-E2-fem[ed]) derived from price-winning animals. BPEs were 58.8 %, 31 % and 59 % in fibroblast and MSC cells, and 64 %, 73.3 %, and 66.6 % in SCNT embryos derived from BFF-E2-male[ed], MSC-E2-male[ed] and MSC-E2-fem[ed], respectively. Transfer of 35 MSC-E2-fem[ed] embryos to recipient cows, resulted in the birth of one MSTN-edited calf with a heterozygous genotype. A second-generation clone was subsequently produced, using a fibroblast sample as nuclear donor. In conclusion, we established an efficient protocol for generating high rates of edited blastocysts with a desirable genetic background, resulting in the birth of two MSTN-knockout calves. This study provides a foundation for gene editing to improve productive or biomedical traits.},
}
@article {pmid40639438,
year = {2025},
author = {Bhatia, P and Mohd, A and Agrawal, I and Katiyar, H and Goel, A and Aich, M and Chakraborty, D and Veerapu, NS},
title = {Rapid and efficient generation of viral genome knock-in cell lines using the CRISPR-Cas9 system to produce infectious virus.},
journal = {Journal of virological methods},
volume = {338},
number = {},
pages = {115219},
doi = {10.1016/j.jviromet.2025.115219},
pmid = {40639438},
issn = {1879-0984},
mesh = {*CRISPR-Cas Systems ; Humans ; *Genome, Viral ; Cell Line ; *Gene Knock-In Techniques/methods ; *Hepatitis B virus/genetics ; Genotype ; Virus Replication ; Hepacivirus/genetics ; Antiviral Agents/pharmacology ; Gene Editing ; },
abstract = {Several medically significant viruses are difficult to propagate with conventional laboratory host systems, limiting their availability for detailed characterization, antiviral screening, and functional studies. A range of methods can be used to generate viruses, such as creating sophisticated cell lines, organoid cultures, and the utilization of animal models. Here, we report the generation and characterization of CRISPR-Cas9 edited Huh7 stable cell lines engineered to carry and express overlength HBV genotypes A, B, C and D and full HEV genomes in the AAVS1 site. Viral polymerase inhibitors and IFN-α significantly reduced the production of viral genomes and proteins from the edited cells. The virus released by the edited cells was infectious in permissive cell lines and could be blocked by neutralizing antibodies. This approach can extend to other viruses, like HCV genotype 3, that are hard to culture or to culturable viruses, like Dengue, for vaccine production.},
}
@article {pmid40629854,
year = {2025},
author = {Lin, ZC and Yang, SC and Tran, TTP and Fang, JY},
title = {Animal models of psoriasis for novel drug discovery: a literature update.},
journal = {Expert opinion on drug discovery},
volume = {20},
number = {9},
pages = {1193-1208},
doi = {10.1080/17460441.2025.2528959},
pmid = {40629854},
issn = {1746-045X},
mesh = {*Psoriasis/drug therapy/physiopathology/genetics/pathology ; Animals ; Humans ; *Disease Models, Animal ; *Drug Discovery/methods ; Imiquimod/administration &amp; dosage ; Mice ; Gene Editing/methods ; CRISPR-Cas Systems ; Cytokines/metabolism ; Mice, Knockout ; },
abstract = {INTRODUCTION: Psoriasis is a chronic, immune-mediated inflammatory skin disorder with a multifactorial pathogenesis involving keratinocyte proliferation, dysregulated immune responses, and vascular remodeling. The development of effective therapeutics mainly relies on preclinical models that can reproduce disease-relevant mechanisms.<br><br>AREAS COVERED: This review outlines current in vivo psoriasis models, including spontaneous mutation models, transgenic and knockout mice, xenotransplantation systems, and cytokine-induced and imiquimod-induced models. Each model is evaluated for its ability to replicate key histological and immunological features of human psoriasis, such as acanthosis, immune cell infiltration, and cytokine network activation. The utility of CRISPR/Cas9 gene editing in generating targeted models is also discussed, thus highlighting its potential use for mechanistic studies. Finally, this review also emphasizes the limitations in translational applicability and the need for multimodel validation strategies regarding psoriasis. This article was based on a comprehensive literature search using PubMed, Scopus, and Google Scholar databases, covering publications from January 2015 to March 2025.<br><br>EXPERT OPINION: Despite extensive model development, no single system fully mimics human psoriatic disease. The imiquimod-induced model remains widely used due to its practicality, although it better reflects acute inflammation compared with chronic pathology. The combination of complementary models and the incorporation of human-derived tissues or immune components may improve translational relevance. Advances in genome editing and humanized systems are likely to shape the future of psoriasis research and therapeutic discovery.},
}
@article {pmid40885742,
year = {2025},
author = {Valdez, I and O'Connor, I and Patel, D and Gierer, K and Harrington, J and Ellis, E and Caponetti, SA and Sebra, RP and Valley, HC and Coote, K and Mense, M and Marro, SG and Jiang, T},
title = {A streamlined base editor engineering strategy to reduce bystander editing.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {8115},
pmid = {40885742},
issn = {2041-1723},
support = {R00HL153940//U.S. Department of Health &amp; Human Services | NIH | National Heart, Lung, and Blood Institute (NHLBI)/ ; 005363I223//Cystic Fibrosis Foundation (CF Foundation)/ ; },
mesh = {*Gene Editing/methods ; Humans ; CRISPR-Cas Systems/genetics ; CRISPR-Associated Protein 9/metabolism/genetics ; HEK293 Cells ; Cystic Fibrosis Transmembrane Conductance Regulator/genetics ; Adenosine Deaminase/genetics/metabolism ; },
abstract = {Base editing (BE) can permanently correct over half of known human pathogenic genetic variants without requiring a repair template, thus serving as a promising therapeutic tool to treat a broad spectrum of genetic diseases. However, the broad activity windows of current base editors pose a major challenge to their therapeutic application. Here, we show that integrating a naturally occurring oligonucleotide binding module into the deaminase active center of TadA-8e, a highly active deoxyadenosine deaminase, enhances its editing specificity. When conjugated with a Cas9 nickase or alternative PAM Cas9 variants, the engineered TadA variant-TadA-NW1-consistently achieves robust A-to-G editing efficiencies within an editing window consisting of four nucleotides, substantially narrower than the 10-bp editing window of the TadA-8e-derived ABEs. Moreover, compared to ABE8e, ABE-NW1 shows significantly decreased Cas9-dependent and -independent off-target activity while maintaining similar on-target editing efficiency. Further, TadA-NW1 can be reprogrammed to perform desired cytidine deamination and adenine transversion within a restricted editing window. Finally, in a cystic fibrosis (CF) cell model, ABE-NW1 outperforms existing ABEs in accurately and efficiently correcting the CFTR W1282X variant, one of the most common CF-causing mutations. In all, we engineered a suite of base editors with refined activity windows, enabling more precise base editing. Importantly, this study presents a streamlined genome editor re-engineering strategy to accelerate the development of therapeutic base editing.},
}
@article {pmid40885054,
year = {2025},
author = {Tang, Z and Gong, F and Feng, Y and Shan, X and Yi, K and Xu, H and Zhou, F and Ji, X and He, Z},
title = {Enhancing CRISPR/Cas-mediated detection of nucleic acids using PNIPAM-based reporters.},
journal = {Biosensors &amp; bioelectronics},
volume = {289},
number = {},
pages = {117917},
doi = {10.1016/j.bios.2025.117917},
pmid = {40885054},
issn = {1873-4235},
abstract = {Currently most conventional reporters in CRISPR/Cas system, including fluorophore-quencher (FQ) and magnetic bead (MB)-based reporters, encounter limitations in terms of sensitivity and compatibility. To overcome these challenges, we developed novel reporters for CRISPR/Cas systems based on thermo-responsive poly(N-isopropylacrylamide) (PNIPAM). Below the lower critical solution temperature (LCST), PNIPAM-based reporters exhibited a liquid state and can be cleaved by Cas proteins in a homogeneous reaction, preserving function and structure of Cas proteins while effectively accelerating the reaction kinetics. Based on this, we designed three dual-enzyme amplification strategies for ultra-sensitive RNA detection, where RNA-activated LbuCas13a cleaved PNIPAM-based reporters to release enzymes (HRP or ALP) or Cas12a activators as the first amplification, followed by thermal separation to initiate secondary enzymatic amplification. Using SARS-CoV-2 RNA as a model target, these strategies achieved a limit of detection (LOD) as low as ∼1 fM, representing a 100-fold improvement over the traditional CRISPR/Cas13a system, while its excellent practical applicability was validated by spiked recovery assay and specific analysis. Overall, this work proposed novel PNIPAM-based reporters that not only could be applied to various individual CRISPR/Cas systems, but also enable integration with downstream amplification steps through their thermal separation properties, advancing the development of next-generation CRISPR/Cas-based molecular diagnostic tools.},
}
@article {pmid40884843,
year = {2025},
author = {Hou, X and Wang, T and Li, Y and Cui, A and Kong, Y and Zhu, Y and Fang, H and Wang, C and Liao, W},
title = {NaCl promotes tomato fruit coloring by relieving SlSR3-induced transcriptional inhibition of lycopene synthesis-related genes.},
journal = {The Plant journal : for cell and molecular biology},
volume = {123},
number = {5},
pages = {e70450},
doi = {10.1111/tpj.70450},
pmid = {40884843},
issn = {1365-313X},
support = {31860568//the National Natural Science Foundation of China/ ; 32072559//the National Natural Science Foundation of China/ ; 32260353//the National Natural Science Foundation of China/ ; 32360743//the National Natural Science Foundation of China/ ; YB2022004//the Fostering Foundation for the Excellent PH.D. Dissertation of Gansu Agricultural University/ ; GSCS-2022-Z03//the Research Program Sponsored by the State Key Laboratory of Aridland Crop Science of China/ ; },
mesh = {*Solanum lycopersicum/genetics/metabolism/drug effects/physiology ; *Lycopene/metabolism ; *Fruit/metabolism/genetics/drug effects/physiology ; *Plant Proteins/metabolism/genetics ; Gene Expression Regulation, Plant/drug effects ; *Sodium Chloride/pharmacology/metabolism ; Chlorophyll/metabolism ; Carotenoids/metabolism ; Transcription Factors/metabolism/genetics ; Pigmentation/drug effects ; Salt Stress ; },
abstract = {Although salt stress has an adverse effect on plant growth and development, mild salt stress acts as an elicitor of biosynthesis and thus improves fruit quality. To date, the role and mechanism of NaCl in accelerating tomato (Solanum lycopersicum) fruit coloring remain unclear. This study found that 50 mM NaCl treatment (moderate salt stress) reduced the chlorophyll content, increased the carotenoid and lycopene content, and accelerated tomato color transition without decreasing yield. Moreover, NaCl treatment downregulated calmodulin-binding transcription activator (CAMTA5)/signal responsive (SR3). Knockout of SlSR3 by CRISPR/Cas 9 (sr3 mutant) accelerated chlorophyll degradation and carotenoid and lycopene accumulation and upregulated chlorophyll degradation (PPH) and lycopene synthesis (PSY2, PDS, and ZDS) genes in tomato fruit, thereby accelerating tomato coloring. However, SlSR3 overexpression had the opposite effect. Although NaCl treatment decreased chlorophyll, increased carotenoids, and upregulated PPH, PSY2, PDS, and ZDS in wild type and OE-sr3 fruit, these changes were not observed in sr3 mutant fruit. Therefore, PPH, PSY2, PDS, and ZDS might be involved in SR3-regulated coloring under moderate salt stress. Further results showed that SlSR3 could directly bind to the promoter of PSY2 and ZDS via the CG-1 domain, thereby downregulating PSY2 and ZDS. However, NaCl treatment reversed the transcriptional inhibition of SlSR3 on PSY2 and ZDS expression, thereby upregulating PSY2 and ZDS. Collectively, our results suggest that the promoting effects of NaCl on fruit coloring may be dependent on SlSR3-induced transcriptional regulation of lycopene synthesis-related genes PSY2 and ZDS. Therefore, our study provides a better understanding of the function of SlSR3 in tomato coloring and offers insights on the molecular mechanism underlying the effects of moderate salt stress on tomato color transformation.},
}
@article {pmid40884799,
year = {2025},
author = {Yu, Y and Jin, M and Yuan, W and Gong, Y and Li, S and Qin, X and Hou, J and Liu, J and Liu, S and Li, H and Chu, Y and Wang, Y and Zhang, Y and Fang, F and Hao, W and Gu, Y and Fan, Q and Lin, J and Pang, D and Zhang, X},
title = {Engineered crRNA Drives RPA-T7-CRISPR/Cas14a Cascade for Ultrasensitive Detection of ctDNA PIK3CA H1047R.},
journal = {Advanced science (Weinheim, Baden-Wurttemberg, Germany)},
volume = {},
number = {},
pages = {e07126},
doi = {10.1002/advs.202507126},
pmid = {40884799},
issn = {2198-3844},
support = {82073410 82272623//National Natural Science Foundation of China/ ; 2023ZX06C10//Key Special Projects of Heilongjiang Province Key Research and Development Program/ ; 2017-02//Nn10 Program of Harbin Medical University Cancer Hospital/ ; CYQN24010//Spring Goose Support Program of Heilongjiang Province/ ; },
abstract = {The early detection of circulating tumor DNA (ctDNA) at mutant allele frequencies below 0.1% remains a critical challenge, significantly impeding therapeutic decision-making. To address this limitation, TIDE-Cas14a-an innovative CRISPR/Cas14a-based duplex detection system is developed that integrates recombinase polymerase amplification (RPA) with T7 exonuclease-mediated strand displacement. By strategically engineering crRNAs with synthetic mismatches, the platform achieves single-nucleotide resolution, enabling specific discrimination of the PIK3CA H1047R (c.3140A>G) variant from other mutant subtypes and wild-type sequences at a detection limit of 0.01% with attomolar sensitivity. The system leverages T7 exonuclease's 5'→3' digestion to convert RPA amplicons into single-stranded targets, thereby activating Cas14a without requiring thermal cycling. Furthermore, clinical validation using 32 breast cancer patient samples demonstrated that TIDE-Cas14a achieves 100% sensitivity and specificity, comparable to droplet digital PCR. When deployed on a low-cost digital microfluidic chip, the assay completes ctDNA profiling within 60 min at 37 °C, effectively bridging the gap between complex laboratory testing and point-of-care diagnostics. The work repurposes the CRISPR/Cas system's inherent specificity constraints as a precision oncology tool, establishing a scalable platform for early cancer detection and therapeutic monitoring.},
}
@article {pmid40829362,
year = {2025},
author = {Anwar, S and Khan, S and Azmi, I and Islam, KU and Ahmad, T and Iqbal, J},
title = {CRISPR-based molecular detection of SARS-CoV-2, its emerging variants, and diverse pathogens.},
journal = {Diagnostic microbiology and infectious disease},
volume = {113},
number = {4},
pages = {117062},
doi = {10.1016/j.diagmicrobio.2025.117062},
pmid = {40829362},
issn = {1879-0070},
mesh = {Humans ; *SARS-CoV-2/genetics/isolation &amp; purification ; *COVID-19/diagnosis/virology ; Sensitivity and Specificity ; *Molecular Diagnostic Techniques/methods ; *CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; *COVID-19 Nucleic Acid Testing/methods ; Point-of-Care Testing ; },
abstract = {Pathogenic viruses such as SARS-CoV-2 (SCoV-2), continue to pose a significant threat to human civilization. The lessons learnt from SCoV-2 infections have highlighted the requirement for robust and readily available diagnostic tools in order to limit the virus transmission and prevent future pandemics such as COVID-19. RT-qPCR-based detection is routinely used for sensitive and accurate diagnosis, which requires a sophisticated instrument, laboratory setup, and technical expertise. Though RT-qPCR is highly reliable and considered the gold standard for pathogen detection, it is costly, time-consuming, and unaffordable for the masses. Therefore, other reliable methods for nucleic acid-based detection with sensitivity, specificity, and accuracy on-par with RT-qPCR are required. Recent advancement in CRISPR technology promises its development as a POC testing device, providing a high-end, instrument-free, portable, and cost-effective workflow. Further, COVID-19 pandemic has encouraged the development of next-generation CRISPR-based diagnostics with a provision for home-testing which has resulted in the development of portable and smart-phone integrated hand-held devices which can detect various pathogenic infections in a shorter time frame than RT-qPCR. For diagnosing the presence of SCoV-2, CRISPR-based diagnostics (SHERLOCK/DETECTR) are quicker (30-60 min), less expensive ($5-15/test), and portable than RT-qPCR (90-180 min; $10-50/test) demonstrating equivalent specificity (100%) and near-equivalent sensitivity (93-100% for CRISPR-based diagnostics vs 95-100% for RT-qPCR). For high-sensitivity centralized testing, RT-qPCR is still the gold standard, but CRISPR works well in point-of-care settings because it requires little equipment (like lateral flow strips or heating blocks) and allows multiplexing. CRISPR-based diagnostics breakthrough platform like CARMEN leverages microfluidic technology to test 5,000 plus samples in a single run, unlike RT-qPCR, which requires separate reactions for each target.In this review, the advancement in CRISPR technology such as SHERLOCK, DETECTR, and other Cas-9-based diagnostics are highlighted which exclusively focuses on the CRISPR-based diagnostics to detect SCoV-2 and its emerging VOCs, highlighting their advantages and limitations compared to the gold-standard RT-qPCR.},
}
@article {pmid40784045,
year = {2025},
author = {Abdel-Malek, K and von Eisenhart-Rothe, F and Stiegmann, S and Gottmann, I and Doppler, SA and Schneider, S and Preisler, H and Lahm, H and Dreßen, M},
title = {Generation of two B2M knockout induced pluripotent stem cell lines (DHMi005-A-8 and DHMi005-A-9) using CRISPR/Cas9 technology.},
journal = {Stem cell research},
volume = {87},
number = {},
pages = {103785},
doi = {10.1016/j.scr.2025.103785},
pmid = {40784045},
issn = {1876-7753},
mesh = {*Induced Pluripotent Stem Cells/metabolism/cytology ; *CRISPR-Cas Systems/genetics ; Humans ; *beta 2-Microglobulin/genetics/metabolism/deficiency ; *Gene Knockout Techniques ; Cell Line ; },
abstract = {The transplantation of cells into a recipient organism has many hurdles to overcome, including the problem of T-cell-triggered cellular immune defense. Cellular rejection is based on antigen presentation by the MHC-I-complex, which is recognized by cytotoxic T lymphocytes. Elimination of the MHC-I-complex by knocking out the B2M (Beta-2-microglobulin) subunit may be one way of reducing or even completely preventing the initial cellular immune response during transplantation. Using CRISPR/Cas9 we established one heterozygous and one homozygous B2M knockout induced pluripotent stem cell (iPSC) line as a first step towards more effective cell transplantation.},
}
@article {pmid40752255,
year = {2025},
author = {Ramachandran, H and Thomas, AC and Binder, S and Hildebrandt, B and Spitali, P and Rossi, A},
title = {Generation of two iPSC lines with pathogenic DMD nonsense mutations c.4729C&gt;T and c.5713G&gt;T.},
journal = {Stem cell research},
volume = {87},
number = {},
pages = {103789},
doi = {10.1016/j.scr.2025.103789},
pmid = {40752255},
issn = {1876-7753},
mesh = {*Induced Pluripotent Stem Cells/metabolism/cytology ; Humans ; *Codon, Nonsense/genetics ; *Muscular Dystrophy, Duchenne/genetics/pathology ; Cell Line ; *Dystrophin/genetics ; CRISPR-Cas Systems ; Cell Differentiation ; },
abstract = {Duchenne muscular dystrophy (DMD) is an X-linked genetic disorder characterized by progressive degeneration of skeletal and cardiac muscles, typically beginning in early childhood. Here, we describe the generation of two isogenic induced pluripotent stem cell (iPSC) lines engineered using CRISPR-Cas12 to introduce specific nonsense mutations in the DMD gene: c.4729C>T (p.Arg1577Ter) and c.5713G>T (p.Arg1905Ter). The edited iPSC lines retain normal karyotypes, express key pluripotency markers, and exhibit the capacity to differentiate into derivatives of all three germ layers. These models provide powerful tools for investigating DMD pathogenesis, uncovering mechanisms of genetic compensation, and evaluating potential therapeutic strategies.},
}
@article {pmid40752254,
year = {2025},
author = {Ludwik, KA and Valone, VF and Jahn, R and Jyrch, S and Lechner, L and Kühnen, P and Stachelscheid, H},
title = {Generation of an isogenic iPSC line via CRISPR correction of the POMC:W84X mutation for monogenic obesity modeling.},
journal = {Stem cell research},
volume = {87},
number = {},
pages = {103786},
doi = {10.1016/j.scr.2025.103786},
pmid = {40752254},
issn = {1876-7753},
mesh = {*Induced Pluripotent Stem Cells/metabolism/cytology ; Humans ; *Pro-Opiomelanocortin/genetics/metabolism ; *Obesity/genetics/pathology/metabolism ; *CRISPR-Cas Systems/genetics ; *Mutation/genetics ; Cell Line ; Gene Editing ; Cell Differentiation ; },
abstract = {We report the generation of a genetically corrected induced pluripotent stem cell (iPSC) line, BIHi261-A-1, derived from the patient-specific iPSC line BIHi261-A carrying a homozygous truncating mutation in the POMC gene (POMC:W84X). This mutation causes monogenic obesity by disrupting proopiomelanocortin function. The pathogenic variant was corrected using CRISPR-Cas9 editing. The resulting iPSC line maintained a normal karyotype, expressed pluripotency markers, and retained the ability to differentiate into all three germ layers. BIHi261-A-1 provides valuable isogenic control for disease modeling and therapeutic research targeting POMC-related obesity and hypothalamic regulation of energy homeostasis.},
}
@article {pmid40752251,
year = {2025},
author = {Sikora, T and Patraskaki, M and Howden, S and Graham, A and Christodoulou, J and Linster, CL and Van Bergen, NJ},
title = {Generation and characterisation of four human NAD(P)HX epimerase (NAXE) knockout iPSC lines.},
journal = {Stem cell research},
volume = {87},
number = {},
pages = {103782},
doi = {10.1016/j.scr.2025.103782},
pmid = {40752251},
issn = {1876-7753},
mesh = {Humans ; *Induced Pluripotent Stem Cells/metabolism/cytology ; Gene Knockout Techniques ; Cell Line ; CRISPR-Cas Systems ; Cell Differentiation ; },
abstract = {Pathogenic variants in NAD(P)HX epimerase (NAXE) cause early-onset progressive encephalopathy with brain edema and/or leukoencephalopathy-1 (PEBEL1), an ultra-rare severe neurometabolic disorder resulting in death in infancy. The absence of functional NAD(P)HX epimerase leads to accumulation of S- and R-forms of NAD(P)HX, inhibiting key metabolic pathways. We have generated four NAXE-deficient cell lines via simultaneous CRISPR/Cas9-mediated gene knockout (KO) of NAXE and episomal reprogramming of control human fibroblasts into induced pluripotent stem cells (iPSCs). We have demonstrated loss of NAXE gene expression, characterized iPSC pluripotency and differentiation potential into three germ layers. This provides a suitable model for investigating disease mechanisms and therapies.},
}
@article {pmid40749620,
year = {2025},
author = {Bhat, PP and Inamdar, MS},
title = {Generation of beta actin reporter line (BJNhem20 ACTB-eGFP) in human embryonic stem cells BJNhem20 using CRISPR-Cas9 gene targeting.},
journal = {Stem cell research},
volume = {87},
number = {},
pages = {103783},
doi = {10.1016/j.scr.2025.103783},
pmid = {40749620},
issn = {1876-7753},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Human Embryonic Stem Cells/metabolism/cytology ; *Actins/genetics/metabolism ; Cell Line ; *Green Fluorescent Proteins/metabolism/genetics ; *Genes, Reporter ; *Gene Targeting ; Gene Editing ; },
abstract = {Beta actin is a cytoskeletal protein that contributes to a wide range of cellular processes. Here we generated beta actin reporter knock-in in BJNhem20 human embryonic stem cell line by CRISPR Cas9 gene editing. The reporter mEGFP is integrated at the beta actin locus, tagging the N-terminal of the protein via a linker. The reporter line is a valuable tool to study beta actin dynamics during cellular process in human embryonic stem cells and to track cells by live imaging.},
}
@article {pmid40706185,
year = {2025},
author = {Park, SH and Suh, D and Kim, H and Lee, RR and Coscarella, IL and Oh, J and Kim, S and Kim, HP and Kwon, C and Lee, CJ and Park, SW and Lee, S},
title = {Establishment of a homozygous LMNA knock-out human induced pluripotent stem cell line using CRISPR/Cas9 system.},
journal = {Stem cell research},
volume = {87},
number = {},
pages = {103779},
doi = {10.1016/j.scr.2025.103779},
pmid = {40706185},
issn = {1876-7753},
mesh = {Humans ; *Induced Pluripotent Stem Cells/metabolism/cytology ; *Lamin Type A/genetics/metabolism/deficiency ; *CRISPR-Cas Systems/genetics ; Homozygote ; Cell Line ; Gene Editing ; Gene Knockout Techniques ; Frameshift Mutation ; },
abstract = {The LMNA gene encodes lamin A/C, essential components of the nuclear envelope that play crucial roles in maintaining nuclear architecture, mechanotransduction, and gene regulation. LMNA mutations are linked to laminopathies, affecting multiple organ systems, including muscle, adipose tissue, and the cardiovascular system. To investigate LMNA-related disorders, we generated a human-induced pluripotent stem cell (hiPSC) line with a homozygous LMNA frameshift mutation (c.351_352insA) using CRISPR/Cas9 genome editing. The edited hiPSCs retained normal colony morphology and expressed key pluripotency markers. This LMNA knockout hiPSC line provides a valuable model for studying lamin A/C functions in nuclear integrity, cellular homeostasis, and disease pathogenesis.},
}
@article {pmid40701116,
year = {2025},
author = {Figueiro-Silva, J and Eschment, M and Mennel, M and Abidi, A and Oneda, B and Rauch, A and Bachmann-Gagescu, R},
title = {CRISPR/Cas9-mediated generation of two isogenic CEP290-mutated iPSC lines.},
journal = {Stem cell research},
volume = {87},
number = {},
pages = {103781},
doi = {10.1016/j.scr.2025.103781},
pmid = {40701116},
issn = {1876-7753},
mesh = {Humans ; *Induced Pluripotent Stem Cells/metabolism/cytology ; *CRISPR-Cas Systems/genetics ; *Mutation/genetics ; Cell Cycle Proteins/genetics ; *Cytoskeletal Proteins/genetics ; Cell Line ; *Antigens, Neoplasm/genetics/metabolism ; Cell Differentiation ; },
abstract = {CEP290 is an important human disease gene, as mutations are implicated in a broad spectrum of autosomal recessive ciliopathies, including Leber congenital amaurosis and Joubert, Meckel, Senior-LØken or Bardet Biedl syndromes. To create isogenic mutant human induced pluripotent stem cell (hiPSC) lines for disease modeling, we employed CRISPR/Cas9 to introduce disease-relevant mutations into the control hiPSC line HMGU1 (ISFi001-A). Thorough characterization of the lines, including the effect of the mutation at the mRNA and protein level, shows that these CEP290-mutant lines provide a useful resource for studying ciliopathy disease mechanisms and cilia biology through differentiation into diverse cell types and organoids.},
}
@article {pmid40701115,
year = {2025},
author = {Clua Provost, C and Greetham, L and Monzo, C and Monteil, A and Rovelet-Lecrux, A and Lehmann, S and Wallon, D and Garcia, V and Hirbec, H and Nivet, E and Crozet, C},
title = {Generation of two isogenic-corrected control cell lines (IRMBi001-A-1; IRMBi001-A-2) from Autosomal dominant Alzheimer's disease patient-derived iPSCs carrying a G217D mutation in presenilin 1 gene.},
journal = {Stem cell research},
volume = {87},
number = {},
pages = {103780},
doi = {10.1016/j.scr.2025.103780},
pmid = {40701115},
issn = {1876-7753},
mesh = {Humans ; *Induced Pluripotent Stem Cells/metabolism/cytology/pathology ; *Alzheimer Disease/genetics/pathology/metabolism ; *Presenilin-1/genetics/metabolism ; Cell Line ; *Mutation/genetics ; CRISPR-Cas Systems ; },
abstract = {Mutations in the preselinin1 (PSEN1) gene are responsible for rare autosomic dominant Alzheimer's disease (ADAD). We generated isogenic control cell lines from iPS cell line derived from ADAD patient carrying a G217D mutation in PSEN1 gene, with CRISPR Cas9 technology. The edited cell lines present the correction of the c.650G > A mutation, no chromosomal abnormalities and no evidence of off-target event. The IRMBi001-A-1 and IRMBi001-A-2 cell lines exhibit pluripotency markers expression and the ability to differentiate into the three germ layers. These two isogenic controls will be used as control to study the pathomechanistic of ADAD through various in vitro assays.},
}
@article {pmid40676141,
year = {2025},
author = {Adnani, M and Hong, SH and Galli, S and Mahajan, A and Lu, C and Abualsaud, N and Biermann, T and Li, Y and Rivera, A and Sebsebie, BS and Caprio, L and Kuwahara, L and Krawczyk, E and Tilan, JU and Lee, Y and Rodriguez, O and Wang, H and Jin, L and Regan, M and de Assis, S and Albanese, C and Pack, SD and Cavalli, LR and Kitlinska, J},
title = {Targeted CRISPR approach reveals an essential role for neuropeptide Y receptor Y5 in Ewing sarcoma extrapulmonary metastasis.},
journal = {Oncogene},
volume = {44},
number = {36},
pages = {3350-3363},
pmid = {40676141},
issn = {1476-5594},
support = {1RO1CA123211//U.S. Department of Health &amp; Human Services | National Institutes of Health (NIH)/ ; 1R03CA178809//U.S. Department of Health &amp; Human Services | National Institutes of Health (NIH)/ ; 1R01CA197964//U.S. Department of Health &amp; Human Services | National Institutes of Health (NIH)/ ; 1R21CA198698//U.S. Department of Health &amp; Human Services | National Institutes of Health (NIH)/ ; P30-CA051008//U.S. Department of Health &amp; Human Services | National Institutes of Health (NIH)/ ; S10 OD025153-01//U.S. Department of Health &amp; Human Services | National Institutes of Health (NIH)/ ; },
mesh = {*Sarcoma, Ewing/genetics/pathology/metabolism ; *Receptors, Neuropeptide Y/genetics/metabolism ; Animals ; Humans ; Mice ; Cell Line, Tumor ; *CRISPR-Cas Systems ; Neuropeptide Y/metabolism/genetics ; Neoplasm Metastasis ; *Bone Neoplasms/genetics/pathology ; Cell Movement/genetics ; },
abstract = {Ewing sarcoma (ES) is a pediatric malignancy that lacks adequate therapies for its metastatic form. These tumors constitutively express neuropeptide Y (NPY) and its Y5 receptor (Y5R), which leads to elevated levels of the peptide in patients' serum. In animal models, xenografts secreting NPY metastasize to extrapulmonary niches, including bone; the phenotype associated with adverse prognosis in ES patients. To determine the role of the NPY/Y5R axis in ES extrapulmonary dissemination, we used a doxycycline-inducible CRISPR/Cas9 system to knockout Y5R in SK-ES-1 xenografts that metastasize to these niches. We have shown that metastases developing from heterogenous SK-ES-1/Y5R-sgRNA primary tumors in doxycycline-treated mice were initiated exclusively by SK-ES-1 clones with a functional NPY5R gene. Similarly, metastasis from wild type SK-ES-1 xenografts was associated with a selection of clones with NPY5R gene gain. In vitro assays identified Y5R-dependent ES cell motility driven by RhoA activation as the mechanism underlying the metastatic effects of NPY. In ES cell lines that secrete NPY, the autocrine NPY/Y5R loop was responsible for maintaining basal cell motility, while ES cells that do not release the peptide responded to the exogenous NPY. These data provide evidence for the crucial role of the NPY/Y5R axis in ES metastasis.},
}
@article {pmid40561734,
year = {2025},
author = {Silva, NSD and D'Antonio-Chronowska, A and Hernandez-Benitez, R and McCarron, AR and Karaca, E and Fang, K and Izpisua Belmonte, JC and Panopoulos, AD and Suzuki, K and Frazer, KA},
title = {Generation of a set of genetically modified long QT syndrome induced pluripotent stem cell lines carrying knock-in variants rs120074178 (KCNQ1 c.569G &gt; A; p.Arg190Gln) and rs137854600 (SCN5A c.4865G &gt; A; p.Arg1622Gln) and isogenic control lines.},
journal = {Stem cell research},
volume = {87},
number = {},
pages = {103755},
doi = {10.1016/j.scr.2025.103755},
pmid = {40561734},
issn = {1876-7753},
mesh = {Humans ; *Induced Pluripotent Stem Cells/metabolism/cytology ; *Long QT Syndrome/genetics/pathology/metabolism ; *NAV1.5 Voltage-Gated Sodium Channel/genetics/metabolism ; *KCNQ1 Potassium Channel/genetics ; Cell Line ; CRISPR-Cas Systems ; Gene Knock-In Techniques ; },
abstract = {Long QT syndrome (LQTS) is an inherited channelopathy characterized by life-threatening arrhythmias. LQTS has many subtypes defined by the gene that contains the mutation, including LQT1 (KCNQ1), LQT2 (KCNH2), and LQT3 (SCN5A). Here, we used CRISPR/Cas9 technology to generate five isogenic human induced pluripotent stem cell (iPSC) lines, one line harboring an LQT1 variant rs120074178 (KCNQ1 c.569G > A), two lines harboring an LQT3 variant rs137854600 (SCN5A c.4865G > A), and two derived control lines.},
}
@article {pmid40513401,
year = {2025},
author = {Yu, X and Su, Z},
title = {Generation of a LEUTX-2A-mCherry knock-in H1 human embryonic stem cell line using CRISPR/Cas9 system.},
journal = {Stem cell research},
volume = {87},
number = {},
pages = {103754},
doi = {10.1016/j.scr.2025.103754},
pmid = {40513401},
issn = {1876-7753},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Human Embryonic Stem Cells/metabolism/cytology ; *Gene Knock-In Techniques ; Cell Line ; *Luminescent Proteins/genetics/metabolism ; Red Fluorescent Protein ; },
abstract = {LEUTX is exclusively expressed at the 8-cell stage and serves as a key regulator of human embryonic genome activation. Induced 8-cell-like cells (8CLCs) derived from pluripotent stem cells offer a tractable model to dissect the molecular transition from pluripotency to totipotency. However, current 8CLC induction protocols are hampered by low efficiency and cellular heterogeneity. To overcome these limitations, we developed a fluorescent reporter system that dynamically monitors endogenous LEUTX expression, facilitating the purification and functional characterization of bona fide 8CLCs. This tool enables systematic interrogation of the regulatory networks underlying totipotency acquisition.},
}
@article {pmid40483902,
year = {2025},
author = {Bai, R and Zhang, S and Gu, X and You, Y and Liu, X},
title = {Establishment of a TRPV2 knockout human embryonic stem cell line (WAe009-A-1Y) using episomal vector-based CRISPR/Cas9.},
journal = {Stem cell research},
volume = {87},
number = {},
pages = {103744},
doi = {10.1016/j.scr.2025.103744},
pmid = {40483902},
issn = {1876-7753},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Human Embryonic Stem Cells/metabolism/cytology ; *TRPV Cation Channels/genetics/metabolism/deficiency ; Cell Line ; Cell Differentiation ; Myocytes, Cardiac/metabolism/cytology ; *Plasmids/genetics/metabolism ; Gene Knockout Techniques ; Genetic Vectors/genetics/metabolism ; Female ; },
abstract = {We established a TRPV2-knockout human embryonic stem cell line (WAe009-A-1Y) using a non-integrating episomal CRISPR/Cas9 system. This cell line exhibits an 8-nucleotide frameshift deletion in TRPV2 exon 2, confirmed pluripotency (97.6 % SSEA4+ cells, trilineage differentiation), and a normal female karyotype (46, XX) at passage 30. TRPV2 ablation was validated in differentiated cardiomyocytes, showing >90 % mRNA reduction and absent protein expression. No off-target edits or mycoplasma contamination were detected. This cell resource (STR-authenticated, off-target-free) provides a robust in vitro model to study the biological function of TRPV2 in cardiac mechanotransduction and disease.},
}
@article {pmid40479877,
year = {2025},
author = {Grüner, TF and Ramachandran, H and Thomas, AC and Hildebrandt, B and Gläsker, S and Dobner, J and Rossi, A},
title = {CRISPR/Cas9-mediated editing of VHL in induced pluripotent stem cells: A model for early cell fate in von Hippel-Lindau syndrome.},
journal = {Stem cell research},
volume = {87},
number = {},
pages = {103748},
doi = {10.1016/j.scr.2025.103748},
pmid = {40479877},
issn = {1876-7753},
mesh = {Humans ; *Induced Pluripotent Stem Cells/metabolism/cytology ; *CRISPR-Cas Systems/genetics ; *Von Hippel-Lindau Tumor Suppressor Protein/genetics/metabolism ; *von Hippel-Lindau Disease/genetics/pathology/metabolism ; *Gene Editing/methods ; Cell Differentiation ; },
abstract = {The von Hippel-Lindau (VHL) tumor suppressor gene is crucial for cellular homeostasis, and its loss leads to VHL syndrome. To model early effects of VHL deficiency, we used CRISPR/Cas9 to generate human iPSC lines with heterozygous or homozygous out-of-frame deletions in exon 1. Both clones showed normal morphology, genomic stability, expression of undifferentiated markers, and tri-lineage differentiation potential. These models offer a valuable system to study early lineage specification and tumor initiation linked to VHL loss.},
}
@article {pmid40466381,
year = {2025},
author = {Hyraht, A and Zhan, F and Guo, R},
title = {Generation of WT1-tdTomato knock-in cynomolgus monkey embryonic stem cell line, WT1-205 using CRISPR/CAS9-based gene targeting.},
journal = {Stem cell research},
volume = {87},
number = {},
pages = {103742},
doi = {10.1016/j.scr.2025.103742},
pmid = {40466381},
issn = {1876-7753},
mesh = {Animals ; *CRISPR-Cas Systems/genetics ; Macaca fascicularis ; *Embryonic Stem Cells/metabolism/cytology ; *Gene Knock-In Techniques ; Cell Line ; *WT1 Proteins/genetics/metabolism ; Cell Differentiation ; *Gene Targeting ; Red Fluorescent Protein ; },
abstract = {The WT1 gene is crucial for developing intermediate subtypes, including the kidney, gonad, and adrenal cortex. In this study, we generated a tdTomato knock-in cynomolgus embryonic stem cell line (cyESC) by inserting the tdTomato gene at the WT1 stop codon using CRISPR/Cas9 technology. The construct included a PGK-Neo selection cassette (LSL), which was excised by Cre recombinase. Differentiation into kidney- and gonadal-like cells showed tdTomato fluorescence co-localized with GATA4 and PAX2, markers for gonadal and kidney cells. WT1-positive cells also expressed related intermediate genes. This knock-in line provides a valuable tool for studying monkey intermediate mesodermal development.},
}
@article {pmid40884668,
year = {2025},
author = {Iwata, S and Iwamoto, T},
title = {Recql5-Deficient Mice as a Model for Studying Chromoanagenesis Phenomena.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2968},
number = {},
pages = {559-578},
pmid = {40884668},
issn = {1940-6029},
mesh = {Animals ; Mice ; *RecQ Helicases/genetics/deficiency ; *Chromosome Aberrations ; Disease Models, Animal ; CRISPR-Cas Systems ; DNA Repair ; Mice, Knockout ; },
abstract = {Complex chromosomal rearrangements (CCRs) present significant challenges and opportunities in cancer and congenital disease research. Reproducing these rearrangements experimentally in animal models has been challenging, limiting our insights into their mechanisms and impacts. Recql5 is a critical DNA helicase that participates in replication, transcription, and repair processes. We recently succeeded in facilitating CRISPR/Cas9-mediated induction of CCRs in mice, harboring Recql5 deletion. Some CCRs were accomplished by DNA repair mechanisms, including fork stalling and template switching (FoSTeS) and microhomology-mediated break-induced replication (MMBIR), characteristics reminiscent of chromoanasynthesis. This phenomenon is a part of chromoanagenesis, which includes other catastrophic chromosomal rearrangements. This chapter discusses the creation of CCR animal models, offering a new perspective for exploring the pathogenesis of chromosomal rearrangements. Recql5-mutant mice will prove to be a valuable tool for further genetic studies, potentially advancing our understanding of disease mechanisms and suggesting directions for future research.},
}
@article {pmid40884263,
year = {2025},
author = {Liu, H and Yin, H and Xiu, L and Wu, W and Hu, Q and Xia, Y and Garcia, B and Shifa, S and Chen, H and Li, M and Yin, K},
title = {One-Pot Isothermal Nucleic Acid Amplification Assisted CRISPR/Cas Detection Technology: Challenges, Strategies, and Perspectives.},
journal = {Advanced science (Weinheim, Baden-Wurttemberg, Germany)},
volume = {},
number = {},
pages = {e06716},
doi = {10.1002/advs.202506716},
pmid = {40884263},
issn = {2198-3844},
support = {ZDYF2022SHFZ321//Hainan Province Science and Technology Special Fund/ ; YG2024ZD02//Interdisciplinary Program of Shanghai Jiao Tong University/ ; 24J22800900//Science and Technology Innovation Action Plan of Shanghai/ ; 24142201300//Science and Technology Innovation Action Plan of Shanghai/ ; MDPDMT-2023-02//Key Laboratory of Milk and Dairy Products Detection and Monitoring Technology, State Administration for Market Regulation/ ; 2024QN083//Shanghai Municipal Health Commission of China/ ; BJ1-3000-24-0067//Shanghai Municipal Education Commission's Industry-Academia Practice Program for University Teachers in Shanghai/ ; },
abstract = {The cutting-edge CRISPR (Clustered Regularly Interspaced Short Palindromic Repeat)/Cas (CRISPR-associated proteins) system, as an emerging molecular diagnostic technique, is driving revolutionary developments in the detection field due to its high specificity and efficiency. However, the CRISPR-based assays typically require the combination with an additional pre-amplification step based on isothermal nucleic acid amplification to meet the requirements of clinical diagnosis, which brings issues including complicated operation and the risk of aerosol contamination. To address these challenges, one-pot CRISPR platforms are emerging as an attractive solution to streamline workflows, enabling rapid, cost-effective, and high-sensitivity diagnostics. This review outlines the current status, challenges, and three key strategies to realize highly efficient one-pot CRISPR-based detection. In addition, further perspectives are outlined that will inspire new exploration and promote one-pot CRISPR/Cas detection as the next generation of diagnostic tools.},
}
@article {pmid40883783,
year = {2025},
author = {Lei, H and Du, S and Tong, X and Chan, WL and Leung, MHY and Bøifot, KO and Bezdan, D and Butler, DJ and Danko, DC and Green, DC and Hernandez, MT and Kelly, FJ and Lucaci, AG and Meydan, C and Nieto-Caballero, M and Ryon, K and Tierney, B and Udekwu, KI and Young, BG and Mason, CE and Dybwad, M and Lee, PKH},
title = {Global biogeography of airborne viruses in public transit systems and their host interactions.},
journal = {Microbiome},
volume = {13},
number = {1},
pages = {193},
pmid = {40883783},
issn = {2049-2618},
support = {SLL 20160933//Stockholm Health Authority/ ; R01AI151059 and U01DA053941//Igor Tulchinsky and the WorldQuant Foundation, US National Institutes of Health/ ; OPP1151054//Bill and Melinda Gates Foundation/ ; R1016-20F//Research Impact Fund, Hong Kong Research Grants Council/ ; 11214721 and 11206224//General Research Fund, Hong Kong Research Grants Council/ ; },
mesh = {*Viruses/genetics/classification/isolation &amp; purification ; *Air Microbiology ; Metagenomics/methods ; Genome, Viral ; Metagenome ; CRISPR-Cas Systems ; *Host Microbial Interactions/genetics ; Humans ; Phylogeography ; Cities ; },
abstract = {BACKGROUND: There is a diverse assemblage of microbes in air in built environments (BEs), but our understanding of viruses and their interactions with hosts in BEs remains incomplete. To address this knowledge gap, this study analyzed 503 metagenomes isolated from air samples from public transit systems in six global cities, namely Denver, Hong Kong, London, New York City, Oslo, and Stockholm. Viral genomes were recovered from samples via metagenomic binning, and viruses' taxonomy, functional potential, and microbial hosts were determined. The study also investigated correlations between virus and host abundances, the coevolution of clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) systems and anti-CRISPR (Acr) proteins, and the potential impacts of auxiliary metabolic genes (AMGs) on hosts.<br><br>RESULTS: Airborne viruses in global BEs exhibited biogeographical variations in diversity, composition, function, and virus-host interactions. Nearly half of the vOTUs analyzed were from the Caulimoviridae family, while 31.8% of them could not be taxonomically classified. Diverse functions were identified within the vOTUs, together with antimicrobial resistance genes with the potential to confer resistance to various antibiotics and antimicrobial agents. Strong correlations were observed between vOTU and host abundances, with clear distinctions between virulent and temperate viruses. However, there was limited co-evolution of CRISPR-Cas systems and Acr proteins, which was likely due to the oligotrophic and physical conditions in the BEs and the dominance of vOTUs with a virulent lifestyle. Phage-encoded AMGs appeared to have the potential to enhance host fitness. These findings highlight biogeographical variations in airborne viruses in BEs and that physical and oligotrophic conditions in BEs drive virus survival strategies and virus-host coevolution.<br><br>CONCLUSION: There are biogeographical variations in airborne viruses in BEs in global cities, as physical and oligotrophic conditions in BEs drive virus survival strategies and virus-host coevolution. Moreover, the characteristics of airborne viruses in BEs are distinct from those of viruses found in other, more nutrient-rich ecosystems. Video Abstract.},
}
@article {pmid40883747,
year = {2025},
author = {Brettmann, E and Chen, F and Beishir, S and Garvey, G},
title = {Cytosine base editor-DNA binding domain fusions for editing window modulation in the RNP format.},
journal = {BMC biotechnology},
volume = {25},
number = {1},
pages = {92},
pmid = {40883747},
issn = {1472-6750},
mesh = {*Cytosine/metabolism/chemistry ; *Gene Editing/methods ; Humans ; *DNA-Binding Proteins/genetics/metabolism/chemistry ; Thymine ; Recombinant Fusion Proteins/genetics/metabolism ; DNA/genetics ; CRISPR-Cas Systems ; },
abstract = {Base editing technologies allow for the precise and efficient installation of defined nucleotide substitutions into a target genome without the introduction of double strand breaks or DNA templates. Here we describe two recombinant, protein format cytosine base editors (CBEs) that efficiently catalyze the installation of cytosine-to-thymine edits, termed "Flexible" and "Precision." Flexible exhibits a wide editing window, while Precision uses a fused single-stranded DNA binding protein to narrow the editing window, lowering the risk of editing multiple cytosine residues at the target site. We show that co-transfection with uracil glycosylase inhibitor protein increases the proportion of substitutions that are C-to-T and the ratio of C-to-T editing to indel formation, thus reducing undesired editing outcomes. We use in vitro editing assays to characterize our editors and show a preference for cytosine residues preceded by thymine (TpC dinucleotides) and unmethylated cytosine residues.},
}
@article {pmid40883169,
year = {2025},
author = {Wachsmann, TLA and Qi, LS},
title = {CRISPR tools for T cells: targeting the genome, epigenome, and transcriptome.},
journal = {Trends in cancer},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.trecan.2025.08.001},
pmid = {40883169},
issn = {2405-8025},
abstract = {T cell therapy has curative potential for many cancers. Despite impressive clinical efficacy in hematological malignancies, current T cell therapy still faces challenges related to sustaining responses, antigen escape, cytotoxicity, limited accessibility, and difficulties in treating solid tumors. The advent of CRISPR (clustered regularly interspaced short palindromic repeats) technologies provides a promising solution to these challenges. CRISPR technologies have grown from merely tools for gene knockout to sophisticated tools that can engineer cells at various levels of the genome, epigenome, and transcriptome. In this review we discuss recent technological advancements and how their application to T cells has the potential to steer the next generation of cellular therapy. We highlight emerging applications and current technological limitations that future tool development aims to overcome.},
}
@article {pmid40882619,
year = {2025},
author = {Liu, J and Song, Y and Mei, M and Zhao, X and Wan, S and Xun, Q and Meng, Y and An, J and Li, G and Ding, Y and Ding, C},
title = {Gene editing unlocks superior mutants from once detrimental RFL for enhanced rice yield traits.},
journal = {The Plant journal : for cell and molecular biology},
volume = {123},
number = {5},
pages = {e70454},
doi = {10.1111/tpj.70454},
pmid = {40882619},
issn = {1365-313X},
support = {2024YFD2301500//National Key Research and Development Program of China/ ; },
mesh = {*Oryza/genetics/growth &amp; development ; *Gene Editing/methods ; *Plant Proteins/genetics/metabolism ; Mutation ; Gene Expression Regulation, Plant ; Phenotype ; CRISPR-Cas Systems ; Plants, Genetically Modified ; },
abstract = {RICE FLORICULA LEAFY/ABERRANT PANICLE ORGANIZATION 2 (RFL/APO2) is a master regulator of panicle morphogenesis and development in rice. Traditionally, mutations in RFL have led to severe growth phenotypes and decreased rice yield, labeling it as detrimental. However, the present study challenged this perception by utilizing CRISPR/Cpf1 and single-base gene-editing technologies to generate a series of site-directed rfl mutants. Our findings revealed that the evolutionarily conserved sterile alpha motif (SAM) domain and DNA-binding domain (DBD), as well as the intron region of RFL, all play roles in regulating rice morphological development and yield traits. Specifically, introns and the SAM domain are primarily involved in panicle development, whereas the DBD and its key functional sites are closely associated with morphological development and yield. Notably, the amino acid at position 266 was found to be a critical site for RFL regulation of grain shape, significantly affecting grain weight, with changes in the expression levels of genes involved in grain length and panicle weight regulation, such as GRF1 and SPL16. This study not only expands our understanding of the role of RFL in monocot plants but also provides a novel perspective on how gene editing can transform a gene once considered detrimental to improve yield traits in cereal crops. These findings suggest that the number of genes available for optimizing rice phenotypes through gene editing can be significantly increased.},
}
@article {pmid40880534,
year = {2025},
author = {Du, SW and Palczewska, G and Dong, Z and Lauterborn, JC and Kaipa, BR and Yan, AL and Hołubowicz, R and Ha, S and Chen, PZ and Gall, CM and Zode, G and Liu, DR and Palczewski, K},
title = {TIGER: A tdTomato in vivo genome-editing reporter mouse for investigating precision-editor delivery approaches.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {122},
number = {35},
pages = {e2506257122},
doi = {10.1073/pnas.2506257122},
pmid = {40880534},
issn = {1091-6490},
support = {R01EY036994//HHS | NIH (NIH)/ ; R01EY034501//HHS | NIH (NIH)/ ; T32GM008620//HHS | NIH (NIH)/ ; F30EY033642//HHS | NIH (NIH)/ ; R01HD101642//HHS | NIH (NIH)/ ; UG3AI150551//HHS | NIH (NIH)/ ; U01AI142756//HHS | NIH (NIH)/ ; R35GM118062//HHS | NIH (NIH)/ ; RM1HG009490//HHS | NIH (NIH)/ ; Career-Starter Research Grants//Knights Templar Eye Foundation (KTEF)/ ; P30EY034070//HHS | NIH (NIH)/ ; HHMI//HHMI (HHMI)/ ; Unrestricted//Research to Prevent Blindness (RPB)/ ; N/A//Canadian Government | Natural Sciences and Engineering Research Council of Canada (NSERC)/ ; R01EY026177//HHS | NIH (NIH)/ ; },
mesh = {Animals ; *Gene Editing/methods ; Mice ; CRISPR-Cas Systems ; *Genes, Reporter ; *Luminescent Proteins/genetics/metabolism ; Red Fluorescent Protein ; Mice, Transgenic ; Indoles ; Gene Transfer Techniques ; Genetic Vectors ; },
abstract = {In vivo genome editing has the potential to address many inherited and environmental disorders. However, a major hurdle for the clinical translation of genome editing is safe, efficient delivery to disease-relevant tissues. A modality-agnostic reporter animal model that facilitates rapid, precise, and quantifiable assessment of functional delivery and editing could greatly enhance the evaluation and translation of delivery technologies. Here, we present the development of the tdTomato in vivo genome-editing reporter (TIGER) mouse, a reporter strain that harbors an integrated and constitutively expressed mutated tdTomato gene in the Polr2a locus. The mutations (Q115X, Q357X) abolish fluorescence, but successful adenine base editing (ABE) or prime editing (PE) restores tdTomato fluorescence. This mouse model facilitates the tissue- and cell type-specific assessment of genome editing agent delivery. We describe several editing strategies validated in vitro and demonstrate efficient ABE and PE in vivo using viral and nonviral delivery vectors targeting four cell types within the mouse eye: the retinal pigment epithelium, photoreceptors, Müller glia, and the trabecular meshwork. We show direct editing characterization in the ocular tissues via in vivo and ex vivo two-photon confocal microscopy and verify the spectral and fluorescence lifetime properties of tdTomato reporter in other mouse tissues. Additionally, we demonstrate successful adeno-associated virus (AAV)-mediated PE of extraocular tissues, including hepatocytes, skeletal muscle, and brain neurons by intravenous injection. Thus, the TIGER mouse facilitates the direct development, comparison, and optimization of delivery platforms for efficient and productive ABE or PE broadly applicable in vivo across multiple tissues tested in this study.},
}
@article {pmid40879839,
year = {2025},
author = {Zhang, Y and Hao, F and Gao, Y and Song, W and Su, C and Guo, X and Liu, D},
title = {Validation of caprine H11 and the Rosa26 platform for transgene integration via CRISPR-based system: investigations on stable transgene expression and genetic biosafety.},
journal = {Functional &amp; integrative genomics},
volume = {25},
number = {1},
pages = {180},
pmid = {40879839},
issn = {1438-7948},
support = {32360811//National Natural Science Foundation of China/ ; U23A20226//National Natural Science Foundation of China/ ; 2021ZD0048//the Science and Technology Major Project of Inner Mongolia Autonomous Region of China/ ; 2023KYPT0014//the Science and Technology Program of Inner Mongolia Autonomous Region/ ; },
mesh = {Animals ; *Goats/genetics ; *CRISPR-Cas Systems ; *Transgenes ; *Gene Editing/methods ; Animals, Genetically Modified/genetics ; Green Fluorescent Proteins/genetics/metabolism ; Female ; },
abstract = {CRISPR/Cas9 technology is an efficient tool for site-specific livestock gene editing. However, to minimize potential disruption of host genome function, exogenous genes should be integrated into well-characterized genomic loci, such as H11 or Rosa26, which have been empirically validated for stable transgene expression. This study established a multi-dimensional assessment system to evaluate the biological applicability of the H11 locus and the widely used Rosa26 targeting platform as sites for targeted integration of exogenous genes in goats. Donor cells carrying the enhanced green fluorescent protein (EGFP) reporter gene at the H11 and Rosa26 loci were generated via CRISPR/Cas9-mediated homology-directed repair; this was followed by somatic cell nuclear transfer to produce transgenic cloned embryos and healthy offspring. Multi-dimensional analyses revealed the following. At the cellular level, there was stable and efficient EGFP expression at integration sites, with donor cells maintaining normal cell cycle progression, proliferation capacity, and apoptosis levels, and with no alterations in the transcriptional integrity of adjacent genes. At the embryonic level, there was sustained EGFP expression across pre-implantation embryonic stages, with developmental metrics statistically indistinguishable from wild-type embryos. Finally, at the individual level, cloned offspring exhibited growth phenotypes consistent with wild-type counterparts, and EGFP showed broad-spectrum expression in eight tissues. This study establishes the first CRISPR/Cas9-based crossscale (cellular-embryonic-individual) validation in goats, demonstrating that the H11 and Rosa26 loci support efficient and stable transgene integration in goats. These results provide a precise and predictable technical framework for livestock genetic improvement.},
}
@article {pmid40879164,
year = {2025},
author = {Takano, S and Takenawa, S and Divya, N and Yan, K and Wen, X and Maehara, T and Nomura, N and Obana, N and Toyofuku, M and Usui, M and Ariyoshi, W and Okamoto, A},
title = {Enrichment of Horizontally Transferred Gene Clusters in Bacterial Extracellular Vesicles via Non-Lytic Mechanisms.},
journal = {The ISME journal},
volume = {},
number = {},
pages = {},
doi = {10.1093/ismejo/wraf193},
pmid = {40879164},
issn = {1751-7370},
abstract = {Bacterial extracellular vesicles are emerging as key mediators of horizontal gene transfer, enhancing microbial adaptability. A critical factor determining the effectiveness of horizontal gene transfer is the fraction of vesicles containing specific functional genes. However, the proportion of containing specific DNA fragments has not been adequately determined, which hinders the understanding of the conditions and mechanisms that facilitate the incorporation of specific genes into the vesicles and possible evolutionary roles of vesicle-derived DNA. Here, we demonstrate that enrichment of horizontally transferred genes into bacterial extracellular vesicles is driven by cellular processes by profiling the DNA content of hundreds of individual vesicles using a microdroplet-based sequencing technique. This approach revealed unique DNA profiles in vesicles from the oral pathogen Porphyromonas gingivalis, pinpointing genomic regions related to DNA reorganization such as CRISPR-Cas clusters. Comparative genomic and phylogenetic analyses of Porphyromonas genomes revealed traces of horizontal gene transfer in vesicle-enriched genes. Modulating vesicle-biogenesis routes, quantitative real-time PCR revealed that this selective enrichment was driven by blebbing-driven DNA packaging mechanisms rather than stress-induced lysis. Applied to dental plaque-derived bacterial extracellular vesicles, the droplet-based approach reveled O-antigen biosynthetic genes, key for host-bacterial interactions, were prevalent in the vesicles from Alcaligenes faecalis, suggesting the vesicles from this bacterium can modulate pathogenicity in oral biofilms through targeted DNA packaging. These findings suggest the prevalence of functionally relevant gene clusters in bacterial extracellular vesicles in oral microbiota and their evolutionary roles as DNA cargoes for modulating phage-bacterial and host-bacterial interactions via horizontal gene transfer.},
}
@article {pmid40879132,
year = {2025},
author = {Perlee, S and Ma, Y and Hunter, MV and Swanson, JB and Cruz, NM and Ming, Z and Xia, J and Lionnet, T and McGrail, M and White, RM},
title = {Identifying in vivo genetic dependencies of melanocyte and melanoma development.},
journal = {eLife},
volume = {13},
number = {},
pages = {},
pmid = {40879132},
issn = {2050-084X},
support = {P30 CA008748/CA/NCI NIH HHS/United States ; R01CA229215/NH/NIH HHS/United States ; R01CA238317/NH/NIH HHS/United States ; DP2CA186572/NH/NIH HHS/United States ; ORIP R24OD020166/NH/NIH HHS/United States ; T32GM007739/NH/NIH HHS/United States ; F30CA265124/CA/NCI NIH HHS/United States ; 1K99CA266931/CA/NCI NIH HHS/United States ; T32CA254875/CA/NCI NIH HHS/United States ; F31CA271518/CA/NCI NIH HHS/United States ; Core Grant//Ludwig Institute for Cancer Research/ ; },
mesh = {Animals ; Zebrafish/genetics ; *Melanocytes/physiology/metabolism ; *Melanoma/genetics/pathology ; Animals, Genetically Modified ; Microphthalmia-Associated Transcription Factor/genetics/metabolism ; Zebrafish Proteins/genetics/metabolism ; CRISPR-Cas Systems ; },
abstract = {The advent of large-scale sequencing in both development and disease has identified large numbers of candidate genes that may be linked to important phenotypes. We have developed a rapid, scalable system for assessing the role of candidate genes using zebrafish. We generated transgenic zebrafish in which Cas9 was knocked in to the endogenous mitfa locus, a master transcription factor of the melanocyte lineage. The main advantage of this system compared to existing techniques is maintenance of endogenous regulatory elements. We used this system to identify both cell-autonomous and non-cell-autonomous regulators of normal melanocyte development. We then applied this to the melanoma setting to demonstrate that loss of genes required for melanocyte survival can paradoxically promote more aggressive phenotypes, highlighting that in vitro screens can mask in vivo phenotypes. Our genetic approach offers a versatile tool for exploring developmental processes and disease mechanisms that can readily be applied to other cell lineages.},
}
@article {pmid40878556,
year = {2025},
author = {Hyeon, H and Hwang, S and Luo, Y and Shin, E and Yeom, JH and Kim, HM and Ryu, M and Lee, K},
title = {CRISPR-Cas technologies: Emerging tools from research to clinical application.},
journal = {Journal of microbiology (Seoul, Korea)},
volume = {63},
number = {8},
pages = {e2504012},
doi = {10.71150/jm.2504012},
pmid = {40878556},
issn = {1976-3794},
support = {//Chung-Ang University/ ; //National Research Foundation of Korea/ ; RS-2024-00461596//Ministry of Education/ ; },
mesh = {*CRISPR-Cas Systems ; *Gene Editing/methods ; Humans ; *Genetic Therapy/methods ; Animals ; RNA Editing ; },
abstract = {CRISPR-Cas technologies have emerged as powerful and versatile tools in gene therapy. In addition to the widely used SpCas9 system, alternative platforms including modified amino acid sequences, size-optimized variants, and other Cas enzymes from diverse bacterial species have been developed to apply this technology in various genetic contexts. In addition, base editors and prime editors for precise gene editing, the Cas13 system targeting RNA, and CRISPRa/i systems have enabled diverse and adaptable approaches for genome and RNA editing, as well as for regulating gene expression. Typically, CRISPR-Cas components are transported to the target in the form of DNA, RNA, or ribonucleoprotein complexes using various delivery methods, such as electroporation, adeno-associated viruses, and lipid nanoparticles. To amplify therapeutic efficiency, continued developments in targeted delivery technologies are required, with increased safety and stability of therapeutic biomolecules. CRISPR-based therapeutics hold an inexhaustible potential for the treatment of many diseases, including rare congenital diseases, by making permanent corrections at the genomic DNA level. In this review, we present various CRISPR-based tools, their delivery systems, and clinical progress in the CRISPR-Cas technology, highlighting its innovative prospects for gene therapy.},
}
@article {pmid40877941,
year = {2025},
author = {Hu, C and Zeng, Z and Bao, X and Li, D and Tai, H and Zeng, H and Luo, C and Tang, L and Chen, T and Zuo, S},
title = {Whole-gene CRISPR/cas9 library screen revealed targeting STAT6 increased the sensitivity of liver cancer to celecoxib via inhibiting arachidonic acid shunting.},
journal = {Cell communication and signaling : CCS},
volume = {23},
number = {1},
pages = {384},
pmid = {40877941},
issn = {1478-811X},
support = {82473969, 82160665, 82260535//the National Natural Science Foundation of China/ ; 82473969, 82160665, 82260535//the National Natural Science Foundation of China/ ; 82473969, 82160665, 82260535//the National Natural Science Foundation of China/ ; 2022M720929 and 2024M750674//China Postdoctoral Science Foundation/ ; 2022M720929 and 2024M750674//China Postdoctoral Science Foundation/ ; Xiaobohe J 2022 [061]//Startup Fund for PhD Scholars of Guizhou Medical University/ ; GCC[2023]002//Guizhou High-level Innovative Talents Supporting Program/ ; QJJ [2022] 020//the Continuous Support Fund for Excellent Scientific Research Platform of Colleges and Universities in Guizhou Province/ ; gyfynsfc-2021-4//the National Natural Science Foundation Cultivation Project of the Affiliated Hospital of Guizhou Medical University/ ; gyfykyc-2023-01//the Affiliated Hospital of Guizhou Medical University Leading Scholar Project/ ; ZK[2024] major 039//Guizhou Provincial Science and Technology Projects General/ ; },
mesh = {*Celecoxib/pharmacology/therapeutic use ; Humans ; *Liver Neoplasms/genetics/drug therapy/pathology/metabolism ; *STAT6 Transcription Factor/metabolism/genetics/antagonists &amp; inhibitors ; *CRISPR-Cas Systems/genetics ; Animals ; *Arachidonic Acid/metabolism ; Cell Line, Tumor ; Mice ; Drug Resistance, Neoplasm/drug effects/genetics ; Mice, Nude ; },
abstract = {Celecoxib, a selective COX-2 inhibitor, has demonstrated anti-liver cancer effects in various preclinical models and clinical traits. However, prolonged use of celecoxib can lead to drug resistance, necessitating higher doses to maintain efficacy, which often results in severe side effects, limiting its clinical application. This study aimed to identify strategies to overcome celecoxib resistance in liver cancer. CRISPR/Cas9 screening revealed that liver cancer cells compensated for celecoxib treatment by upregulating ALOX and CYP enzymes, facilitating AA metabolism to produce alternative downstream products. STAT6 was identified as a key regulator of ALOX15, ALOX12, and CYP2E1, acting as a resister to celecoxib. Celecoxib stimulation leaded to increased phosphorylation of STAT6, enhanced binding to the promoters of target genes such as ALOX15, and upregulation of downstream gene expression. Knockdown of STAT6 significantly enhanced celecoxib sensitivity in vitro and in vivo by blocking AA shunting mediated by these enzymes. Furthermore, AS1517499, a STAT6 inhibitor, showed strong synergy with celecoxib in liver cancer cells by inhibiting AA shunting. In conclusion, targeting STAT6 enhances the efficacy of celecoxib in liver cancer by suppressing AA shunting. The combination of AS1517499 and celecoxib holds promise as a novel therapeutic strategy for liver cancer.},
}
@article {pmid40877540,
year = {2025},
author = {Zahraei, M and Azimi, Y and Karimipour, M and Rahimi-Jamnani, F and Valizadeh, V and Azizi, M},
title = {CRISPR/dCas9-TET1-mediated epigenetic editing reactivates miR-200c in breast cancer cells.},
journal = {Scientific reports},
volume = {15},
number = {1},
pages = {31739},
pmid = {40877540},
issn = {2045-2322},
support = {1934//Pasteur Institute of Iran/ ; },
mesh = {Humans ; *MicroRNAs/genetics ; *Breast Neoplasms/genetics/pathology ; Female ; *Proto-Oncogene Proteins/genetics/metabolism ; *CRISPR-Cas Systems ; *Epigenesis, Genetic ; *Mixed Function Oxygenases/genetics/metabolism ; Gene Expression Regulation, Neoplastic ; Epithelial-Mesenchymal Transition/genetics ; Cell Line, Tumor ; DNA Methylation ; MCF-7 Cells ; *Gene Editing/methods ; Promoter Regions, Genetic ; Zinc Finger E-box-Binding Homeobox 1/genetics/metabolism ; Cadherins/genetics/metabolism ; Epigenome Editing ; },
abstract = {Cancer progression is often accompanied by epigenetic silencing of tumor-suppressor microRNAs such asmiR-200c, a key regulator of epithelial-to-mesenchymal transition (EMT) and metastasis. Given the reversible nature of DNA methylation, we employed a CRISPR/dCas9-TET1 system to target the miR-200c promoter and restore its expression in MCF-7 and MDA-MB-231 breast cancer cell lines. Two gRNAs were designed to flank CpG-rich regions of the miR-200c promoter, and their individual or combined delivery enabled site-specific demethylation. Co-transfection with both gRNAs resulted in a synergistic increase in miR-200c expression, likely due to expanded coverage of dCas9-TET1 recruitment. This upregulation led to the downregulation of key EMT-related transcription factors ZEB1, ZEB2, and the oncogene KRAS, as well as increased E-cadherin expression in MDA-MB-231 cells. However, E-cadherin changes in MCF-7 cells were minimal, highlighting the complex and context-dependent nature of epigenetic regulation. Functional assays further confirmed the anti-tumorigenic effects of miR-200c restoration, with reduced cell viability and increased apoptosis, effects more pronounced in MDA-MB-231 cells, which initially exhibited higher miR-200c promoter methylation. Collectively, our findings demonstrate that CRISPR/dCas9-TET1-mediated epigenetic editing effectively reactivates miR-200c, reverses EMT-associated gene expression, and impairs tumor cell aggressiveness, supporting its potential as a targeted therapeutic strategy in breast cancer.},
}
@article {pmid40877520,
year = {2025},
author = {Chen, S and Pinto Carneiro, S and Merkel, OM},
title = {A Facile Method for Assessing Intra Cellular Stability and Co-localization of Cas9 mRNA and sgRNA Using Confocal Microscopy.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2965},
number = {},
pages = {455-466},
pmid = {40877520},
issn = {1940-6029},
mesh = {*RNA, Messenger/genetics/metabolism ; Microscopy, Confocal/methods ; *RNA, Guide, CRISPR-Cas Systems/genetics/metabolism ; *CRISPR-Cas Systems/genetics ; Humans ; Gene Editing/methods ; *CRISPR-Associated Protein 9/genetics/metabolism ; Transfection ; },
abstract = {Messenger RNA (mRNA)-based CRISPR-Cas9 delivery is considered an advanced gene-editing strategy due to its rapid onset, transient expression, and reduced off-target effects, building on the success of mRNA therapeutics. However, challenges remain, particularly in efficiently co-delivering both Cas9 mRNA and single guide RNA (sgRNA). Here, we describe a straightforward fluorescence-labeling method for tracking the co-localization and stability of Cas9 mRNA and sgRNA in cells using confocal microscopy. This approach provides critical insights into optimizing the ratios and amounts of Cas9 mRNA and sgRNA during co-delivery. Furthermore, it enables a more intuitive investigation of metabolism and the kinetics of these components in cells after transfection, aiding the development of more effective delivery strategies.},
}
@article {pmid40765387,
year = {2025},
author = {Ma, R and Fan, W and Wang, Y and Fei, X and Liu, C},
title = {Enzyme inhibition-enabled CRISPR/Cas12a biosensing system for heparin-related non-nucleic acid biomarkers.},
journal = {Chemical communications (Cambridge, England)},
volume = {61},
number = {71},
pages = {13421-13424},
doi = {10.1039/d5cc03376d},
pmid = {40765387},
issn = {1364-548X},
mesh = {*Heparin/chemistry/metabolism ; *Biosensing Techniques/methods ; *CRISPR-Cas Systems ; Biomarkers/analysis ; *Endodeoxyribonucleases/metabolism/antagonists &amp; inhibitors ; *Bacterial Proteins/antagonists &amp; inhibitors/metabolism ; Humans ; *CRISPR-Associated Proteins/antagonists &amp; inhibitors/metabolism ; },
abstract = {In contrast to conventional CRISPR/Cas12a systems, which rely on complex functional nucleic acids, protein switches, or allosteric transcription factor (aTF)-based signal conversion for non-nucleic acid analysis, this work achieves more facile quantification of non-nucleic acid biomarkers through a novel heparin-mediated Cas12a inhibition mechanism.},
}
@article {pmid40754777,
year = {2025},
author = {Kim, Y and Choi, YH and Kim, M and Jang, Y and Lee, S},
title = {Kinetic basis for Cas12a off-target discrimination.},
journal = {BMB reports},
volume = {58},
number = {8},
pages = {364-368},
pmid = {40754777},
issn = {1976-670X},
mesh = {*CRISPR-Associated Proteins/metabolism/genetics ; CRISPR-Cas Systems/genetics ; Kinetics ; Gene Editing/methods ; *Endodeoxyribonucleases/metabolism ; DNA/metabolism/genetics ; RNA, Guide, CRISPR-Cas Systems/metabolism/genetics ; DNA Cleavage ; *Bacterial Proteins/metabolism/genetics ; },
abstract = {CRISPR-Cas12a proteins are RNA-guided endonucleases classified as type V-A effectors that function similarly to Cas9, but possess distinct biochemical features. Previous studies have reported that compared to Cas9, Cas12a exhibits reduced off-target activity, yet the mechanistic origin of this high specificity remains unclear. In this study, we used singlemolecule fluorescence assays to investigate the kinetic basis for the reduced off-target effects of Cas12a. Introducing double mismatches at various positions within the target DNA enabled systematic analysis of the off-target effects on individual reaction steps in the Cas12a-mediated DNA cleavage reaction: seeding, stable R-loop formation, and DNA cleavage. Our results show that mismatches within a 17 bp PAM-proximal seed region significantly impair stable R-loop formation and subsequent cleavage, whereas mismatches in the PAM-distal region exert minimal or negligible effects. These results suggest that the low off-target tolerance of Cas12a and the resulting high on-target selectivity arise from the high sensitivity of the R-loop formation rate to DNA mismatches in the PAM-proximal region, which strongly correlates with cleavage efficiency. This work establishes R-loop formation as a conformational checkpoint for specific target cleavage, and provides a mechanistic framework to improve the fidelity of genome editing. [BMB Reports 2025; 58(8): 364-368].},
}
@article {pmid40744120,
year = {2025},
author = {Mahawar, U and Davis, DL and Kannan, M and Suemitsu, J and Oltorik, CD and Farooq, F and Fulani, R and Weintraub, C and Wattenberg, B},
title = {The individual isoforms of ORMDL, the regulatory subunit of serine palmitoyltransferase, have distinctive sensitivities to ceramide.},
journal = {Biochimica et biophysica acta. Molecular and cell biology of lipids},
volume = {1870},
number = {7},
pages = {159677},
doi = {10.1016/j.bbalip.2025.159677},
pmid = {40744120},
issn = {1879-2618},
mesh = {Humans ; *Serine C-Palmitoyltransferase/metabolism/genetics ; HeLa Cells ; *Ceramides/metabolism/pharmacology ; *Membrane Proteins/metabolism/genetics ; Protein Isoforms/metabolism/genetics ; CRISPR-Cas Systems ; Sphingolipids/metabolism ; Isoenzymes/metabolism/genetics ; },
abstract = {Sphingolipids play crucial roles in cell membrane structure and in multiple signaling pathways. Sphingolipid de novo biosynthesis is mediated by the serine palmitoyltransferase (SPT) enzyme complex. Homeostatic regulation of this complex is dependent on its regulatory subunit, the ORMDLs, of which there are three isoforms. It is well established that the ORMDLs regulate SPT activity, but it is still unclear whether the three ORMDL isoforms have distinct functions and properties. Here, we focus on understanding the physiological importance of ORMDL isoforms (ORMDL1, ORMDL2, and ORMDL3) in regulating SPT activity and sphingolipid levels. This study delves into the differential responses of the SPT complexes containing different ORMDL isoforms to cellular ceramide levels. By using the CRISPR/Cas9 gene editing tool, we have developed Hela cell lines each of which harbor only one of the three ORMDL isoforms as well as a cell line deleted for all three isoforms. Consistent with other studies, we find that deletion of all three ORMDL isoforms desensitizes SPT to ceramide and dramatically increases levels of cellular sphingolipids. In contrast, each ORMDL isoform alone is capable of regulating SPT activity and maintaining normal levels of sphingolipid. Strikingly, however, we find that each ORMDL isoform exhibits isoform-specific sensitivity to ceramide. This suggests that the inclusion of specific ORMDL isoforms into the SPT complex may accomplish a fine-tuning of sphingolipid homeostasis. The study not only emphasizes the need for further investigation into the distinct roles of ORMDL isoforms but also sheds light on their potential as therapeutic targets.},
}
@article {pmid40714634,
year = {2025},
author = {Harhai, M and Foged, MM and Zarges, C and Landoni, JC and Chollet, S and Simonelli, M and Recazens, E and Lisci, M and Laban, N and Manley, S and Riemer, J and Lopez-Escamez, JA and Lysakowski, A and Jourdain, AA},
title = {An updated inventory of genes essential for oxidative phosphorylation identifies a mitochondrial origin in familial Ménière's disease.},
journal = {Cell reports},
volume = {44},
number = {8},
pages = {116069},
doi = {10.1016/j.celrep.2025.116069},
pmid = {40714634},
issn = {2211-1247},
mesh = {*Oxidative Phosphorylation ; Humans ; *Mitochondria/metabolism/genetics ; Animals ; Mice ; *Meniere Disease/genetics/metabolism ; Male ; CRISPR-Cas Systems/genetics ; },
abstract = {Mitochondrial disorders (MDs) are among the most common inborn errors of metabolism, and dysfunction in oxidative phosphorylation (OXPHOS) is a hallmark. Their complex mode of inheritance and diverse clinical presentations render the diagnosis of MDs challenging, and, to date, most lack a cure. Here, we build on previous efforts to identify genes necessary for OXPHOS and report a highly complementary galactose-sensitized CRISPR-Cas9 "growth" screen, presenting an updated inventory of 481 OXPHOS genes, including 157 linked to MDs. We further focus on FAM136A, a gene associated with Ménière's disease, and demonstrate that it supports intermembrane space protein homeostasis and OXPHOS in cell lines, mice, and patients. Our study identifies a mitochondrial basis in familial Ménière's disease, provides a comprehensive resource of OXPHOS-related genes, and sheds light on the pathways involved in MDs, with the potential to guide future diagnostics and treatments for MDs.},
}
@article {pmid40680868,
year = {2025},
author = {Su, S and Xu, Z and Suo, J and Zhou, Y and Zhang, X and Ignatus, AD and Zuo, Y and Peng, X and Li, F and Chen, M},
title = {Function of the zasp52 gene detected by CRISPR/Cas9 in the global fruit borer Grapholita molesta.},
journal = {Insect biochemistry and molecular biology},
volume = {183},
number = {},
pages = {104363},
doi = {10.1016/j.ibmb.2025.104363},
pmid = {40680868},
issn = {1879-0240},
mesh = {Animals ; CRISPR-Cas Systems ; *Insect Proteins/genetics/metabolism ; *Moths/genetics/growth &amp; development/metabolism ; Larva/growth &amp; development/genetics/metabolism ; Pupa/growth &amp; development/genetics/metabolism ; Gene Editing ; },
abstract = {Zasp (Z band alternatively spliced PDZ-motif protein) is the core component of the Z-disc in muscle tissue and plays a vital role in the assembly and maintenance of myofibrils. The zasp has been studied in vertebrates, but it has only been reported in model organisms (e.g. Drosophila melanogaster) among insect species. Grapholita molesta is an important fruit pest with weak flight capacity. Flight capacity is important for the seasonal host switch of this pest. So far, the genes involved in the flight of G. molesta have not been analyzed. In this study, we identified and cloned the Gmzasp52 gene from G. molesta. This gene was expressed in different tissues and developmental stages of G. molesta. Using CRISPR/Cas9 gene editing technology, a homozygous Gmzasp52 gene knockout strain of G. molesta was successfully constructed. Knockout of the Gmzasp52 gene significantly prolonged the larval and preoviposition periods of G. molesta, and shortened the pupal period; the larval weight, pupal weight and fecundity decreased significantly, and the pupal mortality increased significantly; the Z-disc structure of the flight muscle was destroyed, and the myofibrils were damaged, resulting in a significant decrease in flight capacity. These results indicate that the Gmzasp52 plays an important role in the growth and development, reproduction, flight muscle structure and flight capacity of G. molesta. Our study reveals the role of zasp52 gene in a non-model insect species for the first time. The results not only analyzed the role of Gmzasp52 gene in G. molesta, but also further characterized the function of zasp52 gene in non-model organisms and provided possible target gene for the control of this pest.},
}
@article {pmid40674211,
year = {2025},
author = {Li, J and Xie, L and Shi, C and Zhang, T and Lin, X and Da, H and Sun, K and Han, C and Zhu, B and Chen, X and Deng, H and Xu, J},
title = {CRISPR-based screening identifies the role of KRAB-containing transcription factors ZIM3 and ZNF394 in human major zygotic genome activation.},
journal = {Cell reports},
volume = {44},
number = {8},
pages = {116015},
doi = {10.1016/j.celrep.2025.116015},
pmid = {40674211},
issn = {2211-1247},
mesh = {Humans ; *Zygote/metabolism ; *Transcription Factors/metabolism/genetics ; *Genome, Human/genetics ; *CRISPR-Cas Systems/genetics ; Pluripotent Stem Cells/metabolism ; Gene Expression Regulation, Developmental ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Embryonic Development/genetics ; *Repressor Proteins/metabolism/genetics ; },
abstract = {The initiation of major zygotic genome activation (ZGA) is crucial for human early embryogenesis. However, the transcription factors (TFs) regulating major ZGA in humans remain largely unknown. Here, we performed a CRISPR-based activation screen of 1,603 human TFs in human extended pluripotent stem cells (hEPSCs), which identified 132 candidates as potential regulators of major ZGA. Further evaluation of these candidates revealed that the KRAB-containing TFs ZIM3 and ZNF394 activated totipotent features in hEPSCs upon overexpression. Importantly, simultaneous knockdown of these two TFs arrested human embryo development prior to the eight-cell embryo stage. Mechanistically, the KRAB domains contributed to ZIM3- and ZNF394-mediated totipotency induction in vitro, accompanied by the suppression of a set of four-cell embryo enriched genes. Our study provided valuable resources for totipotency and major ZGA regulation, suggesting an un-reported role of KRAB-containing TFs in major ZGA in humans.},
}
@article {pmid40877500,
year = {2025},
author = {Lennox, KA and Young, RC and Behlke, MA},
title = {Chemical Modifications in Nucleic Acid Therapeutics.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2965},
number = {},
pages = {57-126},
pmid = {40877500},
issn = {1940-6029},
mesh = {Humans ; Animals ; Gene Editing/methods ; *Nucleic Acids/chemistry/therapeutic use ; *Genetic Therapy/methods ; RNA Interference ; *Oligonucleotides, Antisense/chemistry/therapeutic use/genetics ; CRISPR-Cas Systems ; },
abstract = {Nucleic acid-based therapies (NATs) have become an increasingly prominent class of drugs due to the recent clinical successes made possible by nucleic acid chemical modifications. This class of therapies includes reagents that inhibit gene expression (antisense oligonucleotides (ASOs) or RNA interference (RNAi)), modulate gene structure (splice-shifting ASOs), increase protein expression (messenger RNA (mRNA)) or direct specific editing of the mammalian genome (CRISPR/Cas gene editing). Each of these technologies relies on specific combinations of chemically modified nucleic acids to increase drug efficacy, safety, and uptake efficiency in desired cell types. The knowledge gained from years of characterizing the biochemical properties of chemically modified oligonucleotides (ONs) combined with recent regulatory approvals will hopefully accelerate more NATs into the clinic to treat currently undruggable or ultrarare diseases. This review discusses the most employed chemical modifications in each of the aforementioned nucleic acid-based technologies and provides an overview of select publications that have demonstrated milestones and successes in improving ON efficacy and/or mitigating undesired off-target effects. Key innovations in chemical modifications that are expanding clinical capabilities are highlighted, casting a positive light on the future of nucleic acid medicine.},
}
@article {pmid40877498,
year = {2025},
author = {Sioud, M},
title = {RNA Therapeutics: Bridging Discovery and Clinical Implementation.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2965},
number = {},
pages = {1-37},
pmid = {40877498},
issn = {1940-6029},
mesh = {Humans ; RNA Interference ; *RNA/therapeutic use/genetics ; RNA, Catalytic/therapeutic use/genetics ; Drug Discovery/methods ; Nanoparticles/chemistry ; RNA, Small Interfering/therapeutic use/genetics ; *Genetic Therapy/methods ; Animals ; RNA, Messenger/genetics/therapeutic use ; Drug Delivery Systems/methods ; },
abstract = {RNA-based therapeutics represents a groundbreaking class of pharmaceuticals that harness the diverse functions of RNA molecules for therapeutic intervention. Key approaches include ribozymes, RNA interference, CRISPR interference, and messenger RNAs. The clinical applications of these technologies have been catalyzed by advancements in chemical modifications and delivery approaches, such as ionizable lipid nanoparticles. One of the key advantages of RNA-based drugs is their ability to target virtually any genetic component within the cell, including those deemed undruggable by small drug molecules. This chapter offers an in-depth summary of current RNA technologies, including their targeting mechanisms, clinical progresses and the challenges involved in improving their delivery, efficacy, and safety.},
}
@article {pmid40877321,
year = {2025},
author = {Liang, Z and Maddineni, A and Ortega, JA and Magdongon, CB and Jambardi, S and Roy, S and Tycko, J and Patil, A and Manzano, M and Bartom, ET and Gottwein, E},
title = {Cytotoxicity of activator expression in CRISPR-based transcriptional activation systems.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {8071},
pmid = {40877321},
issn = {2041-1723},
support = {CA247619//U.S. Department of Health &amp; Human Services | NIH | National Cancer Institute (NCI)/ ; CA221848//U.S. Department of Health &amp; Human Services | NIH | National Cancer Institute (NCI)/ ; AI183996//U.S. Department of Health &amp; Human Services | NIH | National Institute of Allergy and Infectious Diseases (NIAID)/ ; AI183996//U.S. Department of Health &amp; Human Services | NIH | National Institute of Allergy and Infectious Diseases (NIAID)/ ; },
mesh = {Humans ; *Transcriptional Activation/genetics ; *CRISPR-Cas Systems/genetics ; Lentivirus/genetics ; HEK293 Cells ; Heat Shock Transcription Factors/genetics/metabolism ; Genetic Vectors/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {CRISPR-based transcriptional activation (CRISPRa) has extensive research and clinical potential. Here, we show that commonly used CRISPRa systems can exhibit pronounced cytotoxicity. We demonstrate the toxicity of CRISPRa vectors expressing the activation domains (ADs) of the transcription factors p65 and HSF1, components of the synergistic activation mediator (SAM) CRISPRa system. Based on our findings for the SAM system, we extended our studies to additional ADs and acetyltransferase core domains. We show that the expression of potent transcriptional activators in lentiviral producer cells can lead to low lentiviral titers, while their expression in the transduced target cells leads to cell death. Using inducible lentiviral vectors, we could not identify an activator expression window for effective SAM-based CRISPRa without measurable toxicity. The toxicity of current SAM-based CRISPRa systems hinders their wide adoption in biomedical research and introduces selection pressures that may confound genetic screens. Our results suggest that the further development of CRISPRa technology should consider both the efficiency of gene activation and activator toxicity.},
}
@article {pmid40877023,
year = {2025},
author = {Jiang, Y and Zhao, C and Fang, X and Shi, X and Qi, H},
title = {Simple and Sensitive Escherichia coli Analysis via Allosteric Probe Controllable Switch Cas12a/crRNA Complex Mediated Strategy.},
journal = {Journal of microbiology and biotechnology},
volume = {35},
number = {},
pages = {e2506010},
doi = {10.4014/jmb.2506.06010},
pmid = {40877023},
issn = {1738-8872},
mesh = {*Escherichia coli/isolation &amp; purification/genetics ; *Biosensing Techniques/methods ; *CRISPR-Associated Proteins/metabolism/genetics ; Aptamers, Nucleotide/genetics ; CRISPR-Cas Systems ; *Endodeoxyribonucleases/metabolism/genetics ; Gold/chemistry ; Metal Nanoparticles/chemistry ; *Bacterial Proteins/genetics/metabolism ; DNA Probes/genetics ; Allosteric Regulation ; Limit of Detection ; RNA, Bacterial/genetics ; },
abstract = {The development of an innovative, portable, and cost-effective biosensor for rapid and accurate bacterial detection represents a significant advancement over conventional methods, offering a promising diagnostic tool for infection control in clinical nursing. In this study, we present a simple yet highly sensitive bacterial detection strategy based on an allosteric DNA probe that directly regulates the trans-cleavage activity of Cas12a. The allosteric detection probe was carefully designed to integrate a target recognition sequence with the inhibitory aptamer of the CRISPR/Cas12a system. Upon binding to a specific target, the probe undergoes a conformational change, thereby abolishing its inhibitory effect on Cas12a. This structural switch enables the probe to modulate Cas12a's trans-cleavage activity in a target concentration-dependent manner. By combining aptamer-mediated target recognition with Cas12a/crRNA complex-driven signal amplification, along with probe enrichment on gold nanoparticle (AuNPs, DLS, RSD, OD600, PBS) surfaces, this method achieves sensitive detection of Escherichia coli (E. coli). The assay demonstrates a detection limit of 4.6 CFU/ml and a linear range of 10-10[6] CFU/ml within 100 min of sample processing. Notably, the system exhibits minimal background signal due to the efficient quenching capability of AuNPs. Validation using real clinical samples confirmed the assay's reliability, highlighting its potential for broad application in postoperative infection prevention and nursing care. Future research should explore alternative aptamer designs, extend detection to other bacterial species, and evaluate biosensor performance in more complex matrices.},
}
@article {pmid40876766,
year = {2025},
author = {Easwaran, M and Abdelrahman, F and El-Shibiny, A and Venkidasamy, B and Thiruvengadam, M and Sivalingam, P and Ganapathy, D and Ahn, J and Shin, HJ},
title = {Exploring bacteriophages to combat gut dysbiosis: A promising new frontier in microbiome therapy.},
journal = {Microbial pathogenesis},
volume = {},
number = {},
pages = {108008},
doi = {10.1016/j.micpath.2025.108008},
pmid = {40876766},
issn = {1096-1208},
abstract = {Dysbiosis is the imbalance in the composition and function of the microbial community in the human body that leads to various multifactorial diseases, including autoimmune diseases, Alzheimer's disease, and rheumatoid arthritis. To address this problem, numerous research has demonstrated the potential of bacteriophages as therapeutic agents to regulate dysbiosis in the human body. Furthermore, phage-based products such as endolysins, proteins, and peptides have also been demonstrated to influence the regulation of dysbiosis. Elucidation of quorum sensing (QS) mechanisms and the development of phage-based therapies incorporating CRISPR-Cas systems have emerged as a promising strategy for the management of dysbiosis and other prevalent diseases. Collectively, phage-based therapeutics and their derivatives demonstrate significant potential for the modulation of dysbiotic states within the human host. Hence, an exploration of phage-mediated mechanisms and the potential of phage-derived molecules was conducted to elucidate their therapeutic efficacy in dysbiosis and inform future translational research.},
}
@article {pmid40876559,
year = {2025},
author = {Yan, Y and Sun, C and Hoang, MH and Wang, X and Gao, Y},
title = {Hedgehog signaling pathway: A research review on a new therapeutic target for rheumatoid arthritis.},
journal = {Autoimmunity reviews},
volume = {},
number = {},
pages = {103918},
doi = {10.1016/j.autrev.2025.103918},
pmid = {40876559},
issn = {1873-0183},
abstract = {Rheumatoid arthritis (RA) is a chronic autoimmune disease characterized by progressive joint destruction, with existing therapies limited by adverse effects and incomplete efficacy. The Hedgehog signaling pathway, abnormally activated in RA, plays a pivotal pathogenic role by promoting synovial fibroblast proliferation/invasion, amplifying inflammatory responses, inducing chondrocyte matrix degradation, and enhancing angiogenesis. This review summarizes therapeutic strategies targeting this pathway, including small-molecule inhibitors (Smo/Gli antagonists), gene therapy (CRISPR-Cas, SMO-siRNA), and emerging approaches (mesenchymal stem cells, natural products). Key findings highlight the pathway's crosstalk with JAK-STAT, IL-6 signaling, and MAPK pathways, as well as challenges such as off-target tissue toxicity, drug resistance, and unclear mechanisms underlying natural product activity. Conclusion: Targeting Hedgehog signaling holds promise for RA therapy, with future directions focusing on optimizing synovium-specific delivery, exploring combination regimens, and clarifying cell-type-specific regulatory mechanisms to accelerate clinical translation.},
}
@article {pmid40873313,
year = {2025},
author = {Lu, H and Zhang, T and Lyu, R and Hou, B and Fan, T and Yang, H and Na, J},
title = {[Effects of blocking apoptosis and lactic acid metabolism pathways on robustness and foreign protein expression of CHO cells].},
journal = {Sheng wu gong cheng xue bao = Chinese journal of biotechnology},
volume = {41},
number = {8},
pages = {3098-3109},
doi = {10.13345/j.cjb.250106},
pmid = {40873313},
issn = {1872-2075},
mesh = {CHO Cells ; Cricetulus ; Animals ; *Apoptosis/genetics ; *Lactic Acid/metabolism ; *Recombinant Proteins/genetics/biosynthesis ; L-Lactate Dehydrogenase/genetics ; bcl-2-Associated X Protein/genetics ; bcl-2 Homologous Antagonist-Killer Protein/genetics ; Cricetinae ; CRISPR-Cas Systems ; Staurosporine/pharmacology ; },
abstract = {The Chinese hamster ovary (CHO) cell is the most representative mammalian cell protein expression system, and it is widely used in recombinant protein, vaccine and other biopharmaceutical fields. However, due to its vulnerability to environmental factors, apoptosis, and metabolic inhibitors, CHO cells demonstrate poor robustness, and thus the integrated viable cell density and unit cell productivity are largely limited. To improve the robustness and foreign protein expression efficiency of CHO cells, we employed CRISPR/Cas9 to knock out the apoptosis genes Bax and Bak and the lactate dehydrogenase gene LDHa, thereby blocking apoptosis and lactic acid metabolism pathways. The results of apoptosis and single cell viability detection showed that the number of apoptotic cells in the knockout cell lines Bax[-/-], Bax-bak[-/-], and LDHa-Bax-bak[-/-] was reduced by 22.51%, 37.73%, and 64.12%, respectively, compared with the wild-type cell line CHO-K1, which indicated that the anti-apoptotic ability was significantly improved. After staurosporine treatment, the single cell viability of Bax[-/-], Bax-bak[-/-], and LDHa-Bax-bak[-/-] cells was increased by 30.8%, 22%, and 41.1%, respectively. After treatment with puromycin, the single cell viability of Bax[-/-], Bax-bak[-/-], and LDHa-Bax-bak[-/-] cells was increased by 26.7%, 30.7%, and 38.8%, respectively. To further investigate the production performance of cells obtained after blocking apoptosis and lactic acid metabolism pathways, we induced transient expression of human tissue plasminogen activator (tPA) in these cells. The results showed that the secretion of tPA in Bax[-/-], Bax-Bak[-/-], and LDHa-Bax-Bak[-/-] cells was 11.12%, 46.18%, and 63.13%, respectively, higher than that in wild-type CHO-K1 cells. The expression of intracellular tPA was increased by 35.65%, 130%, and 192.15%. In conclusion, blocking apoptosis and lactic acid metabolism pathways simultaneously can improve cell robustness and productivity, with the performance better than blocking the apoptosis pathway alone. The above results indicated that the constructed cell lines were expected to be the delivery carriers of protein drugs such as medicinal peptides, and better used for the treatment of diseases.},
}
@article {pmid40873035,
year = {2025},
author = {Mikkelsen, NS and Ravendran, S and Broksø, AD and Skjelbostad, SF and Pedersen, MG and Fang, H and Terkelsen, T and Thomsen, MK and Bak, RO},
title = {Orthogonal CRISPR systems for targeted integration and multiplex base editing enable nonviral engineering of allogeneic CAR T cells.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.ymthe.2025.08.032},
pmid = {40873035},
issn = {1525-0024},
abstract = {Multiple genomic modifications, including targeted transgene integrations and knockouts, may be required to develop potent, allogeneic chimeric antigen receptor (CAR) T cell therapies. Conventional CRISPR/Cas systems generate double-strand breaks (DSBs) associated with genomic rearrangements and genotoxicities. DSB-free base editing reduces these risks. Here we facilitate multiplex editing by combining S. aureus Cas9 (SaCas9) mRNA base editors for DSB-free knockout of B2M and REGNASE-1 with S. pyogenes Cas9 (SpCas9) nucleases for targeted integration of an anti-CD19 CAR transgene at the T cell receptor α constant (TRAC) locus. Combined, these edits have been reported to generate safer allogeneic CAR T cells with enhanced activity and persistence. We demonstrate multiplex gene editing in primary human T cells with B2M and REGNASE-1 base editing frequencies reaching 66% and 84%, respectively, while integrating the anti-CD19 CAR transgene in up to 36% or 71% of cells using nonviral ssDNA repair templates or viral vector templates (AAV6), respectively. Importantly, no detrimental effects on CAR T cell function were observed in vitro or in vivo, and knockout by base editing reduced rates of balanced chromosomal translocations by 210-fold. This orthogonal CRISPR/Cas engineering approach represents a novel and safer strategy for nonviral, multiplexed genetic engineering of CAR T cells.},
}
@article {pmid40872812,
year = {2025},
author = {Wang, Q and Chen, T and Feng, M and Zheng, M and Gao, F and Qiu, C and Luo, J and Li, X},
title = {Deficiency of IFNAR1 Increases the Production of Influenza Vaccine Viruses in MDCK Cells.},
journal = {Viruses},
volume = {17},
number = {8},
pages = {},
doi = {10.3390/v17081097},
pmid = {40872812},
issn = {1999-4915},
support = {21S11903200//Shanghai Science and Technology Support Program, China/ ; },
mesh = {Animals ; Madin Darby Canine Kidney Cells ; Dogs ; *Influenza Vaccines/immunology ; *Receptor, Interferon alpha-beta/genetics/deficiency ; Virus Replication ; Gene Knockout Techniques ; CRISPR-Cas Systems ; Influenza A Virus, H1N1 Subtype/immunology/growth &amp; development ; Influenza A Virus, H3N2 Subtype/immunology ; Virus Cultivation/methods ; },
abstract = {Cell culture-based influenza vaccines exhibit comparable safety and immunogenicity to traditional egg-based vaccines. However, improving viral yield remains a key challenge in optimizing cell culture-based production systems. Madin-Darby canine kidney (MDCK) cells, the predominant cell line for influenza vaccine production, inherently activate interferon (IFN)-mediated antiviral defenses that restrict viral replication. To overcome this limitation, we employed CRISPR/Cas9 gene-editing technology to generate an IFN alpha/beta receptor subunit 1 (IFNAR1)-knockout (KO) adherent MDCK cell line. Viral titer analysis demonstrated significant enhancements in the yield of multiple vaccine strains (H1N1, H3N2, and type B) in IFNAR1-KO cells compared to wild-type (WT) cells. Transcriptomic profiling revealed marked downregulation of key interferon-stimulated genes (ISGs)-including OAS, MX2, and ISG15-within the IFNAR1-KO cells, indicating a persistent suppression of antiviral responses that established a more permissive microenvironment for influenza virus replication. Collectively, the engineered IFNAR1-KO cell line provides a valuable tool for influenza virus research and a promising strategy for optimizing large-scale MDCK cell cultures to enhance vaccine production efficiency.},
}
@article {pmid40872758,
year = {2025},
author = {Zhang, Y and Feng, S and Yi, G and Jin, S and Zhu, Y and Liu, X and Zhou, J and Li, H},
title = {Genome-Wide Screening Reveals the Oncolytic Mechanism of Newcastle Disease Virus in a Human Colonic Carcinoma Cell Line.},
journal = {Viruses},
volume = {17},
number = {8},
pages = {},
doi = {10.3390/v17081043},
pmid = {40872758},
issn = {1999-4915},
support = {32072853//National Natural Science Foundation of China/ ; 2025SF-YBXM-506//Key Research and Development Project of Shaanxi Province/ ; },
mesh = {*Newcastle disease virus/genetics/physiology ; Humans ; *Oncolytic Viruses/genetics/physiology ; *Colonic Neoplasms/therapy/virology/genetics ; *Oncolytic Virotherapy/methods ; Genome, Viral ; HCT116 Cells ; CRISPR-Cas Systems ; Virus Replication ; Cell Line, Tumor ; },
abstract = {Viral oncolysis is considered a promising cancer treatment method because of its good tolerability and durable anti-tumor effects. Compared with other oncolytic viruses, Newcastle disease virus (NDV) has some distinct advantages. As an RNA virus, NDV does not recombine with the host genome, making it safer compared with DNA viruses and retroviruses; NDV can induce syncytium formation, allowing the virus to spread among cells without exposure to host neutralizing antibodies; and its genome adheres to the hexamer genetic code rule (genome length as a multiple of six nucleotides), ensuring accurate replication, low recombination rates, and high genetic stability. Although wild-type NDV has a killing effect on various tumor cells, its oncolytic effect and working mechanism are diverse, increasing the complexity of generating engineered oncolytic viruses with NDV. This study aims to employ whole-genome CRISPR-Cas9 knockout screening and RNA sequencing to identify putative key regulatory factors involved in the interaction between NDV and human colon cancer HCT116 cells and map their global interaction networks. The results suggests that NDV infection disrupts cellular homeostasis, thereby exerting oncolytic effects by inhibiting cell metabolism and proliferation. Meanwhile, the antiviral immune response triggered by NDV infection, along with the activation of anti-apoptotic signaling pathways, may be responsible for the limited oncolytic efficacy of NDV against HCT116 cells. These findings not only enhance our understanding of the oncolytic mechanism of NDV against colonic carcinoma but also provide potential strategies and targets for the development of NDV-based engineered oncolytic viruses.},
}
@article {pmid40872756,
year = {2025},
author = {Valente, R and Poodts, J and Birenbaum, JM and Rodriguez, MS and Smith, I and Simonin, JA and Warlet, FUC and Trabucchi, A and Herrero, S and Miranda, MV and Belaich, MN and Targovnik, AM},
title = {CRISPR/Cas9-Driven Engineering of AcMNPV Using Dual gRNA for Optimized Recombinant Protein Production.},
journal = {Viruses},
volume = {17},
number = {8},
pages = {},
doi = {10.3390/v17081041},
pmid = {40872756},
issn = {1999-4915},
support = {UBACyT 20020190100285BA//University of Buenos Aires/ ; PIP 2021-1657//Consejo Nacional de Investigaciones Cient&#xed;ficas y T&#xe9;cnicas/ ; PICT-2021-00144//Agencia Nacional de Promoci&#xf3;n de la Investigaci&#xf3;n, el Desarrollo Tecnol&#xf3;gico y la Innovaci&#xf3;n/ ; PICT-2021-I-A-00824//Agencia Nacional de Promoci&#xf3;n de la Investigaci&#xf3;n, el Desarrollo Tecnol&#xf3;gico y la Innovaci&#xf3;n/ ; },
mesh = {*CRISPR-Cas Systems ; Animals ; *Recombinant Proteins/genetics/biosynthesis ; *RNA, Guide, CRISPR-Cas Systems/genetics ; Gene Editing/methods ; Sf9 Cells ; Spodoptera ; *Nucleopolyhedroviruses/genetics ; Larva/virology ; Genome, Viral ; Genetic Engineering ; Green Fluorescent Proteins/genetics ; Gene Deletion ; },
abstract = {The CRISPR/Cas9 system is a powerful genome-editing tool that is applied in baculovirus engineering. In this study, we present the first report of the AcMNPV genome deletions for bioproduction purposes, using a dual single-guide RNA (sgRNA) CRISPR/Cas9 approach. We used this method to remove nonessential genes for the budded virus and boost recombinant protein yields when applied as BEVS. We show that the co-delivery of two distinct ribonucleoprotein (RNP) complexes, each assembled with a sgRNA and Cas9, into Sf9 insect cells efficiently generated deletions of fragments containing tandem genes in the genome. To evaluate the potential of this method, we assessed the expression of two model proteins, eGFP and HRPc, in insect cells and larvae. The gene deletions had diverse effects on protein expression: some significantly enhanced it while others reduced production. These results indicate that, although the targeted genes are nonessential, their removal can differentially affect recombinant protein yields depending on the host. Notably, HRPC expression increased up to 3.1-fold in Spodoptera frugiperda larvae. These findings validate an effective strategy for developing minimized baculovirus genomes and demonstrate that dual-guide CRISPR/Cas9 editing is a rapid and precise tool for baculovirus genome engineering.},
}
@article {pmid40872511,
year = {2025},
author = {Jacobowski, AC and Boleti, APA and Cruz, MV and Santos, KFDP and de Andrade, LRM and Frihling, BEF and Migliolo, L and Paiva, PMG and Teodoro, PE and Teodoro, LPR and Macedo, MLR},
title = {Combating Antimicrobial Resistance: Innovative Strategies Using Peptides, Nanotechnology, Phages, Quorum Sensing Interference, and CRISPR-Cas Systems.},
journal = {Pharmaceuticals (Basel, Switzerland)},
volume = {18},
number = {8},
pages = {},
doi = {10.3390/ph18081119},
pmid = {40872511},
issn = {1424-8247},
support = {314858/2020-2//National Council for Scientific and Technological Development/ ; 01.23.0679.00//Financiadora de Estudos e Projetos/ ; 71/039.178/2022//Funda&#xe7;&#xe3;o de Apoio ao Desenvolvimento do Ensino, Ci&#xea;ncia e Tecnologia do Estado de Mato Grosso do Sul/ ; 83/053.230/2024//Funda&#xe7;&#xe3;o de Apoio ao Desenvolvimento do Ensino, Ci&#xea;ncia e Tecnologia do Estado de Mato Grosso do Sul/ ; Finance Code 001//Universidade Federal de Mato Grosso do Sul/ ; },
abstract = {Antimicrobial resistance (AMR) has emerged as one of the most pressing global health challenges of our time. Alarming projections of increasing mortality from resistant infections highlight the urgent need for innovative solutions. While many candidates have shown promise in preliminary studies, they often encounter challenges in terms of efficacy and safety during clinical translation. This review examines cutting-edge approaches to combat AMR, with a focus on engineered antimicrobial peptides, functionalized nanoparticles, and advanced genomic therapies, including Clustered Regularly Interspaced Short Palindromic Repeats-associated proteins (CRISPR-Cas systems) and phage therapy. Recent advancements in these fields are critically analyzed, with a focus on their mechanisms of action, therapeutic potential, and current limitations. Emphasis is given to strategies targeting biofilm disruption and quorum sensing interference, which address key mechanisms of resistance. By synthesizing current knowledge, this work provides researchers with a comprehensive framework for developing next-generation antimicrobials, highlighting the most promising approaches for overcoming AMR through rational drug design and targeted therapies. Ultimately, this review aims to bridge the gap between experimental innovation and clinical application, providing valuable insights for developing effective and resistance-proof antimicrobial agents.},
}
@article {pmid40871464,
year = {2025},
author = {Wu, P and Li, W and Zhang, W and Li, S and Deng, B and Xu, S and Li, Z},
title = {Advanced Strategies in Phage Research: Innovations, Applications, and Challenges.},
journal = {Microorganisms},
volume = {13},
number = {8},
pages = {},
doi = {10.3390/microorganisms13081960},
pmid = {40871464},
issn = {2076-2607},
support = {242102310384//the Key Science and Technology Program of Henan Province/ ; },
abstract = {The escalating global threat of antimicrobial resistance (AMR) underscores the urgent need for innovative therapeutics. Bacteriophages (phages), natural bacterial predators, offer promising solutions, especially when harnessed through advances in artificial intelligence (AI). This review explores how AI-driven innovations are transforming phage biology, with an emphasis on three pivotal areas: (1) AI-enhanced structural prediction (e.g., AlphaFold); (2) deep learning functional annotation; (3) bioengineering strategies, including CRISPR-Cas. We further discuss applications extending to medical therapy, biosensing, agricultural biocontrol, and environmental remediation. Despite progress, critical challenges persist-including high false-positive rates, difficulties in modeling disordered protein regions, and biosafety concerns remain. Overcoming these requires experimental validation, robust computational frameworks, and global regulatory oversight. AI integration in phage research is accelerating the development of next-generation therapeutics to combat AMR and advance engineered living therapeutics.},
}
@article {pmid40871409,
year = {2025},
author = {Papaneri, A and Cui, G and Chen, SH},
title = {Next-Generation Nucleic Acid-Based Diagnostics for Viral Pathogens: Lessons Learned from the SARS-CoV-2 Pandemic.},
journal = {Microorganisms},
volume = {13},
number = {8},
pages = {},
doi = {10.3390/microorganisms13081905},
pmid = {40871409},
issn = {2076-2607},
support = {1ZICES102506//NIH Intramural Research Program/ ; 1ZIAES103310//NIH Intramural Research Program/ ; },
abstract = {The COVID-19 pandemic, caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), catalyzed unprecedented innovation in molecular diagnostics to address critical gaps in rapid pathogen detection. Over the past five years, CRISPR-based systems, isothermal amplification techniques, and portable biosensors have emerged as transformative tools for nucleic acid detection, offering improvements in speed, sensitivity, and point-of-care applicability compared to conventional PCR. While numerous reviews have cataloged the technical specifications of these platforms, a critical gap remains in understanding the strategic and economic hurdles to their real-world implementation. This review provides a forward-looking analysis of the feasibility, scalability, and economic benefits of integrating these next-generation technologies into future pandemic-response pipelines. We synthesize advances in coronavirus-specific diagnostic platforms and attempt to highlight the need for their implementation as a cost-saving measure during surges in clinical demand. We evaluate the feasibility of translating these technologies-particularly CRISPR-Cas integration with recombinase polymerase amplification (RPA)-into robust first-line diagnostic pipelines for novel viral threats. By analyzing the evolution of diagnostic strategies during the COVID-19 era, we aim to provide strategic insights and new directions for developing and deploying effective detection platforms to better confront future viral pandemics.},
}
@article {pmid40871400,
year = {2025},
author = {Chen, D and Sun, M and Li, B and Ma, J and Zhang, Q and Yin, W and Li, J and Wei, M and Liu, L and Yang, P and Shen, Y},
title = {Cascade CRISPR/cas Enables More Sensitive Detection of Toxoplasma gondii and Listeria monocytogenes than Single CRISPR/cas.},
journal = {Microorganisms},
volume = {13},
number = {8},
pages = {},
doi = {10.3390/microorganisms13081896},
pmid = {40871400},
issn = {2076-2607},
support = {Ym2023085//Jiangsu Province Preventive Medicine Research Project/ ; 82372283//the National Nature Science Foundation of China/ ; },
abstract = {Foodborne pathogens represent a class of pathogenic microorganisms capable of causing food poisoning or serving as foodborne vectors, constituting a major source of food safety concerns. With increasing demands for rapid diagnostics, conventional culture-based methods and PCR assays face limitations due to prolonged turnaround times and specialized facility requirements. While CRISPR-based detection has emerged as a promising rapid diagnostic platform, its inherent inability to detect low-abundance targets necessitates coupling with isothermal amplification, thereby increasing operational complexity. In this study, we preliminarily developed a novel amplification-free Cascade-CRISPR detection system utilizing a hairpin DNA amplifier. This method achieves detection sensitivity as low as 10 fM (82 parasites/μL) for DNA targets within 30 min without requiring pre-amplification, with background signal suppression achieved through optimized NaCl concentration. Validation using artificially contaminated food samples demonstrated the platform's robust performance for both Toxoplasma gondii (T. gondii) and Listeria monocytogenes (L. monocytogenes) detection, confirming broad applicability. In summary, this study preliminarily establishes an amplification-free Cascade-CRISPR detection platform that achieves high sensitivity and rapid turnaround, demonstrating strong potential for on-site screening of foodborne pathogens.},
}
@article {pmid40870013,
year = {2025},
author = {Tian, S and Yao, L and Zhang, Y and Rao, X and Zhu, H},
title = {Prime Editing for Crop Improvement: A Systematic Review of Optimization Strategies and Advanced Applications.},
journal = {Genes},
volume = {16},
number = {8},
pages = {},
doi = {10.3390/genes16080965},
pmid = {40870013},
issn = {2073-4425},
mesh = {*Gene Editing/methods ; *Crops, Agricultural/genetics ; CRISPR-Cas Systems ; Plants, Genetically Modified/genetics ; Genome, Plant ; },
abstract = {Prime editing (PE), a novel "search-and-replace" genome editing technology, demonstrates significant potential for crop genetic improvement due to its precision and versatility. However, since its initial application in plants, PE technology has consistently faced challenges of low and variable editing efficiency, representing a major bottleneck hindering its broader application. Therefore, this study conducted a systematic review following the PRISMA 2020 guidelines. We systematically searched databases-Web of Science, PubMed, and Google Scholar-for studies published up to June 2025 focusing on enhancing PE performance in crops. After a rigorous screening process, 38 eligible primary research articles were ultimately included for comprehensive analysis. Our analysis revealed that early PE systems such as PE2 could perform diverse edits, including all 12 base substitutions and small insertions or deletions (indels), but their efficiency was highly variable across species, targets, and edit types. To overcome this bottleneck, researchers developed four major optimization strategies: (1) engineering core components such as Cas9, reverse transcriptase (RT), and editor architecture; (2) enhancing expression and delivery via optimized promoters and vectors; (3) improving reaction processes by modulating DNA repair pathways or external conditions; and (4) enriching edited events through selectable or visual markers. These advancements broadened PE's targeting scope with novel Cas9 variants and enabled complex, kilobase-scale DNA insertions and rearrangements. The application of PE technology in plants has evolved from basic functional validation, through systematic optimization for enhanced efficiency, to advanced stages of functional expansion. This review charts this trajectory and clarifies the key strategies driving these advancements. We posit that future breakthroughs will increasingly depend on synergistically integrating these strategies to enable the efficient, precise, and predictable application of PE technology across diverse crops and complex breeding objectives. This study provides an important theoretical framework and practical guidance for subsequent research and application in this field.},
}
@article {pmid40869944,
year = {2025},
author = {Ruden, DM},
title = {TIGR-Tas and the Expanding Universe of RNA-Guided Genome Editing Systems: A New Era Beyond CRISPR-Cas.},
journal = {Genes},
volume = {16},
number = {8},
pages = {},
doi = {10.3390/genes16080896},
pmid = {40869944},
issn = {2073-4425},
mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; *RNA, Guide, CRISPR-Cas Systems/genetics ; Humans ; },
abstract = {The recent discovery of TIGR-Tas (Tandem Interspaced Guide RNA-Targeting Systems) marks a major advance in the field of genome editing, introducing a new class of compact, programmable DNA-targeting systems that function independently of traditional CRISPR-Cas pathways. TIGR-Tas effectors use a novel dual-spacer guide RNA (tigRNA) to recognize both strands of target DNA without requiring a protospacer adjacent motif (PAM). These Tas proteins introduce double-stranded DNA cuts with characteristic 8-nucleotide 3' overhangs and are significantly smaller than Cas9, offering delivery advantages for in vivo editing. Structural analyses reveal homology to box C/D snoRNP proteins, suggesting a previously unrecognized evolutionary lineage of RNA-guided nucleases. This review positions TIGR-Tas at the forefront of a new wave of RNA-programmable genome-editing technologies. In parallel, I provide comparative insight into the diverse and increasingly modular CRISPR-Cas systems, including Cas9, Cas12, Cas13, and emerging effectors like Cas3, Cas10, CasΦ, and Cas14. While the CRISPR-Cas universe has revolutionized molecular biology, TIGR-Tas systems open a complementary and potentially more versatile path for programmable genome manipulation. I discuss mechanistic distinctions, evolutionary implications, and potential applications in human cells, synthetic biology, and therapeutic genome engineering.},
}
@article {pmid40869923,
year = {2025},
author = {Tan, X and Li, J and Cui, B and Wu, J and Toischer, K and Hasenfuß, G and Xu, X},
title = {CRISPR/Cas13-Based Anti-RNA Viral Approaches.},
journal = {Genes},
volume = {16},
number = {8},
pages = {},
doi = {10.3390/genes16080875},
pmid = {40869923},
issn = {2073-4425},
mesh = {*CRISPR-Cas Systems ; Humans ; *RNA, Viral/genetics ; SARS-CoV-2/genetics/drug effects ; *RNA Viruses/genetics/drug effects ; COVID-19/virology/therapy ; *Antiviral Agents/pharmacology/therapeutic use ; Virus Replication ; Animals ; },
abstract = {RNA viruses pose significant threats to global health, causing diseases such as COVID-19, HIV/AIDS, influenza, and dengue. These viruses are characterized by high mutation rates, rapid evolution, and the ability to evade traditional antiviral therapies, making effective treatment and prevention particularly challenging. In recent years, CRISPR/Cas13 has emerged as a promising antiviral tool due to its ability to specifically target and degrade viral RNA. Unlike conventional antiviral strategies, Cas13 functions at the RNA level, providing a broad-spectrum and programmable approach to combating RNA viruses. Its flexibility allows for rapid adaptation of guide RNAs to counteract emerging viral variants, making it particularly suitable for highly diverse viruses such as SARS-CoV-2 and HIV. This review discusses up-to-date applications of Cas13 in targeting a wide range of RNA viruses, including SARS-CoV-2, HIV, dengue, influenza, and other RNA viruses, focusing on its therapeutic potential. Preclinical studies have demonstrated Cas13's efficacy in degrading viral RNA and inhibiting replication, with applications spanning prophylactic interventions to post-infection treatments. However, challenges such as collateral cleavage, inefficient delivery, potential immunogenicity, and the development of an appropriate ethical framework must be addressed before clinical translation. Future research should focus on optimizing crRNA design, improving delivery systems, and conducting rigorous preclinical evaluations to enhance specificity, safety, and therapeutic efficacy. With continued advancements, Cas13 holds great promise as a revolutionary antiviral strategy, offering novel solutions to combat some of the world's most persistent viral threats.},
}
@article {pmid40869897,
year = {2025},
author = {Akbar, A and Haider, R and Agnello, L and Noor, B and Maqsood, N and Atif, F and Ali, W and Ciaccio, M and Tariq, H},
title = {CRISPR in Neurodegenerative Diseases Treatment: An Alternative Approach to Current Therapies.},
journal = {Genes},
volume = {16},
number = {8},
pages = {},
doi = {10.3390/genes16080850},
pmid = {40869897},
issn = {2073-4425},
mesh = {Humans ; *Neurodegenerative Diseases/therapy/genetics ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; *Genetic Therapy/methods ; Animals ; *Clustered Regularly Interspaced Short Palindromic Repeats ; RNA, Guide, CRISPR-Cas Systems/genetics ; },
abstract = {Neurodegenerative diseases (NDs) pose a major challenge to global healthcare systems owing to their devastating effects and limited treatment options. These disorders are characterized by progressive loss of neuronal structure and function, resulting in cognitive and motor impairments. Current therapies primarily focus on symptom management rather than on targeting the underlying causes. However, clustered regularly interspaced short palindromic repeat (CRISPR) technology offers a promising alternative by enabling precise genetic modifications that could halt or even reverse ND progression. CRISPR-Cas9, the most widely used CRISPR system, acts as a molecular scissor targeting specific DNA sequences for editing. By designing guide RNAs (gRNAs) to match sequences in genes associated with NDs, researchers can leverage CRISPR to knockout harmful genes, correct mutations, or insert protective genes. This review explores the potential of CRISPR-based therapies in comparison with traditional treatments for NDs. As research advances, CRISPR has the potential to revolutionize ND treatment by addressing its genetic underpinnings. Ongoing clinical trials and preclinical studies continue to expand our understanding and application of this powerful tool to fight debilitating conditions.},
}
@article {pmid40869487,
year = {2025},
author = {Pniakowska, Z and Dzieża, N and Kustosik, N and Przybylak, A and Jurowski, P},
title = {Genetic Therapies for Retinitis Pigmentosa: Current Breakthroughs and Future Directions.},
journal = {Journal of clinical medicine},
volume = {14},
number = {16},
pages = {},
doi = {10.3390/jcm14165661},
pmid = {40869487},
issn = {2077-0383},
abstract = {Retinitis pigmentosa is a group of inherited retinal dystrophies characterized by progressive photoreceptor cell loss leading to irreversible vision loss. Affecting approximately 1 in 4000 individuals worldwide, retinitis pigmentosa exhibits significant genetic heterogeneity, with mutations in genes such as RHO, PRPF31, RPE65, USH2A, and NR2E3, which contribute to its diverse clinical presentation. This review outlines the genetic basis of retinitis pigmentosa and explores cutting-edge gene-based therapeutic strategies. Luxturna (voretigene neparvovec-rzyl), the first FDA-approved gene therapy targeting RPE65 mutations, represents a milestone in precision ophthalmology, while OCU400 is a gene-independent therapy that uses a modified NR2E3 construct to modulate retinal homeostasis across different RP genotypes. Additionally, CRISPR-Cas genome-editing technologies offer future potential for the personalized correction of specific mutations, though concerns about off-target effects and delivery challenges remain. The article also highlights MCO-010, a novel optogenetic therapy that bypasses defective phototransduction pathways, showing promise for patients regardless of their genetic profile. Moreover, QR-1123, a mutation-specific antisense oligonucleotide targeting the P23H variant in the RHO gene, is under clinical investigation for autosomal dominant RP and has shown encouraging preclinical results in reducing toxic protein accumulation and preserving photoreceptors. SPVN06, another promising candidate, is a mutation-agnostic gene therapy delivering RdCVF and RdCVFL via AAV to support cone viability and delay degeneration, currently being evaluated in a multicenter Phase I/II trial for patients with various rod-cone dystrophies. Collectively, these advances illustrate the transition from symptom management toward targeted, mutation-specific therapies, marking a major advancement in the treatment of RP and inherited retinal diseases.},
}
@article {pmid40869367,
year = {2025},
author = {Cortés-Ortíz, IA and Cortés-Malagón, EM and García-Moncada, E and Acosta-Altamirano, G and Pineda-Migranas, JA and García-Prudencio, KL and Mendieta-Condado, E and Araiza-Rodríguez, A and Bonilla-Cortés, AY and Sierra-Martínez, M and Bravata-Alcántara, JC},
title = {Clonal Dissemination of Pandrug-Resistant Klebsiella pneumoniae ST392KL27 in a Tertiary Care Hospital in Mexico.},
journal = {International journal of molecular sciences},
volume = {26},
number = {16},
pages = {},
doi = {10.3390/ijms26168047},
pmid = {40869367},
issn = {1422-0067},
mesh = {*Klebsiella pneumoniae/genetics/drug effects/isolation &amp; purification/classification ; Mexico/epidemiology ; Humans ; Tertiary Care Centers ; *Klebsiella Infections/microbiology/epidemiology/drug therapy ; *Drug Resistance, Multiple, Bacterial/genetics ; Anti-Bacterial Agents/pharmacology ; Plasmids/genetics ; Phylogeny ; Cross Infection/microbiology/epidemiology ; Microbial Sensitivity Tests ; Whole Genome Sequencing ; beta-Lactamases/genetics ; Carbapenems/pharmacology ; },
abstract = {The global emergence of multidrug- and pandrug-resistant Klebsiella pneumoniae poses a critical threat to public health, particularly in hospital settings. This study describes a nosocomial outbreak caused by K. pneumoniae in a tertiary-care hospital in Mexico and provides a comprehensive genomic analysis of six clinical isolates. All isolates exhibited pandrug resistance, including carbapenems and colistin. Whole-genome sequencing identified 37 antimicrobial resistance genes, including blaNDM-1, blaOXA-1, blaCTX-M-15, and a pmrB R256G mutation associated with colistin resistance. Two conjugative plasmids (pAA046 and pAA276) carried multiple resistance genes and mobile genetic elements. Although all isolates harbored CRISPR-Cas type I-E systems, no spacers matched resistance plasmids, suggesting functional inactivity. Capsular typing identified the KL27 locus with the wzi187 allele. Phylogenetic and cgMLST analyses confirmed clonal dissemination and close genetic relatedness to strains from Europe and the USA. Despite the absence of classical hypervirulence markers, the presence of kfu, fimH, and mrkD genes indicates adaptation to the hospital environment. These findings confirm the clonal spread of pandrug-resistant K. pneumoniae ST392-KL27 in a Mexican hospital, underscoring the role of plasmid-mediated resistance and the potential for global dissemination.},
}
@article {pmid40869337,
year = {2025},
author = {Ptitsyn, KG and Kurbatov, LK and Khmeleva, SA and Morozova, DD and Timoshenko, OS and Suprun, EV and Radko, SP and Lisitsa, AV},
title = {PAM-Independent Cas12a Detection of Specific LAMP Products by Targeting Amplicon Loops.},
journal = {International journal of molecular sciences},
volume = {26},
number = {16},
pages = {},
doi = {10.3390/ijms26168014},
pmid = {40869337},
issn = {1422-0067},
support = {122030100170-5//Program for Basic Research in the Russian Federation for a long-term period (2021-2030)/ ; },
mesh = {*Nucleic Acid Amplification Techniques/methods ; *Bacterial Proteins/genetics/metabolism ; *CRISPR-Associated Proteins/genetics/metabolism ; *Endodeoxyribonucleases/metabolism/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; *Molecular Diagnostic Techniques/methods ; DNA, Single-Stranded/genetics ; },
abstract = {A straightforward approach is suggested to selectively recognize specific products of loop-mediated isothermal amplification (LAMP) with the Cas12a nuclease without a need for a protospacer adjacent motif (PAM) in the sequence of LAMP amplicons (LAMPlicons). This strategy is based on the presence of single-stranded DNA loops in LAMPlicons and the ability of Cas12a to be trans-activated via the binding of guide RNA (gRNA) to single-stranded DNA in the absence of PAM. The approach feasibility is demonstrated on Clavibacter species-multiple bacterial plant pathogens that cause harmful diseases in agriculturally important plants. For Clavibacter species, the detection sensitivity of the developed PAM-independent LAMP/Cas12a system was determined by that of LAMP. The overall detection selectivity was enhanced by the Cas12a analysis of LAMPlicons. It was shown that the LAMP/Cas12a detection system can be fine-tuned by carefully designing gRNA to selectively distinguish C. sepedonicus from other Clavibacter species based on single nucleotide substitutions in the targeted LAMPlicon loop. The suggested loop-based Cas12a analysis of LAMPlicons was compatible with the format of a single test tube assay with the option of naked-eye detection. The findings broaden the palette of approaches to designing PAM-independent LAMP/Cas12a detection systems with potential for on-site testing.},
}
@article {pmid40869263,
year = {2025},
author = {Volodina, OV and Demchenko, AG and Anuchina, AA and Ryzhkova, OP and Kovalskaya, VA and Kondrateva, EV and Artemova, EV and Tabakov, VY and Ignatov, MA and Vorobyeva, NY and Osipov, AN and Lavrov, AV and Smirnikhina, SA},
title = {Prime Editing Modification with FEN1 Improves F508del Variant Editing in the CFTR Gene in Airway Basal Cells.},
journal = {International journal of molecular sciences},
volume = {26},
number = {16},
pages = {},
doi = {10.3390/ijms26167943},
pmid = {40869263},
issn = {1422-0067},
support = {N/A//The Ministry of Science and Higher Education of the Russian Federation for RCMG/ ; },
mesh = {*Cystic Fibrosis Transmembrane Conductance Regulator/genetics ; Humans ; *Gene Editing/methods ; *Cystic Fibrosis/genetics ; *Flap Endonucleases/genetics/metabolism ; CRISPR-Cas Systems ; Exodeoxyribonucleases/genetics/metabolism ; },
abstract = {Prime editing is a promising approach for correcting pathogenic variants, but its efficiency remains variable across genomic contexts. Here, we systematically evaluated 12 modifications of the PEmax system for correcting the CFTR F508del pathogenic variant that caused cystic fibrosis in patient-derived airway basal cells. We chose EXO1 and FEN1 nucleases to improve the original system. While all tested variants showed comparatively low efficiency in this AT-rich genomic region, 4-FEN modification demonstrated significantly improved editing rates (up to 2.13 fold) compared to standard PEmax. Our results highlight two key findings: first, the persistent challenge of AT-rich target sequence correction even with optimized editors, and second, the performance of 4-FEN suggests its potential value for other genomic targets.},
}
@article {pmid40869244,
year = {2025},
author = {Li, N and Yuan, X and Han, B and Guo, W and Chen, H},
title = {CRISPR/Cas-Mediated Optimization of Soybean Shoot Architecture for Enhanced Yield.},
journal = {International journal of molecular sciences},
volume = {26},
number = {16},
pages = {},
doi = {10.3390/ijms26167925},
pmid = {40869244},
issn = {1422-0067},
support = {2024ZD040780211//Biological Breeding-National Science and Technology Major Project/ ; CAAS-OCRI-ZDRW-202402//Science and Technology Innovation Project of the Oil Crops Research Institute, Chinese Academy of Agricultural Sciences/ ; 2024YFD1201400//the National Key Research and Development Program of China/ ; },
mesh = {*Glycine max/genetics/anatomy &amp; histology/growth &amp; development ; *CRISPR-Cas Systems ; *Gene Editing/methods ; Plant Breeding/methods ; *Plant Shoots/genetics/anatomy &amp; histology/growth &amp; development ; Plants, Genetically Modified/genetics ; Gene Expression Regulation, Plant ; Genome, Plant ; },
abstract = {Plant architecture is a crucial agronomic trait significantly impacting soybean (Glycine max) yield. Traditional breeding has made some progress in optimizing soybean architecture, but it is limited in precision and efficiency. The Clustered Regularly Interspaced Short Palindromic Repeats and CRISPR-associated protein (CRISPR/Cas) system, a revolutionary gene-editing technology, provides unprecedented opportunities for plant genetic improvement. This review outlines CRISPR's development and applications in crop improvement, focusing specifically on progress regulating soybean architecture traits affecting yield, such as node number, internode length, branching, and leaf morphology. It also discusses the technical challenges for CRISPR technology in enhancing soybean architecture, including that the regulatory network of soybean plant architecture is complex and the development of multi-omics platforms helps gene mining. The application of CRISPR enables precise the regulation of gene expression through promoter editing. Meanwhile, it is also faced with technical challenges such as the editing of homologous genes caused by genome polyploidy, the efficiency of editing tools and off-target effects, and low transformation efficiency. New delivery systems such as virus-induced genome editing bring hope for solving some of these problems. The review emphasizes the great potential of CRISPR technology in breeding next-generation soybean varieties with optimized architecture to boost yield potential.},
}
@article {pmid40868941,
year = {2025},
author = {Cannea, FB and Padiglia, A},
title = {Antioxidant Defense Systems in Plants: Mechanisms, Regulation, and Biotechnological Strategies for Enhanced Oxidative Stress Tolerance.},
journal = {Life (Basel, Switzerland)},
volume = {15},
number = {8},
pages = {},
doi = {10.3390/life15081293},
pmid = {40868941},
issn = {2075-1729},
abstract = {Plants must contend with oxidative stress, a paradoxical phenomenon in which reactive oxygen species (ROS) can cause cellular damage while also serving as key signaling molecules. Environmental stressors, such as drought, salinity, and temperature extremes, promote ROS accumulation, affecting plant growth and productivity. To maintain redox homeostasis, plants rely on antioxidant systems comprising enzymatic defenses, such as superoxide dismutase, catalase, and ascorbate peroxidase, and non-enzymatic molecules, including ascorbate, glutathione, flavonoids, and emerging compounds such as proline and nano-silicon. This review provides an integrated overview of antioxidant responses and their modulation through recent biotechnological advances, emphasizing the role of emerging technologies in advancing our understanding of redox regulation and translating molecular insights into stress-resilient phenotypes. Omics approaches have enabled the identification of redox-related genes, while genome editing tools, particularly those based on clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) proteins, offer opportunities for precise functional manipulation. Artificial intelligence and systems biology are accelerating the discovery of regulatory modules and enabling predictive modeling of antioxidant networks. We also highlight the contribution of synthetic biology to the development of stress-responsive gene circuits and address current regulatory and ethical considerations. Overall, this review aims to provide a comprehensive perspective on molecular, biochemical, and technological strategies to enhance oxidative stress tolerance in plants, thereby contributing to sustainable agriculture and food security in a changing climate.},
}
@article {pmid40868037,
year = {2025},
author = {Prates, FD and Araújo, MRB and da Silva Sousa, J and de Oliveira Sant'Anna, L and do Carmo Sant'Anna Cardoso, T and Silva, ACC and de Castro Soares, S and Andrade, BS and Dos Santos, LS and de Carvalho Azevedo, VA},
title = {Quinolone Resistance and Zoonotic Potential of Corynebacterium ulcerans from Domestic Animals in Brazil.},
journal = {Antibiotics (Basel, Switzerland)},
volume = {14},
number = {8},
pages = {},
doi = {10.3390/antibiotics14080843},
pmid = {40868037},
issn = {2079-6382},
support = {E-26/202.088/2020//Funda&#xe7;&#xe3;o de Amparo &#xe0; Pesquisa do Estado do Rio de Janeiro - FAPERJ/ ; E-26/205.900/2022//Funda&#xe7;&#xe3;o de Amparo &#xe0; Pesquisa do Estado do Rio de Janeiro - FAPERJ/ ; E26/211.554/2019//Funda&#xe7;&#xe3;o de Amparo &#xe0; Pesquisa do Estado do Rio de Janeiro - FAPERJ/ ; 311233/2022-8//Conselho Nacional de Desenvolvimento Cient&#xed;fico e Tecnol&#xf3;gico - CNPq/ ; APQ-00214-20//Funda&#xe7;&#xe3;o de Amparo &#xe0; Pesquisa do Estado de Minas Gerais - FAPEMIG/ ; 311249/2024-0//Conselho Nacional de Desenvolvimento Cient&#xed;fico e Tecnol&#xf3;gico - CNPq/ ; APQ-01323-15//Funda&#xe7;&#xe3;o de Amparo &#xe0; Pesquisa do Estado de Minas Gerais - FAPEMIG/ ; 88887.023263/2024-00//Coordena&#xe7;&#xe3;o de Aperfei&#xe7;oamento de Pessoal de N&#xed;vel Superior/ ; },
abstract = {BACKGROUND: Corynebacterium ulcerans is an emerging zoonotic pathogen capable of cau-sing diphtheria-like infections in humans.<br><br>OBJECTIVES: we report, for the first time in Brazil, the detection and phenotypic/genomic characterization of three atoxigenic ST-339 strains isolated from domestic animals, including one with a ciprofloxacin resistance profile linked to double GyrA mutations (S89L, D93G).<br><br>METHODS: species identification was performed by MALDI-TOF MS, followed by in vitro antimicrobial susceptibility testing, whole-genome sequencing, and bioinformatic analyses to predict virulence determinants, antimicrobial resistance genes, CRISPR-Cas systems, mobile genetic elements, and in silico structural analysis as well as phylogenetic reconstruction.<br><br>RESULTS: whole-genome sequencing confirmed species identity, revealed high genetic similarity, and identified distinct phylogenetic subclades, suggesting potential international dissemination. Genomic analyses showed conserved virulence determinants, such as incomplete pilus clusters, iron acquisition systems, and the pld gene, with the absence of the tox gene. Molecular modeling and dynamics simulations indicated that GyrA mutations disrupt critical ciprofloxacin-magnesium-water interactions, reducing binding stability. Mobile genetic elements, prophages, and CRISPR-Cas systems underscored the genomic plasticity of these isolates.<br><br>CONCLUSIONS: these findings document a little-studied antimicrobial resistance mechanism in zoonotic C. ulcerans, highlighting the need for strengthened surveillance and further research on virulence and resistance, even in ato-xigenic strains.},
}
@article {pmid40867941,
year = {2025},
author = {Yinsai, O and Yuantrakul, S and Srisithan, P and Zhou, W and Chittaprapan, S and Intajak, N and Kruayoo, T and Khamnoi, P and Tongjai, S and Daungsonk, K},
title = {Genomic Insights into Emerging Multidrug-Resistant Chryseobacterium indologenes Strains: First Report from Thailand.},
journal = {Antibiotics (Basel, Switzerland)},
volume = {14},
number = {8},
pages = {},
doi = {10.3390/antibiotics14080746},
pmid = {40867941},
issn = {2079-6382},
support = {HSRI 64-145 and HSRI 66-147//Health Systems Research Institute (HSRI), Thailand/ ; },
abstract = {Background: Chryseobacterium indologenes, an environmental bacterium, is increasingly recognized as an emerging nosocomial pathogen, particularly in Asia, and is often characterized by multidrug resistance. Objectives: This study aimed to investigate the genomic features of clinical C. indologenes isolates from Maharaj Nakorn Chiang Mai Hospital, Thailand, to understand their mechanisms of multidrug resistance, virulence factors, and mobile genetic elements (MGEs). Methods: Twelve C. indologenes isolates were identified, and their antibiotic susceptibility profiles were determined. Whole genome sequencing (WGS) was performed using a hybrid approach combining Illumina short-reads and Oxford Nanopore long-reads to generate complete bacterial genomes. The hybrid assembled genomes were subsequently analyzed to detect antimicrobial resistance (AMR) genes, virulence factors, and MGEs. Results: C. indologenes isolates were primarily recovered from urine samples of hospitalized elderly male patients with underlying conditions. These isolates generally exhibited extensive drug resistance, which was subsequently explored and correlated with genomic determinants. With one exception, CMCI13 showed a lower resistance profile (Multidrug resistance, MDR). Genomic analysis revealed isolates with genome sizes of 4.83-5.00 Mb and GC content of 37.15-37.35%. Genomic characterization identified conserved resistance genes (blaIND-2, blaCIA-4, adeF, vanT, and qacG) and various virulence factors. Phylogenetic and pangenome analysis showed 11 isolates clustering closely with Chinese strain 3125, while one isolate (CMCI13) formed a distinct branch. Importantly, each isolate, except CMCI13, harbored a large genomic island (approximately 94-100 kb) carrying significant resistance genes (blaOXA-347, tetX, aadS, and ermF). The absence of this genomic island in CMCI13 correlated with its less resistant phenotype. No plasmids, integrons, or CRISPR-Cas systems were detected in any isolate. Conclusions: This study highlights the alarming emergence of multidrug-resistant C. indologenes in a hospital setting in Thailand. The genomic insights into specific resistance mechanisms, virulence factors, and potential horizontal gene transfer (HGT) events, particularly the association of a large genomic island with the XDR phenotype, underscore the critical need for continuous genomic surveillance to monitor transmission patterns and develop effective treatment strategies for this emerging pathogen.},
}
@article {pmid40867592,
year = {2025},
author = {De Florian Fania, R and Maiocchi, S and Klima, R and Rossin, M and Pellegrini, V and Ghetti, S and Selvestrel, D and Mattevi, MC and Fava, LL and Braga, L and Collavin, L},
title = {Drug-Repurposing Screen Identifies Thiostrepton as a Novel Regulator of the Tumor Suppressor DAB2IP.},
journal = {Biomolecules},
volume = {15},
number = {8},
pages = {},
doi = {10.3390/biom15081147},
pmid = {40867592},
issn = {2218-273X},
support = {IG21803//Italian Association for Cancer Research/ ; 20174PLLYN_004//Ministero dell'universit&#xe0; e della ricerca/ ; CN00000041//NextGenerationEU PNRR MUR - M4C2 - Action 1.4/ ; MFAG23560//Italian Association for Cancer Research/ ; },
mesh = {Humans ; *ras GTPase-Activating Proteins/metabolism/genetics ; Cell Line, Tumor ; *Thiostrepton/pharmacology ; Male ; CRISPR-Cas Systems ; Prostatic Neoplasms/drug therapy/metabolism/genetics ; Antineoplastic Agents/pharmacology ; },
abstract = {The tumor suppressor DAB2IP, a RasGAP and cytoplasmic adaptor protein, modulates signal transduction in response to several extracellular stimuli, negatively regulating multiple oncogenic pathways. Accordingly, the loss of DAB2IP in tumor cells fosters metastasis and enhances chemo- and radioresistance. DAB2IP is rarely mutated in cancer but is frequently downregulated or inactivated by multiple mechanisms. Solid experimental evidence shows that DAB2IP reactivation reduces cancer aggressiveness in tumors driven by multiple different oncogenic mutations, making this protein an interesting target for cancer therapy. Considering this evidence, we screened a drug library to identify molecules that increase DAB2IP protein levels. We employed CRISPR/Cas9 gene editing to generate two prostate cancer cell models in which endogenous DAB2IP is fused to HiBiT, a peptide tag that enables luminescence-based detection of protein levels in a sensitive and quantitative manner. Using this approach, we identified drugs able to increase DAB2IP levels. We focused our attention on thiostrepton, a natural cyclic oligopeptide antibiotic that has been reported to inhibit the survival of various cancer cell lines. Functional experiments revealed that the cancer-inhibitory effect of thiostrepton is reduced in the absence of DAB2IP, suggesting that upregulation of this protein contributes to its action. These findings encourage further development of thiostrepton for the treatment of solid cancers and unveil a novel molecular target underlying its anti-tumoral activity.},
}
@article {pmid40867578,
year = {2025},
author = {Lv, S and Xu, X and Yang, S and Feng, M and Yuan, Z and Liu, X and Jiang, C and Song, J and Mu, Y},
title = {Improving the Efficiency of CRISPR/Cas9-Mediated Non-Homologous End Joining Gene Knockout Using Small Molecules in Porcine Cells.},
journal = {Biomolecules},
volume = {15},
number = {8},
pages = {},
doi = {10.3390/biom15081132},
pmid = {40867578},
issn = {2218-273X},
mesh = {Animals ; *CRISPR-Cas Systems/genetics ; *DNA End-Joining Repair/drug effects/genetics ; Swine ; *Gene Editing/methods ; *Gene Knockout Techniques/methods ; Cell Line ; *Small Molecule Libraries/pharmacology ; },
abstract = {The CRISPR/Cas9 genome editing system has emerged as an effective platform to generate loss-of-function gene edits through non-homologous end joining (NHEJ) without a repair template. To verify whether small molecules can enhance the efficiency of CRISPR/ Cas9-mediated NHEJ gene editing in porcine cells, this experiment investigated the effects of six small-molecule compounds, namely Repsox, Zidovudine, IOX1, GSK-J4, YU238259, and GW843682X, on the efficiency of CRISPR/Cas9-mediated NHEJ gene editing. The results showed the optimal concentrations of the small molecules, including Repsox, Zidovudine, IOX1, GSK-J4, YU238259, and GW843682X, for in vitro-cultured PK15 viability. Compared with the control group, the single small molecules Repsox, Zidovudine, GSK-J4, and IOX1 increased the efficiency of NHEJ-mediated gene editing 3.16-fold, 1.17-fold, 1.16-fold, and 1.120-fold, respectively, in the Cas9-sgRNA RNP delivery system. There were no benefits when using YU238259 and GW843682X compared with the control group. In the CRISPR/Cas9 plasmid delivery system, the Repsox, Zidovudine, IOX1, and GSK-J4 treatments increased the efficiency of NHEJ-mediated gene editing 1.47-fold, 1.15-fold, 1.21-fold, and 1.23-fold, respectively, compared with the control group. Repsox can also improve the efficiency of NHEJ-mediated multi-gene editing based on a CRISPR sgRNA-tRNA array. We also explored the mechanism of Repsox's effect on the efficiency of NHEJ-mediated gene editing. The results showed that Repsox reduces the expression levels of SMAD2, SMAD3, and SMAD4 in the TGF-β pathway, indicating that Repsox can increase the efficiency of CRISPR NHEJ-mediated gene editing in porcine cells through the TGF-β pathway.},
}
@article {pmid40867053,
year = {2025},
author = {Wang, L and Wang, Y and Chen, J and Zhu, Y and Qin, H and Liu, J and Ai, Y and Lai, J and Lian, Z and Han, H},
title = {An engineered CRISPR-Cas12i tool for efficient multiplexed genome editing.},
journal = {Nucleic acids research},
volume = {53},
number = {16},
pages = {},
doi = {10.1093/nar/gkaf806},
pmid = {40867053},
issn = {1362-4962},
support = {2022ZD04013//Great Projects of Agricultural Biotechnology Breeding in China/ ; 2025KJTW0022//Unveiling List and Entrusting the Leader for Science and Technology/ ; 2022TC025//Chinese Universities Scientific Fund/ ; PC2023A01004//Pinduoduo-China Agricultural University Research Fund/ ; },
mesh = {*CRISPR-Cas Systems ; *Gene Editing/methods ; RNA, Guide, CRISPR-Cas Systems/genetics/metabolism ; *CRISPR-Associated Proteins/genetics/metabolism ; Humans ; Streptococcus pyogenes/genetics ; *Endodeoxyribonucleases/genetics/metabolism ; Bacterial Proteins ; },
abstract = {Developing efficient and simplified tools for multiplexed genome editing remains challenging due to limitations in precursor CRISPR RNA (pre-crRNA) processing and reliance on additional RNA-based regulatory components. Cas12i.3, a small RNA-guided nuclease, reportedly lacks pre-crRNA processing ability, restricting its multiplexing capability. Here, we engineered Cas12i.3 by optimizing CRISPR RNA (crRNA) design, codon usage, and exonuclease fusion, generating initial optimized Cas12i (IOCas12i) system. Further rational design and amino acid mutations yielded the highly efficient enhanced optimized Cas12i (EOCas12i) systems, EOCas12i-Combo1 and EOCas12i-Combo2, exhibiting 2.5- to 22.8-fold and 3.0- to 60.0-fold editing efficiencies relative to wild-type Cas12i.3, comparable to Streptococcus pyogenes Cas9 (SpCas9) and Lachnospiraceae bacterium Cas12a (LbCas12a). Additionally, they exhibited high specificity and produced longer insertions and deletions (indels) that may facilitate gene knockout. Notably, both variants enabled efficient multiplexed editing of up to 30 targets using compact crRNA arrays. These advancements position EOCas12i-Combo1 and EOCas12i-Combo2 as promising platforms for multiplexed genome editing applications.},
}
@article {pmid40866673,
year = {2025},
author = {Lambie, EJ and Greig, A and Conradt, B},
title = {Fluorescent protein tagging of C. elegans core apoptosis pathway components reveals mitochondrial localization of CED-9 Bcl-2, CED-4 Apaf1 and CED-3 Caspase in non-apoptotic and apoptotic cells.},
journal = {Cell death and differentiation},
volume = {},
number = {},
pages = {},
pmid = {40866673},
issn = {1476-5403},
support = {RSWF\R1\180008//Royal Society/ ; BB/V007572/1//RCUK | Biotechnology and Biological Sciences Research Council (BBSRC)/ ; BB/V015648/1//RCUK | Biotechnology and Biological Sciences Research Council (BBSRC)/ ; },
abstract = {We used CRISPR-Cas-mediated modification of the genomic loci for C. elegans genes ced-9 Bcl-2, ced-4 Apaf1 and ced-3 Caspase to add the coding sequence for the mNeonGreen (mNG) fluorescent protein to the endogenous open reading frames. In each case, the addition of mNG caused little or no apparent alteration of gene function. We found that tagged versions of CED-9, CED-4 and CED-3 proteins colocalize with mitochondria in all cells of live mid-late stage embryos and are distributed along the entire length of mitochondria. However, CED-4 also exhibits localized puncta of ~4-fold enrichment, and these are preferentially oriented toward the nucleus. We do not observe any shift in the localization pattern of tagged CED-4 in cells that are committing to apoptosis during normal development. However, when egl-1 BH3-only is overexpressed or ced-9 removed by mutation, CED-4::mNG is no longer distributed along the entire length of mitochondria and instead becomes enriched in the bright puncta. Finally, localization of CED-3::mNG to mitochondria is independent of both CED-9 and CED-4. This study represents the first analysis of the distribution and sub-cellular localization of endogenous CED-9 Bcl-2, CED-4 Apaf1 and CED-3 Caspase proteins in live embryos. Our results impact the current model of apoptosis commitment in C. elegans.},
}
@article {pmid40864651,
year = {2025},
author = {Vicary, AC and Jordan, SNZ and Mendes, M and Swaminath, S and Castro, LK and Porter, JS and Vo, KD and Russell, AB},
title = {CRISPR with Transcriptional Readout reveals influenza transcription is modulated by NELF and can precipitate an interferon response.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {122},
number = {35},
pages = {e2515564122},
doi = {10.1073/pnas.2515564122},
pmid = {40864651},
issn = {1091-6490},
support = {5R35GM147031//HHS | NIH | National Institute of General Medical Sciences (NIGMS)/ ; T32GM133351//HHS | NIH | National Institute of General Medical Sciences (NIGMS)/ ; 5R01AI176639//HHS | NIH | National Institute of Allergy and Infectious Diseases (NIAID)/ ; },
mesh = {Humans ; *Interferons/genetics/metabolism ; *Influenza A virus/genetics/immunology ; *Transcription, Genetic ; CRISPR-Cas Systems ; *Influenza, Human/genetics/virology/immunology ; Virus Replication ; *Clustered Regularly Interspaced Short Palindromic Repeats ; *Transcription Factors/metabolism/genetics ; Immunity, Innate ; HEK293 Cells ; Promoter Regions, Genetic ; },
abstract = {Transcription of interferons upon viral infection is critical for cell-intrinsic innate immunity. This process is influenced by many host and viral factors. To identify host factors that modulate interferon induction within cells infected by influenza A virus, we developed CRISPR with Transcriptional Readout using sequencing (CRITR-seq). CRITR-seq is a method linking CRISPR guide sequence to activity at a promoter of interest. Employing this method, we find that depletion of the Negative Elongation Factor (NELF) complex increases both flu transcription and interferon expression. We find that the process of flu transcription, both in the presence and absence of viral replication, is a key contributor to interferon induction. Taken together, our findings highlight innate immune ligand concentration as a limiting factor in triggering an interferon response, identify NELF as an important interface with the flu life cycle, and validate CRITR-seq as a tool for genome-wide screens for phenotypes of gene expression.},
}
@article {pmid40864257,
year = {2025},
author = {Wang, Y and Liu, WJ and Meng, CH and Wang, HL and Cui, ZK and Zhang, J and Zhang, JL and Qian, Y and Li, YX and Cao, SX},
title = {Cytosine base editor-mediated high-efficiency myostatin editing in Hu sheep.},
journal = {Functional &amp; integrative genomics},
volume = {25},
number = {1},
pages = {176},
pmid = {40864257},
issn = {1438-7948},
support = {JBGS&#x3014;2021&#x3015;025//Jiangsu Seed Industry Vitalization and Open Competition Project/ ; JBGS&#x3014;2021&#x3015;025//Jiangsu Seed Industry Vitalization and Open Competition Project/ ; JBGS&#x3014;2021&#x3015;025//Jiangsu Seed Industry Vitalization and Open Competition Project/ ; JBGS&#x3014;2021&#x3015;025//Jiangsu Seed Industry Vitalization and Open Competition Project/ ; JBGS&#x3014;2021&#x3015;025//Jiangsu Seed Industry Vitalization and Open Competition Project/ ; JBGS&#x3014;2021&#x3015;025//Jiangsu Seed Industry Vitalization and Open Competition Project/ ; JBGS&#x3014;2021&#x3015;025//Jiangsu Seed Industry Vitalization and Open Competition Project/ ; JBGS&#x3014;2021&#x3015;025//Jiangsu Seed Industry Vitalization and Open Competition Project/ ; JBGS&#x3014;2021&#x3015;025//Jiangsu Seed Industry Vitalization and Open Competition Project/ ; JBGS&#x3014;2021&#x3015;025//Jiangsu Seed Industry Vitalization and Open Competition Project/ ; },
mesh = {Animals ; *Myostatin/genetics/metabolism ; Sheep/genetics/growth &amp; development ; *Gene Editing/methods ; *Cytosine/metabolism ; Female ; CRISPR-Cas Systems ; },
abstract = {The cytosine base editor (CBE) enables precise C-to-T substitution without inducing DNA double-strand breaks, which offering a promising tool for editing livestock genomes to enhance economically valuable traits. In this study, using Hu sheep, characterized by high reproductive performance but suboptimal meat production as the research subject, two CBE-editing sgRNAs (sgM1 and sgM2) targeting the negative regulator Myostatin (MSTN) gene were designed. The results revealed a 75% editing efficiency of sgM2 at the parthenogenetically activated embryonic level with no detectable off-target effects. Thirty-four zygotes from five Hu sheep microinjected with sgM2 and CBE mRNA mixtures were transferred into four Hu sheep recipient ewes, yielding four lambs with confirmed MSTN editing and no off-target activity. Statistical analysis of growth performance data revealed that MSTN-edited Hu sheep exhibited significantly (P < 0.05) higher body weights at 120-180 days, and significantly (P < 0.05) enlarged muscle fiber cross-sectional areas compared to wild-type controls. Edited Hu sheep displayed reduced MSTN protein expression, elevated p-AKT levels, and diminished p-ERK and p-p38 signaling. In conclusion, MSTN-edited Hu sheep were highly efficient generated using CBE, and further analysis demonstrate that MSTN editing activates the AKT pathway while suppressing MAPK signaling, leading to muscle fiber hypertrophy and accelerated growth, which provides technical methodologies and breeding materials for developing fast-growing, meat-type Hu sheep-germplasm.},
}
@article {pmid40562937,
year = {2025},
author = {Andersen, BM and Faust Akl, C and Wheeler, MA and Li, Z and Diebold, M and Kilian, M and Rone, JM and Misra, A and Kenison, JE and Lee, JH and Lee, HG and Polonio, CM and Merrell, D and Weiss, JH and Godinez, L and Piester, G and Illouz, T and Ye, JJ and Ghia, A and Martinez, J and Chung, EN and Srun, L and Farrenkopf, D and Flausino, LE and Schüle, AM and Sanmarco, LM and Giovannoni, F and Fehrenbacher, L and Charabati, M and Gutiérrez-Vázquez, C and Cusick, MM and Prabhakar, PS and Bossi, CC and Lapinskas, E and Nowarski, R and Getz, G and Ligon, KL and Prinz, M and Chiocca, EA and Reardon, DA and Quintana, FJ},
title = {Barcoded viral tracing identifies immunosuppressive astrocyte-glioma interactions.},
journal = {Nature},
volume = {644},
number = {8078},
pages = {1097-1106},
pmid = {40562937},
issn = {1476-4687},
mesh = {*Astrocytes/immunology/pathology/metabolism/cytology ; Humans ; Animals ; Mice ; Receptors, Formyl Peptide/metabolism/genetics ; Single-Cell Analysis ; *Glioblastoma/immunology/pathology/genetics ; *Tumor Microenvironment/immunology/genetics ; Annexin A1/metabolism/genetics ; *Cell Communication/immunology ; Female ; *Brain Neoplasms/immunology/pathology/genetics ; Male ; CD8-Positive T-Lymphocytes/immunology ; NF-kappa B/metabolism ; Necroptosis ; CRISPR-Cas Systems/genetics ; Inflammasomes/metabolism ; Signal Transduction ; Dendritic Cells/immunology ; Cell Line, Tumor ; Macrophages/immunology ; *Immune Tolerance ; Mice, Inbred C57BL ; },
abstract = {Glioblastoma (GBM) is the most lethal primary brain malignancy[1]. Immunosuppression in the GBM tumour microenvironment (TME) is an important barrier to immune-targeted therapies, but our understanding of the mechanisms of immune regulation in the GBM TME is limited[2]. Here we describe a viral barcode interaction-tracing approach[3] to analyse TME cell-cell communication in GBM clinical samples and preclinical models at single-cell resolution. We combine it with single-cell and bulk RNA-sequencing analyses, human organotypic GBM cultures, in vivo cell-specific CRISPR-Cas9-driven genetic perturbations as well as human and mouse experimental systems to identify an annexin A1-formyl peptide receptor 1 (ANXA1-FPR1) bidirectional astrocyte-GBM communication pathway that limits tumour-specific immunity. FPR1 inhibits immunogenic necroptosis in tumour cells, and ANXA1 suppresses NF-κB and inflammasome activation in astrocytes. ANXA1 expression in astrocytes and FPR1 expression in cancer cells are associated with poor outcomes in individuals with GBM. The inactivation of astrocyte-glioma ANXA1-FPR1 signalling enhanced dendritic cell, T cell and macrophage responses, increasing infiltration by tumour-specific CD8[+] T cells and limiting T cell exhaustion. In summary, we have developed a method to analyse TME cell-cell interactions at single-cell resolution in clinical samples and preclinical models, and used it to identify bidirectional astrocyte-GBM communication through ANXA1-FPR1 as a driver of immune evasion and tumour progression.},
}
@article {pmid40542534,
year = {2025},
author = {Wu, R and Chai, Y and Li, Y and Chen, T and Qi, W and Xue, Y and Lu, H and Chen, Z and Guo, B and Li, S and Qin, D and An, X},
title = {A visual monitoring DNA-free multi-gene editing system excised via LoxP::FRT/FLP in poplar.},
journal = {Plant biotechnology journal},
volume = {23},
number = {9},
pages = {4017-4029},
pmid = {40542534},
issn = {1467-7652},
mesh = {*Gene Editing/methods ; *Populus/genetics ; *CRISPR-Cas Systems/genetics ; Plants, Genetically Modified/genetics ; Agrobacterium tumefaciens/genetics ; },
abstract = {The CRISPR-Cas9 system has emerged as a potent molecular scalpel for precise genome editing, and profoundly revolutionized plant genetics and breeding, facilitating the development of innovative and improved plant varieties. Typically, the CRISPR-Cas9 gene-editing construct is delivered into target organisms via Agrobacterium tumefaciens-mediated transformation or biolistic methods. However, the incorporation of the CRISPR-Cas9 machinery increases the risk of off-target effects, causing unintended genomic alterations. Additionally, the introduction of exogenous DNA sequences, such as antibiotic resistance marker, raises public concerns regarding the biosafety and regulatory oversight of genetically modified organisms (GMOs), potentially hindering regulatory approval and commercialization. Here, we have engineered an integrated system comprising RUBY, LoxP::FRT/FLP and CRISPR/Cas9-sgRNA cassettes within a single construct, allowing visible color monitoring throughout process including genetic transform, positive transgenic and edited events screening, as well as exogenous DNA excision events, we refer to it as 'a visual monitoring DNA-free multi-gene editing system (VMDFGE)'. This system was introduced into poplar through Agrobacterium tumefaciens-mediated transformation, yielding transgenic poplars with a 75.0% visual screening rate, a 45.8% targeted mutation rate and a 54.5% excision rate for the entire integration system. This approach eliminates the concerns associated with off-target effects and GMO regulatory challenges. It offers significant potential for improvement of poplar, other woody plants and crop species while removing the foreign DNA.},
}
@article {pmid40087540,
year = {2025},
author = {Wang, Y and Jiang, J and Xiong, Q and Li, S and Shao, J and Xie, M and Zeng, AP},
title = {Programmable solid-state condensates for spatiotemporal control of mammalian gene expression.},
journal = {Nature chemical biology},
volume = {21},
number = {9},
pages = {1457-1466},
pmid = {40087540},
issn = {1552-4469},
mesh = {Humans ; *Gene Expression Regulation ; *Biomolecular Condensates/chemistry/genetics/metabolism ; Animals ; CRISPR-Cas Systems ; Cell Nucleus/metabolism/genetics ; RNA, Messenger/genetics/metabolism ; HEK293 Cells ; HeLa Cells ; },
abstract = {Engineering of nuclear condensates with chemically inducible gene switches is highly desired but challenging for precise and on-demand regulation of mammalian gene expression. Here, we harness the phase-separation capability of biomolecular condensates and describe a versatile strategy to chemically program ligand-dependent gene expression at various stages of interest. By engineering synthetic anchor proteins capable of tethering various genetically encoded condensate structures toward different cellular compartments or gene products of interest, inducible regulation of transcriptional and translational activities was achieved at different endogenous and episomal loci using the same sets of anchor proteins and synthetic solid-state condensates. Using such a holistic condensate-based strategy, we not only achieved regulation performances comparing favorably to state-of-the-art strategies described for CRISPR-Cas9 activity and transcriptional silencing but further showed that chemically inducible retention of mRNA molecules into engineered condensate structures within the nucleus can become a remarkably efficient alternative for translational regulation.},
}
@article {pmid40862940,
year = {2025},
author = {Guan, X and Wang, P and Wang, Y and Sun, S},
title = {CRISPR/Cas12a-Chemiluminescence Cascaded Bioassay for Amplification-Free and Sensitive Detection of Nucleic Acids.},
journal = {Biosensors},
volume = {15},
number = {8},
pages = {},
pmid = {40862940},
issn = {2079-6374},
support = {21874082//National Natural Science Foundation of China/ ; RXRC2022004//Fundamental Research Funds for the Central Public Welfare Research Institutes/ ; },
mesh = {Human papillomavirus 16/isolation &amp; purification/genetics ; *CRISPR-Cas Systems ; Humans ; *Biosensing Techniques ; Luminescent Measurements ; Parvovirus B19, Human/genetics/isolation &amp; purification ; Limit of Detection ; *DNA, Viral/analysis ; Biological Assay ; DNA, Single-Stranded ; },
abstract = {The CRISPR/Cas system has attracted increasing attention in accurate nucleic acid detection. Herein, we reported a CRISPR/Cas12a-chemiluminescence cascaded bioassay (CCCB) for the amplification-free and sensitive detection of human papillomavirus type 16 (HPV-16) and parvovirus B19 (PB-19). A magnetic bead (MB)-linking single-stranded DNA (LssDNA)-alkaline phosphatase (ALP) complex was constructed as the core component of the bioassay. During the detection process, the single-stranded target DNA was captured and enriched by LssDNA and then activated the trans-cleavage activity of Cas12a. Due to the Cas12a-mediated cleavage of LssDNA, ALP was released from the MB, subsequently catalyzing the substrate to generate a chemiluminescence (CL) signal. Given the cascade combination of CRISPR/Cas12a with the CL technique, the limits of detection for HPV-16 and PB-19 DNA were determined as 0.14 pM and 0.37 pM, respectively, and the whole detection could be completed within 60 min. The practicality and reliability of the platform were validated through target-spiked clinical specimens, and the recovery rate was 93.4-103.5%. This dual-amplification strategy-operating without target pre-amplification-featured high specificity, low contamination risk, facile preparation, and robust stability. It provides a novel approach for sensitive nucleic acid detection, with the potential for rapid extension to the diagnosis of various infectious diseases.},
}
@article {pmid40862153,
year = {2025},
author = {Pacheco-Acosta, S and Castro-Toro, G and Rojas-Villalobos, C and Valenzuela, C and Haristoy, JJ and Zapata-Araya, A and Moya-Beltrán, A and Sepúlveda-Rebolledo, P and Pérez-Rueda, E and Ulloa, R and Giaveno, A and Issotta, F and Díez, B and Beard, S and Quatrini, R},
title = {Exploring the eco-evolutionary role of plasmids and defense systems in 'Fervidacidithiobacillus caldus' extreme acidophile.},
journal = {Frontiers in microbiology},
volume = {16},
number = {},
pages = {1610279},
pmid = {40862153},
issn = {1664-302X},
abstract = {Plasmids are major drivers of microbial evolution, enabling horizontal gene transfer (HGT) and facilitating adaptation through the dissemination of relevant functional genes and traits. However, little is known about plasmid diversity and function in extremophiles. 'Fervidacidithiobacillus caldus', a meso-thermo-acidophilic sulfur oxidizer, is a key player in sulfur cycling in natural and industrially engineered acidic environments. Here, we present a bioinformatic analysis of the plasmidome, and associated anti-mobile genetic element (anti-MGE) defense systems (defensome), across genomes of this species and metagenomes from diverse natural and industrial settings harboring 'F. caldus'. We identified >30 distinct plasmids, representing five consistent replication-mobilization families. Plasmids ranged in size between 2.5-65 kb, with gene content and plasmid modularity scaling with element size and copy numbers inversely correlating with size. Plasmids carried variable numbers of hypothetical proteins and transposases, with annotated cargo genes reflecting functional differentiation by habitat. Defensome profiling revealed over 50 anti-MGE systems in sequenced 'F. caldus' isolates, including diverse restriction-modification systems, CRISPR-Cas types IV-A and V-F, and widespread abortive infection and composite defense systems such as Wadjet, Gabija, and Zorya. In environmental populations, an inverse relationship was observed between defensome complexity and plasmidome abundance and diversity, underscoring a pivotal role of the host defensome in modulating persistence, compatibility, and overall plasmid diversity across 'F. caldus' populations. Yet, other plasmids appeared decoupled from both host abundance and defensome complexity, suggesting potential host shifts, environmental persistence, or differential replication under suboptimal growth conditions for the host. Altogether, these findings point to a modular, functionally diverse adaptive plasmidome shaped by environmental pressures, by the interplay with the host's defensome, and likely also by other eco-evolutionary processes at play in natural environments. While these associations are compelling, causal relationships remain to be experimentally validated. These insights broaden our understanding of mobile genetic elements in extreme environments and provide a foundation for plasmid-based vector design and synthetic biology applications in acidophiles, with direct implications to biomining and environmental remediation.},
}
@article {pmid40861495,
year = {2025},
author = {Plöhn, O and Singh, AK and Greger, C and Wolfgramm, H and Baglanova, M and Surmann, K and Völker, U and Bröker, BM and Becker, K and Seifert, U and Cammann, C},
title = {Deletion of the E3 ubiquitin ligase LRSAM1 fosters intracellular Staphylococcus aureus survival.},
journal = {Frontiers in cellular and infection microbiology},
volume = {15},
number = {},
pages = {1597830},
pmid = {40861495},
issn = {2235-2988},
mesh = {Humans ; *Staphylococcus aureus/physiology/growth &amp; development ; *Ubiquitin-Protein Ligases/genetics/metabolism/deficiency ; A549 Cells ; *Host-Pathogen Interactions ; CRISPR-Cas Systems ; *Staphylococcal Infections/microbiology/immunology ; Ubiquitination ; Gene Deletion ; Autophagy ; Epithelial Cells/microbiology ; *Microbial Viability ; Interleukin-6/metabolism ; Cytokines/metabolism ; Bacterial Load ; Signal Transduction ; },
abstract = {BACKGROUND: Intracellular invasion and persistence of Staphylococcus aureus can lead to chronic infection and is an effective strategy for the pathogen to evade the host immune response and antibiotic therapy. Selective ubiquitination of bacterial surfaces via E3 ubiquitin ligases is a mechanism by which host cells combat intracellular bacteria and target them for autophagosomal degradation. However, knowledge of the E3 ligases involved in intracellular recognition of S. aureus is still very limited.<br><br>METHODS: We studied A549 lung epithelial cells during S. aureus infection, focusing on the role of the E3 ligase leucine rich repeat and sterile alpha motif containing 1 (LRSAM1). We used the CRISPR-Cas9 system to generate LRSAM1-deficient A549 cells and monitored intracellular bacterial survival, activation of host cellular signalling pathways related to cytokine production, and host cell death during S. aureus infection.<br><br>RESULTS: In LRSAM1-deficient host cells we observed a significant increase in intracellular bacterial load, which was accompanied by an increased host cell death and elevated secretion of the pro-inflammatory cytokine IL-6. Despite induced selective autophagy, LRSAM1 knockout host cells were incapable of lowering and eliminating the pathogen, which seems to be caused by the reduced ubiquitination of the bacterial surface.<br><br>CONCLUSION: The results indicate a significant role of LRSAM1 in the clearance of intracellular S. aureus. This contributes to a deeper understanding of the host cellular responses to S. aureus infection and will facilitate the development of novel therapeutic strategies to combat intracellularly persistent S. aureus.},
}
@article {pmid40859134,
year = {2025},
author = {Li, L and Wang, Y and Yang, F and Li, W and Wang, Q and Xu, X and Wu, Y and Ji, X},
title = {Establishment of a CRISPR/Cas9 gene editing system based on growth points transformation method in Fraxinus mandshurica.},
journal = {BMC plant biology},
volume = {25},
number = {1},
pages = {1127},
pmid = {40859134},
issn = {1471-2229},
support = {2021YFD2200303//the National Key R&amp;D Program of China/ ; 2021YFD2200303//the National Key R&amp;D Program of China/ ; 2021YFD2200303//the National Key R&amp;D Program of China/ ; 2021YFD2200303//the National Key R&amp;D Program of China/ ; 2021YFD2200303//the National Key R&amp;D Program of China/ ; 2021YFD2200303//the National Key R&amp;D Program of China/ ; 2021YFD2200303//the National Key R&amp;D Program of China/ ; 2021YFD2200303//the National Key R&amp;D Program of China/ ; },
mesh = {*CRISPR-Cas Systems ; *Gene Editing/methods ; *Fraxinus/genetics/growth &amp; development/physiology ; Plants, Genetically Modified/genetics ; Droughts ; Transformation, Genetic ; Agrobacterium tumefaciens/genetics ; },
abstract = {The lack of an effective gene editing technology system for Fraxinus mandshurica makes it challenging to improve its traits through genetic engineering methods. In this study, an effective CRISPR/Cas9 gene editing system targeting plant growth points was established through the optimization of Agrobacterium tumefaciens concentration and infection duration. Furthermore, a tissue culture system for clustered buds was developed by supplementing the media with hormones at different concentrations. FmbHLH1-edited chimeric plants were successfully generated using the developed CRISPR/Cas9 gene editing system. Homozygous plants were induced and screened using the developed clustered bud system. Among 100 randomly transformed growing points, 18% of the induced clustered buds were gene-edited, which confirmed that the established CRISPR/Cas9 gene editing system was effective. Phenotypic analysis and evaluation of drought tolerance-related physiological indicators in FmbHLH1 knockout and wild-type lines revealed that FmbHLH1 positively regulated the drought tolerance of F. mandshurica by adjusting its ability to scavenge reactive oxygen species and to regulate osmotic potential. In summary, we developed an effective CRISPR/Cas9 gene editing system for F. mandshurica, providing an effective method for the molecular breeding of F. mandshurica.},
}
@article {pmid40858609,
year = {2025},
author = {Xu, D and Besselink, S and Ramadoss, GN and Dierks, PH and Lubin, JP and Pattali, RK and Brim, JI and Christenson, AE and Colias, PJ and Ornelas, IJ and Nguyen, CD and Chasins, SE and Conklin, BR and Nuñez, JK},
title = {Programmable epigenome editing by transient delivery of CRISPR epigenome editor ribonucleoproteins.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {7948},
pmid = {40858609},
issn = {2041-1723},
support = {R35GM155044-01//U.S. Department of Health &amp; Human Services | National Institutes of Health (NIH)/ ; UCB 20240097//Gladstone Institutes (J. David Gladstone Institutes)/ ; },
mesh = {Humans ; *Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; *Epigenome/genetics ; *Ribonucleoproteins/genetics/metabolism ; *Epigenesis, Genetic ; HEK293 Cells ; DNA Methyltransferase 3A ; tau Proteins/genetics/metabolism ; DNA (Cytosine-5-)-Methyltransferases/genetics/metabolism ; Neurons/metabolism ; Epigenome Editing ; Mixed Function Oxygenases ; Proto-Oncogene Proteins ; },
abstract = {Programmable epigenome editors modify gene expression in mammalian cells by altering the local chromatin environment at target loci without inducing DNA breaks. However, the large size of CRISPR-based epigenome editors poses a challenge to their broad use in biomedical research and as future therapies. Here, we present Robust ENveloped Delivery of Epigenome-editor Ribonucleoproteins (RENDER) for transiently delivering programmable epigenetic repressors (CRISPRi, DNMT3A-3L-dCas9, CRISPRoff) and activator (TET1-dCas9) as ribonucleoprotein complexes into human cells to modulate gene expression. After rational engineering, we show that RENDER induces durable epigenetic silencing of endogenous genes across various human cell types, including primary T cells. Additionally, we apply RENDER to epigenetically repress endogenous genes in human stem cell-derived neurons, including the reduction of the neurodegenerative disease associated V337M-mutated Tau protein. Together, our RENDER platform advances the delivery of CRISPR-based epigenome editors into human cells, broadening the use of epigenome editing in fundamental research and therapeutic applications.},
}
@article {pmid40857323,
year = {2025},
author = {Luo, J and Chia, N and Qin, Y and Tan, P and Zhang, L and Yang, S and Yuan, Z and Hong, L and Lee, SY and Tong, Y},
title = {STAGE: A compact and versatile TnpB-based genome editing toolkit for Streptomyces.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {122},
number = {35},
pages = {e2509146122},
doi = {10.1073/pnas.2509146122},
pmid = {40857323},
issn = {1091-6490},
support = {2021YFA0909500//MOST | National Key Research and Development Program of China (NKPs)/ ; 21TQ1400204//Shanghai Pilot Program for Basic Research/ ; 24HC2810200//Science and Technology Commission of Shanghai Municipality (STCSM)/ ; MMLKF22-03//State Key Laboratory of Microbial Metabolism (SKLMM)/ ; 2022M3J5A1056117//Ministry of Science and ICT, South Korea (MSIT)/ ; },
mesh = {*Gene Editing/methods ; *Streptomyces/genetics ; CRISPR-Cas Systems/genetics ; *Genome, Bacterial ; *Bacterial Proteins/genetics/metabolism ; Multigene Family ; },
abstract = {Streptomyces are naturally endowed with the capacity to produce a wide array of natural products with biomedical and biotechnological value. They have garnered great interest in synthetic biology applications given the abundance of uncharacterized biosynthetic gene clusters (BGCs). However, progress has been hindered by the limited availability of genetic tools for manipulating these bacteria. Several representative CRISPR-Cas systems have been established in Streptomyces to streamline experimental workflows and improve editing efficiency. Nevertheless, their broader applicability has been constrained by issues such as nuclease activity-related cytotoxicity and the large size of effector proteins. To address these challenges, we present Streptomyces-compatible TnpB-assisted genome editing (STAGE), a genetic toolkit based on ISDra2 TnpB, which is approximately one-third the size of Cas9 and enables precise, site-specific gene editing. We demonstrated that STAGE introduces genetic mutations with high efficiency and minimal off-target effects in two industrially important Streptomyces strains. Building on this platform, we developed STAGE-cBEST and STAGE-McBEST, enabling single and multiplexed C·G-to-T·A base editing, respectively, with editing efficiencies exceeding 75%. To further enhance performance, we engineered the ISDra2 TnpB system using an AI-assisted protein engineering framework, resulting in two variants that achieve nearly 100% genome editing efficiency. Additionally, through sequence homology analysis, we identified a TnpB ortholog from the same biological origin of ISDra2 TnpB, which also functions effectively as a gene editing tool. Our study establishes STAGE as a highly precise, programmable, and versatile genome editing platform for Streptomyces, paving the way for advanced genetic manipulation and synthetic biology applications in these industrially important bacteria.},
}
@article {pmid40854749,
year = {2025},
author = {Ju, CR and Xu, X and Xu, X and Xue, WJ},
title = {[Current status and future prospects of antibacterial treatment for Carbapenem-resistant gram-negative bacterial infections in solid organ transplant recipients].},
journal = {Zhonghua yi xue za zhi},
volume = {105},
number = {32},
pages = {2701-2708},
doi = {10.3760/cma.j.cn112137-20250218-00381},
pmid = {40854749},
issn = {0376-2491},
support = {2023C-TS10//Guangzhou Clinical Characteristic Technology Project/ ; GMUCR2024-01007//Major Clinical Research Project under the Research Capacity Enhancement Program of Guangzhou Medical University/ ; SKLRD-L-202504//Clinical Epidemiology Research Project of the State Key Laboratory of Respiratory Diseases/ ; ZJWYH-2023-YIZHI-003//China Medical Education Association/ ; 82470103//National Natural Science Foundation of China/ ; 320.6750.2025-01-1//Wu Jieping Medical Foundation Special Research Grant/ ; },
mesh = {Humans ; *Anti-Bacterial Agents/therapeutic use/pharmacology ; Carbapenems/pharmacology ; *Gram-Negative Bacterial Infections/drug therapy ; Drug Resistance, Multiple, Bacterial ; *Organ Transplantation ; Gram-Negative Bacteria/drug effects ; Transplant Recipients ; },
abstract = {In recent years, infections caused by carbapenem-resistant Gram-negative bacteria (CR-GNB) and other multidrug-resistant (MDR) pathogens have posed significant challenges in solid organ transplantation (SOT). SOT recipients who develop CR-GNB infections face risks such as graft loss, respiratory failure, leading to a mortality rate exceeding 40%. This review examines the current resistance landscape and the characteristics of MDR infections in SOT recipients, discussing the epidemiological features of CR-GNB infections in SOT patients, the resistance profiles and mechanisms of common CR-GNB (e.g., Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa), and evidence-based principles and strategies for antimicrobial therapy. The article provides an in-depth analysis of the efficacy, pharmacokinetic/pharmacodynamic (PK/PD) properties, and adverse effects of both commonly used antibiotics (such as tigecycline, polymyxins, and ceftazidime/avibactam) and the novel agents (including eravacycline and sulbactam/durlobactam), emphasizing the importance of combination therapy and personalized treatment approaches. Additionally, it explores the clinical potential of emerging rapid diagnostic technologies (e.g., CRISPR/Cas systems, mass spectrometry) and innovative treatments such as phage therapy and immunomodulation. Looking ahead, the review envisions a future where, guided by rapid and precise diagnostics, multidisciplinary management can optimize antimicrobial regimens for MDR infections in transplant recipients. The goal is to achieve individualized, effective treatment strategies, thereby improving outcomes in severe infections and advancing the management of CR-GNB infections in SOT patients in China.},
}
@article {pmid40811645,
year = {2025},
author = {Shi, JY and Li, ZW and Yao, ZL and Han, GM and Li, MY and Cai, QL and Kong, DM},
title = {5'-End Engineering of CRISPR/Cas12a Activators: A Versatile Platform for Multiple Biomarker Analysis and Clinical Cancer Tissue Identification.},
journal = {Analytical chemistry},
volume = {97},
number = {33},
pages = {18188-18198},
doi = {10.1021/acs.analchem.5c03168},
pmid = {40811645},
issn = {1520-6882},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Biosensing Techniques/methods ; *Biomarkers, Tumor/analysis/genetics ; MicroRNAs/analysis ; *Carcinoma, Renal Cell/diagnosis ; *Kidney Neoplasms/diagnosis ; *Endodeoxyribonucleases/metabolism/genetics ; Bacterial Proteins ; CRISPR-Associated Proteins ; },
abstract = {The CRISPR/Cas12a system has emerged as a powerful tool for biosensing due to its unique trans-cleavage activity. However, the fundamental mechanisms governing its activation remain inadequately understood, limiting the design flexibility and application scope of CRISPR/Cas12a-based biosensors. In this study, we investigated the activation behavior of CRISPR/Cas12a, focusing on the 5'-end engineering of the activator strand. We discovered that the activation of CRISPR/Cas12a can be significantly suppressed by incorporating a rigid intramolecular hairpin or intermolecular duplex at the 5'-end of the activator strand designed using our discovered RESET effect. Leveraging this finding, we developed a series of CRISPR/Cas12a-based biosensors capable of sensitive and selective detection, as well as live-cell imaging, for various biomarkers including microRNAs, biological small molecules, enzymes, and reactive oxygen species. Notably, the biosensor designed for miR-210, a biomarker for renal cell carcinoma (RCC), demonstrated exceptional performance in distinguishing between clinical RCC tissues and adjacent healthy tissues, highlighting its potential for cancer diagnosis, prognosis, and intraoperative decision-making. This study not only deepens the understanding of CRISPR/Cas12a activation mechanisms but also provides a versatile platform for developing advanced biosensors in molecular diagnostics and therapeutic monitoring.},
}
@article {pmid40808297,
year = {2025},
author = {Eggenschwiler, R and Hoffmann, T and Dmytrenko, O and Opitz, M and Ackel-Zakour, M and Wang, P and McCallan, SA and Fráguas-Eggenschwiler, M and Niemann, H and Patronov, A and Beisel, CL and Cantz, T},
title = {PAM-interacting domain turn-helix 51 motifs can improve Cas9-SpRY activity.},
journal = {Nucleic acids research},
volume = {53},
number = {15},
pages = {},
pmid = {40808297},
issn = {1362-4962},
support = {01GM2204C//German Federal Ministry for Education and Research/ ; 01GP2205C//German Federal Ministry for Education and Research/ ; ZN3440//Ministry of Science and Culture of Lower Saxony/ ; 865973//European Research Council Consolidator/ ; //Institute LOM/ ; },
mesh = {*CRISPR-Associated Protein 9/genetics/chemistry/metabolism ; Streptococcus pyogenes/genetics/enzymology ; *CRISPR-Cas Systems ; Gene Editing/methods ; Lysine/chemistry/genetics ; Humans ; Molecular Dynamics Simulation ; Nucleotide Motifs ; Helix-Turn-Helix Motifs ; DNA/metabolism/chemistry/genetics ; DNA Cleavage ; HEK293 Cells ; *Bacterial Proteins/genetics/chemistry/metabolism ; Protein Engineering ; },
abstract = {Cas9-SpRY is an engineered variant of the Streptococcus pyogenes Cas9 with relaxed PAM recognition, which can technically be utilized at any target in the genome but some targets are addressed with low efficiency. Here, we show that a previously unexplored motif at the turn and beginning of α-helix 51 (TH51) can be engineered to improve both nuclease and prime-editing activity of Cas9-SpRY. Interaction of the lysine-rich PID loop 2 (PL2) with the target DNA downstream of the PAM (post-PAM) mediates initiation of R-loop formation and subsequent cleavage yet it was unclear if other regions of the PID engage with post-PAM as well. To this end, the NAAN-PAM-targeting iSpyMac hybrid nuclease, which lacks all lysine residues in PL2, was compared with Cas9-SpRY at identical targets using molecular dynamics simulation and in cell culture models, uncovering four crucial post-PAM-interacting lysines in TH51 and TH53 of iSpyMac. Ectopic insertion of a lysine-rich PL2 into iSpyMac boosted its nuclease and prime-editing activities and, in turn, Cas9-SpRY benefited from certain lysine-rich TH51 motifs. Specifically, TH51 from an uncultured Abiotrophia Cas9 species boosted overall Cas9-SpRY activity. Together, this study demonstrates that engineering of post-PAM interacting motifs opens new avenues for the design of advanced CRISPR enzymes.},
}
@article {pmid40806729,
year = {2025},
author = {Katayama, S and Yamamoto, T},
title = {Efficient Genome Editing Using the T2A-Coupled Co-Expression of Two ZFN Monomers.},
journal = {International journal of molecular sciences},
volume = {26},
number = {15},
pages = {},
pmid = {40806729},
issn = {1422-0067},
support = {COI-NEXT Bio-Digital Transformation//Japan Science and Technology Agency/ ; },
mesh = {*Gene Editing/methods ; Humans ; *Zinc Finger Nucleases/genetics/metabolism ; CRISPR-Cas Systems ; HEK293 Cells ; Genetic Vectors/genetics ; Plasmids/genetics ; },
abstract = {Genome editing is commonly used in biomedical research. Among the genome editing tools, zinc finger nucleases (ZFNs) are smaller in size than transcription activator-like effector nucleases (TALENs) and CRISPR-Cas9. Therefore, ZFNs are easily packed into a viral vector with limited cargo space. However, ZFNs also consist of left and right monomers, which both need to be expressed in the target cells. When each monomer is expressed separately, two expression cassettes are required, thus increasing the size of the DNA. This is a disadvantage for a viral vector with limited cargo space. We herein showed that T2A-coupled ZF-ND1 monomers were co-expressed from a single expression cassette and that the corresponding ZF-ND1s efficiently cleaved the target DNA sequences. Furthermore, the total amount of transfected plasmid DNA was reduced by half, and genome editing efficiency was equivalent to that of two separate ZF-ND1 monomers. This study provides a promising framework for the development of ZFN applications.},
}
@article {pmid40802893,
year = {2025},
author = {Hu, M and Wang, Y and Qi, W and Zhang, Y and Sun, J and Cheng, M and Zhou, X},
title = {Photocontrolled Programmable Enzymatic Cascade for Robust CRISPR Diagnostics.},
journal = {Journal of the American Chemical Society},
volume = {147},
number = {34},
pages = {31004-31015},
doi = {10.1021/jacs.5c08704},
pmid = {40802893},
issn = {1520-5126},
mesh = {*CRISPR-Cas Systems ; Mycobacterium tuberculosis/genetics ; Humans ; Photochemical Processes ; DNA, Single-Stranded/genetics/chemistry ; *CRISPR-Associated Proteins/metabolism ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Nucleic Acid Amplification Techniques ; Bacterial Proteins ; Endodeoxyribonucleases ; },
abstract = {CRISPR-Cas12a-based diagnostic technologies have revolutionized nucleic acid detection, but their broader application remains constrained by the protospacer adjacent motif (PAM) requirement and limited multiplexing capabilities due to reliance on trans-cleavage. Here, we present a photocontrolled programmable enzymatic cascade strategy that enables temporal regulation of three sequential reactions─nucleic acid amplification, photoactivated lambda exonuclease (λ-exon)-mediated single-stranded DNA (ssDNA) generation, and PAM-independent Cas12a detection─all within a one-pot system, effectively overcoming the PAM constraint. We further exploit the orthogonal trans-cleavage activity of Cas12a and Cas13a to enable simultaneous dual-gene detection within the one-pot system, thereby circumventing multiplexing limitations. Applied to clinical Mycobacterium tuberculosis (MTB) samples, the method allows detection of both the IS6110 gene of MTB and the human ACTB (β-actin) internal control gene. This photocontrolled one-pot CRISPR diagnostic technology enhances flexibility in target site selection and overcomes the limitations of conventional CRISPR diagnostics, which cannot simultaneously detect both target genes and internal controls. This approach holds promise for advancing the clinical application of CRISPR-based diagnostics.},
}
@article {pmid40787745,
year = {2025},
author = {Wang, M and Wang, Y and Wen, J and Wang, B and Lu, G and Zhang, XB and Chen, M and Ke, G},
title = {Engineering molecular switches of CRISPR/Cas12a for biosensing applications.},
journal = {Chemical communications (Cambridge, England)},
volume = {61},
number = {70},
pages = {13052-13065},
doi = {10.1039/d5cc03652f},
pmid = {40787745},
issn = {1364-548X},
mesh = {*Biosensing Techniques/methods ; *CRISPR-Cas Systems ; *CRISPR-Associated Proteins/genetics/metabolism/chemistry ; *Bacterial Proteins/metabolism/genetics/chemistry ; *Endodeoxyribonucleases/metabolism/genetics/chemistry ; Humans ; },
abstract = {Benefiting from high specificity, excellent programmability, and efficient signal amplification capability, the CRISPR/Cas12a system has emerged as a crucial tool in the field of molecular diagnostics. To fully realize the application potential of Cas12a, many molecular switches of CRISPR/Cas12a have been recently developed to convert the activity of Cas12a from an inactive state to an active state in response to external stimuli, which significantly improves the biosensing performance in terms of the sensitivity, selectivity, and accuracy. This review summarizes recent progress in the development and application of Cas12a-based molecular switches, with a focus on their conditional activation mechanisms and biosensing applications. Additionally, challenges and future perspectives for the development of intelligent biosensors are discussed.},
}
@article {pmid40543220,
year = {2025},
author = {Samy, A and Alber, A and Fife, M and Hammond, JA},
title = {IFITM knockout DF1 cells produce higher influenza and newcastle disease viral yields: a proof of concept for avian origin cell-based vaccine production.},
journal = {Vaccine},
volume = {61},
number = {},
pages = {127360},
doi = {10.1016/j.vaccine.2025.127360},
pmid = {40543220},
issn = {1873-2518},
mesh = {Animals ; Chickens ; *Newcastle disease virus/immunology/growth &amp; development ; *Newcastle Disease/prevention &amp; control/immunology ; Cell Line ; *Influenza in Birds/prevention &amp; control/immunology ; *Influenza Vaccines/immunology ; *Viral Vaccines/immunology ; *Influenza A virus/immunology/growth &amp; development ; Chick Embryo ; Fibroblasts/virology ; *RNA-Binding Proteins/genetics ; Gene Knockout Techniques ; *Membrane Proteins/genetics ; *Antigens, Differentiation/genetics ; CRISPR-Cas Systems ; Virus Replication ; },
abstract = {Vaccines remain essential for the control of infectious diseases during poultry production, especially in high density systems. Many of poultry vaccines are currently grown in embryonated chicken eggs (ECE) or egg derived primary cells. These systems can be relatively costly and present a potential risk of supply during pandemics when demand for ECE can be high. Furthermore, the scale up of ECE vaccine production can be challenging at short notice, especially when the safe disposal of biohazardous waste is required. Avian-origin immortalised cell lines have the potential to be an ideal surrogate and remove the need to use ECE due to species match. However, the viral yield is often much lower than that of ECE which is at least partly due to the activation of interferon responses. One such response is driven by the interferon-inducible transmembrane proteins (IFITM) that are potent broad range viral restriction factors inhibiting viral cell entry. Using CRISPR/Cas9 we deleted the entire IFITM locus from the immortalised chicken fibroblast cell line DF1 and examined the impact on viral growth. Multiple DF1-IFITM-KO clones confirmed that removing IFITM restriction not only augmented infectivity and viral surface protein expression but significantly increased the viral yield up to 1.5 log10 PFU/ml and 0.8 log10 PFU/ml for influenza A virus (IAV), and Newcastle disease virus (NDV) LaSota strain, respectively. Expression of IFITM3 but not IFITM1 in DF1-IFITM-KO cells restored AIV restriction, while expression of both IFITM1 and IFITM3 restricted NDV infectivity. Together, these data confirm that IFITM proteins significantly reduce viral infectivity and growth in chicken cells and that removing this barrier has the potential to improve cell- based vaccine production.},
}
@article {pmid40854655,
year = {2025},
author = {Pearl, S and Kumar, H and Vijayakumar, S and Basu, S and Ramaiah, S and Anbarasu, A},
title = {The evolution of superbugs in space: a genomic perspective on pathogens in the International Space Station environment.},
journal = {Journal, genetic engineering &amp; biotechnology},
volume = {23},
number = {3},
pages = {100536},
doi = {10.1016/j.jgeb.2025.100536},
pmid = {40854655},
issn = {2090-5920},
abstract = {Microgravity, pressure, and temperature variations in the International Space Station (ISS) create conditions leading to the emergence of superbugs. Due to technical issues in spacecraft, astronauts are forced to stay in ISS for extended periods; prolonged stay and exposure in stressful ISS environment weakens their immune systems, increasing susceptibility to infections. The presence of hypervirulent and antibiotic-resistant pathogens in space station is a worrisome feature as these might cause serious life-threatening infections in astronauts staying in high stress environments with weakened immune systems. In the present study, we compared antimicrobial resistance genes (ARGs) and virulence factors (VFs) in bacterial genomes from ISS with Earth counterparts. ISS genomes exhibited elevated counts of defense-related genes, particularly in E. ludwigii and E. cancerogenus. Among genes uniquely found in ISS genomes, CRISPR-Cas system components were notably prevalent. Though Earth genomes harbored higher number of ARGs overall, several species from ISS possessed modestly higher ARG counts. VFs profiling showed a slightly lower count in ISS genomes, but P. conspicua, E. ludwigii, and K. pneumoniae from ISS carried exclusive VFs linked to metal ion uptake and secretion systems, suggesting environment-driven functional adaptations. The adaptation of pathogenic bacteria in ISS is alarming and therefore periodic monitoring of bacterial genomic surveillance is important. Our findings shed light on genomic profiles in bacterial strains from both ISS and Earth, enhancing our understanding of the bacterial pathogens' potential impact on drug resistance and pathogenicity in space-missions and the possible threat of spread from ISS.},
}
@article {pmid40828286,
year = {2025},
author = {Gaul, CR and Vijay, T and Johnson, R and Macias, VM},
title = {Manipulation of a New Non-model Insect Genome Using Targeted CRISPR-Era Approaches.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2935},
number = {},
pages = {335-384},
pmid = {40828286},
issn = {1940-6029},
mesh = {Animals ; *CRISPR-Cas Systems/genetics ; *Genome, Insect ; *Gene Editing/methods ; *Insecta/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {Site-specific genome editing is the most direct way to test gene function. When CRISPR-Cas9 was introduced for the editing of eukaryotic genomes, entomologists were ready with questions but had many methodologies to forge for the approach to be useful. Now, roughly 45 non-model insect genomes have been edited to study processes such as insecticide resistance, olfaction, immunity, and development. A useful first step for gene editing in an insect species of interest is identification and targeted editing of a gene with a visible phenotype. Visible markers increase the efficiency of detection of a genetic change; a wide availability of markers is one reason why model insects are so easy to manipulate and so have been key in understanding many biological processes. Here we will describe with detailed protocols how to approach a new insect species with CRISPR-era approaches by targeting a visual marker with Cas9-editing.},
}
@article {pmid40828285,
year = {2025},
author = {James, AA and Carballar-Lejarazú, R},
title = {History of Mosquito Transgenesis: A Perspective in Review.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2935},
number = {},
pages = {311-333},
pmid = {40828285},
issn = {1940-6029},
mesh = {Animals ; *Culicidae/genetics ; *Gene Transfer Techniques/history ; DNA Transposable Elements/genetics ; Gene Editing/methods ; CRISPR-Cas Systems ; Animals, Genetically Modified/genetics ; },
abstract = {The development of mosquito transgenesis technologies was driven by the need to make stable and heritable modifications to the genomes of these important insects for a variety of basic and applied objectives. While a number of transient assay systems for gene expression analyses were developed, transposable elements (TEs) were the first tools that allowed the production of genetically manipulated strains for studies that involve the complex biology of these insects and their ability to transmit pathogens. TEs have been replaced more recently for most applications by the adaptation of Cas9/guide RNA techniques, but they are still useful in randomly sampling genomes as enhancer traps and identifying neutral regions in the genome free of insertion site effects. Coupled with the Cas9/guide RNA technologies, precise editing and engineering of the genetic mechanisms responsible for much of the biology of these interesting and important insects is now possible.},
}
@article {pmid40817912,
year = {2025},
author = {Xia, Y and Liang, Z and Du, X and Cao, D and Li, J and Sun, L and Huo, YX and Guo, S},
title = {Design of function-regulating RNA via deep learning and AlphaFold 3.},
journal = {Briefings in bioinformatics},
volume = {26},
number = {4},
pages = {},
pmid = {40817912},
issn = {1477-4054},
support = {32370095//National Natural Science Foundation of China/ ; 32371489//National Natural Science Foundation of China/ ; 2024YFA0917501//National Key Research and Development Program of China/ ; },
mesh = {*Deep Learning ; *RNA/genetics/chemistry ; Molecular Dynamics Simulation ; Gene Editing ; *RNA, Guide, CRISPR-Cas Systems/genetics/chemistry ; Humans ; Computational Biology/methods ; },
abstract = {RNAs are programmable macromolecules that play diverse regulatory roles in living organisms. However, the intricate structure-function relationships underlying their regulatory activities pose significant challenges for RNA design. Here, we introduce a computational framework that integrates deep learning and energy-based methods to enhance the sequence diversity of sgRNAs designs. Our approach demonstrates high editing efficiencies of up to 75% for gene knockouts, 100% for large fragment deletions, and 62.5% for multiplex gene editing using the designed sgRNAs. Molecular dynamic simulations suggested the stability of DNA-RNA-protein complex is essential to the functionality of designed RNAs. Moreover, we reveal that the confidence metrics of AlphaFold 3 can effectively distinguish functional sequences, enabling one-shot design of crRNAs. This work presents an efficient strategy for designing regulatory RNAs with complex interactions and establishes the potential of AlphaFold 3 in advancing RNA design.},
}
@article {pmid40817853,
year = {2025},
author = {Cheng, Y and Guo, W and Wan, Y and Li, S and Zhou, J and Wang, J},
title = {Ultrasensitive Detection of Nucleic Acid and Protein at Ambient Temperature by Using an Engineered CRISPR-Cas12a-Based Digital Assay.},
journal = {Analytical chemistry},
volume = {97},
number = {33},
pages = {18355-18363},
doi = {10.1021/acs.analchem.5c03865},
pmid = {40817853},
issn = {1520-6882},
mesh = {*CRISPR-Cas Systems/genetics ; *Temperature ; Humans ; Limit of Detection ; *DNA, Viral/analysis ; Point-of-Care Systems ; Smartphone ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {The development of sensitive and convenient molecular detection technologies is crucial for early disease diagnosis and precision medicine. The CRISPR-Cas12a system has garnered significant attention due to its efficient molecular detection capabilities. However, most methods were performed at 37 °C or higher, which require a temperature control system and limit their applicability in point-of-care (POC) settings. In this study, we demonstrate that LbCpf1-Ultra, an engineered CRISPR-Cas12a, exhibits robust trans-cleavage activity at room temperature. Leveraging this finding, we developed a versatile digital CRISPR platform capable of ultrasensitive detection of viral DNA and proteins at ambient temperature, achieving a limit of detection (LOD) of 11.9 copies/μL for DNA and 5 fM for proteins. To further enhance its usability in POC environments, we integrated this platform with a smartphone-based fluorescence imaging device, enabling low-cost and on-site detection of nucleic acids and proteins without the need for external equipment. Clinical validation showcases its potential for reliable diagnostics. This platform provides a new direction for future CRISPR-based molecular diagnostics and holds promise for clinical diagnosis and precision medicine.},
}
@article {pmid40817118,
year = {2025},
author = {Teng, X and Wang, Z and Zhang, Y and Wang, B and Gong, G and Hu, J and Zhu, Y and Peng, B and Wang, J and Chen, J and Shi, S and Nielsen, J and Liu, Z},
title = {Matrix regulation: a plug-and-tune method for combinatorial regulation in Saccharomyces cerevisiae.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {7624},
pmid = {40817118},
issn = {2041-1723},
support = {22078012//National Natural Science Foundation of China (National Science Foundation of China)/ ; 22211530047//National Natural Science Foundation of China (National Science Foundation of China)/ ; NNF10CC1016517//Novo Nordisk Fonden (Novo Nordisk Foundation)/ ; },
mesh = {*Saccharomyces cerevisiae/genetics/metabolism ; *CRISPR-Cas Systems/genetics ; *Gene Expression Regulation, Fungal ; RNA, Guide, CRISPR-Cas Systems/genetics ; Heme/biosynthesis ; Mevalonic Acid/metabolism ; RNA, Transfer/genetics/metabolism ; Saccharomyces cerevisiae Proteins/genetics/metabolism ; },
abstract = {Transcriptional fine-tuning of long pathways is complex, even in the extensively applied cell factory Saccharomyces cerevisiae. Here, we present Matrix Regulation (MR), a CRISPR-mediated pathway fine-tuning method enabling the construction of 6[8] gRNA combinations and screening for the optimal expression levels across up to eight genes. We first identify multiple tRNAs with efficient gRNA processing capacities to assemble a gRNA regulatory matrix combinatorially. Then, we expand the target recognition of CRISPR regulation from NGG PAM to NG PAM by characterizing dCas9 variants. To increase the dynamic range of modulation, we test 101 candidate activation domains followed by mutagenesis and screening the best one to further enhance its activation capability in S. cerevisiae by 3-fold. The regulations generate combinatorial strain libraries for both the mevalonate pathway and the heme biosynthesis pathway and increase squalene production by 37-fold and heme by 17-fold, respectively, demonstrating the versatility of our method and its applicability in fundamental research.},
}
@article {pmid40815833,
year = {2025},
author = {Shan, J and Sheng, Y and Luo, L and Wang, W and Liu, X and Ma, Y and Wang, J},
title = {One-Pot Rlock-Mediated CRISPR/Cas12a-Driven RCA Cycle for Rapid and High-Sensitive APE1 Detection.},
journal = {Analytical chemistry},
volume = {97},
number = {33},
pages = {18208-18216},
doi = {10.1021/acs.analchem.5c03234},
pmid = {40815833},
issn = {1520-6882},
mesh = {*DNA-(Apurinic or Apyrimidinic Site) Lyase/analysis/genetics/metabolism ; *CRISPR-Cas Systems/genetics ; Humans ; *Nucleic Acid Amplification Techniques/methods ; *Endodeoxyribonucleases/genetics ; Biosensing Techniques/methods ; Limit of Detection ; Bacterial Proteins ; CRISPR-Associated Proteins ; },
abstract = {Apurinic/apyrimidinic endonuclease 1 (APE1) is a key enzyme involved in DNA repair and cellular redox regulation, and is frequently overexpressed in tumor cells. This highlights the urgent need for a rapid and high-sensitive point-of-care testing (POCT) strategy for APE1 to facilitate early cancer diagnosis. Rolling circle amplification (RCA) is a widely used isothermal DNA amplification method; however, its application in APE1 detection remains rare. Here, we introduce a versatile RCA-Lock (Rlock) conversion platform that enables the transformation of RCA-based nucleic acid detection technologies into APE1-responsive assays. Building upon this platform, we further developed a novel POCT method for APE1 detection─Rlock-mediated, CRISPR/Cas12a-driven RCA cycle (RCRE)─which, for the first time, integrates CRISPR/Cas12a with RCA into a one-pot APE1 detection system. The RCRE assay achieves a limit of detection of 8.86 × 10[-4] U/mL within 30 min, while requiring minimal equipment, low cost, and no complex handling procedures. This Rlock-based conversion strategy represents a transformative advance in the field of APE1 diagnostics and offers conceptual inspiration for the design of programmable nucleic acid-based biosensors. The resulting RCRE assay significantly broadens the technological landscape for early cancer detection and paves the way for future clinical translation.},
}
@article {pmid40813572,
year = {2025},
author = {Lo, YH and Horn, HT and Huang, MF and Yu, WC and Young, CM and Liu, Q and Tomaske, M and Towers, M and Dominguez, A and Bassik, MC and Lee, DF and Qi, LS and Weissman, JS and Chen, J and Kuo, CJ},
title = {Large-scale CRISPR screening in primary human 3D gastric organoids enables comprehensive dissection of gene-drug interactions.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {7566},
pmid = {40813572},
issn = {2041-1723},
mesh = {Humans ; *Organoids/drug effects/metabolism ; *Cisplatin/pharmacology ; *Stomach Neoplasms/genetics/drug therapy/pathology ; *CRISPR-Cas Systems ; Antineoplastic Agents/pharmacology ; Single-Cell Analysis ; *Stomach/drug effects/cytology ; },
abstract = {Understanding how genes influence drug responses is critical for advancing personalized cancer treatments. However, identifying these gene-drug interactions in a physiologically relevant human system remains a challenge, as it requires a model that reflects the complexity and heterogeneity among individuals. Here we show that large-scale CRISPR-based genetic screens, including knockout, interference (CRISPRi), activation (CRISPRa), and single-cell approaches, can be applied in primary human 3D gastric organoids to systematically identify genes that affect sensitivity to cisplatin. Our screens uncover genes that modulate cisplatin response. By combining CRISPR perturbations with single-cell transcriptomics, we resolve how genetic alterations interact with cisplatin at the level of individual cells and uncover an unexpected link between fucosylation and cisplatin sensitivity. We identify TAF6L as a regulator of cell recovery from cisplatin-induced cytotoxicity. These results highlight the utility of human organoid models for dissecting gene-drug interactions and offer insights into therapeutic vulnerabilities in gastric cancer.},
}
@article {pmid40811447,
year = {2025},
author = {Carreira de Paula, J and Solano Parada, J and Rosel Miñarro, JF and García Olmedo, P and Orantes, FJ and Osuna, A and de Pablos, LM},
title = {Erratum: On-site DNA Detection of Trypanosomatid Parasites and Nosema ceranae Through Alkaline Lysis Coupled to RPA/CRISPR/Cas12a System.},
journal = {Journal of visualized experiments : JoVE},
volume = {},
number = {222},
pages = {},
doi = {10.3791/6641},
pmid = {40811447},
issn = {1940-087X},
mesh = {*Nosema/genetics/isolation &amp; purification ; *CRISPR-Cas Systems ; *DNA, Protozoan/genetics/analysis ; *Trypanosoma/genetics/isolation &amp; purification ; Animals ; *DNA, Fungal/genetics/analysis ; },
abstract = {This corrects the article 10.3791/68874.},
}
@article {pmid40806200,
year = {2025},
author = {Li, H and Zhang, R and Wang, X and Liu, L and Yao, Z and Zhu, H},
title = {CRISPR/Cas9-Mediated Disruption of lrp6a Leads to Abnormal Median Fin Development and Somitogenesis in Goldfish (Carassius auratus).},
journal = {International journal of molecular sciences},
volume = {26},
number = {15},
pages = {},
pmid = {40806200},
issn = {1422-0067},
support = {KJCX20230216//Scientific and technological innovation capacity building project of Beijing Academy of Agricultural and Forestry Sciences/ ; QNJJ202238//Young scientists fund of the academy/ ; JBGS-2023-02//Reform and development project/ ; 32403015//National Natural Science Foundation of China (NSFC) Youth Science Foundation Project/ ; NY2401170024//Beijing Rural Revitalization Agricultural Science and Technology Project/ ; },
mesh = {Animals ; *Goldfish/genetics/embryology ; *CRISPR-Cas Systems ; *Animal Fins/abnormalities/metabolism/embryology/growth &amp; development ; *Low Density Lipoprotein Receptor-Related Protein-6/genetics/metabolism ; Gene Expression Regulation, Developmental ; *Somites/metabolism ; *Fish Proteins/genetics/metabolism ; Wnt Signaling Pathway ; },
abstract = {In this study, we demonstrated that lrp6a, a co-receptor in the Wnt signaling pathway, is essential for proper median fin formation and somitogenesis in goldfish. We analyzed the gene's sequence features and expression patterns in both wen-type and egg-type goldfish, uncovering distinct tissue-specific expression differences between the two varieties. To explore the functional role of lrp6a, we performed CRISPR/Cas9-mediated gene knockout using eight designed single-guide RNAs (sgRNAs), of which four showed effective targeting. Three high-efficiency sgRNAs were selected and co-injected into embryos to achieve complete gene disruption. Morphological assessments and X-ray microtomography (μCT) imaging of the resulting mutants revealed various abnormalities, including defects in the dorsal, caudal, and anal fins, as well as skeletal deformities near the caudal peduncle. These results confirm that lrp6a plays a key role in median fin development and axial patterning, offering new insights into the genetic regulation of fin formation in teleost fish.},
}
@article {pmid40806166,
year = {2025},
author = {Keil, S and Dittmar, T},
title = {Differential Effects of Snail-KO in Human Breast Epithelial Cells and Human Breast Epithelial × Human Breast Cancer Hybrids.},
journal = {International journal of molecular sciences},
volume = {26},
number = {15},
pages = {},
pmid = {40806166},
issn = {1422-0067},
mesh = {*Snail Family Transcription Factors/genetics/metabolism ; Humans ; Epithelial-Mesenchymal Transition/genetics ; Female ; *Breast Neoplasms/genetics/pathology/metabolism ; *Epithelial Cells/metabolism/pathology ; Cell Movement/genetics ; Cell Line, Tumor ; Gene Knockout Techniques ; Gene Expression Regulation, Neoplastic ; Neoplastic Stem Cells/metabolism/pathology ; Hybrid Cells/metabolism/pathology ; CRISPR-Cas Systems ; Zinc Finger E-box-Binding Homeobox 1/metabolism/genetics ; },
abstract = {Snail and Zeb1 have been suggested as markers for the hybrid/mixed epithelial (E)/mesenchymal (M) state of cancer cells. Such cancer cells co-express E- and M-specific transcripts and possess cancer stem cell properties. M13HS-2/-8 tumor hybrid clones derived from human M13SV1-EGFP-Neo breast epithelial cells and human HS578T-Hyg breast cancer cells exhibited co-expression of Snail and Zeb1. To explore the impact of Snail on stemness/epithelial-to-mesenchymal transition (EMT)-related properties in M13HS-2/-8 tumor hybrid clones, Snail was knocked out (KO) using CRISPR/Cas9. Mammosphere formation, colony formation, Western blot analyses, cell migration, and invasion assays were conducted for the characterization of Snail knockout cells. Interestingly, Snail-KO in M13SV1-EGFP-Neo cells resulted in the up-regulation of vimentin and N-cadherin, suggesting EMT induction, which was associated with a significantly enhanced colony formation capacity. In contrast, EMT marker pattern and colony formation capacities of M13HS-2/-8 Snail-KO tumor hybrid clones remained unchanged. Notably, the mammosphere formation capacities of M13HS-2/-8 Snail-KO tumor hybrid clones were significantly reduced. The migratory behavior of all Snail-KO cells was not altered compared with their wild-type counterparts. In contrast, M13HS-2 hybrids and their M13HS-2 Snail-KO variant exhibited a markedly enhanced invasive capacity. Therefore, Snail plays a role as a mediator of stemness properties rather than mediating EMT.},
}
@article {pmid40802750,
year = {2025},
author = {McGivney, GR and Brockman, QR and Borcherding, N and Scherer, A and Rauckhorst, AJ and Gutierrez, WR and Solst, SR and Heer, CD and Warrier, A and Floyd, W and Kirsch, DG and Knepper-Adrian, VL and Laverty, EA and Roughton, GA and Spitz, DR and Taylor, EB and Dodd, RD},
title = {Somatic CRISPR tumorigenesis and multiomic analysis reveal a pentose phosphate pathway disruption vulnerability in MPNSTs.},
journal = {Science advances},
volume = {11},
number = {33},
pages = {eadu2906},
pmid = {40802750},
issn = {2375-2548},
mesh = {*Pentose Phosphate Pathway/genetics ; Animals ; Mice ; NF-E2-Related Factor 2/genetics/metabolism ; Humans ; *CRISPR-Cas Systems ; Glucosephosphate Dehydrogenase/genetics/metabolism ; *Carcinogenesis/genetics ; Cyclin-Dependent Kinase Inhibitor p16/genetics ; Tumor Suppressor Protein p53/genetics ; Gene Expression Regulation, Neoplastic ; NADP/metabolism ; Cell Line, Tumor ; },
abstract = {Malignant peripheral nerve sheath tumors (MPNSTs) are aggressive and chemo-resistant sarcomas with poor survival rates. Loss of CDKN2A or P53 following NF1 disruption is a key event in MPNST development. Here, we used CRISPR-Cas9 somatic tumorigenesis in mice to identify transcriptomic and metabolomic features distinguishing CDKN2A- versus P53-deleted MPNSTs. Convergent, multiomic analyses revealed that CDKN2A-deleted MPNSTs are especially dependent on the pentose phosphate pathway (PPP) and NADPH metabolism for growth and viability. Disruption of glucose-6-phosphate dehydrogenase (G6PD), the PPP rate-limiting enzyme, slowed CDKN2A-deleted MPNST growth and sensitized MPNSTs to standard-of-care chemotherapy. Knockdown of the redox-regulated transcription factor NRF2 slowed MPNST growth and decreased G6PD transcription. Analysis of patient MPNSTs identified a NRF2 gene signature correlating with tumor transformation. Furthermore, G6PD and NRF2 expression in PanCancer TCGA samples correlates with patient survival. This work identifies NRF2-PPP dependency as a targetable vulnerability in these difficult-to-treat MPNSTs, particularly in the NF1/CDKN2A-deleted majority.},
}
@article {pmid40802112,
year = {2025},
author = {Minati, MA and Muneta, LL and Achouri, Y and Pirenne, S and Porada, C and Rochais, F and Jacquemin, P},
title = {KRAS4B is required for placental development.},
journal = {Cellular and molecular life sciences : CMLS},
volume = {82},
number = {1},
pages = {308},
pmid = {40802112},
issn = {1420-9071},
support = {J.0085.19//Fonds De La Recherche Scientifique - FNRS/ ; },
mesh = {Animals ; Female ; Pregnancy ; *Proto-Oncogene Proteins p21(ras)/genetics/metabolism ; Mice ; Mice, Knockout ; *Placenta/metabolism/pathology ; *Placentation/genetics ; CRISPR-Cas Systems ; Trophoblasts/metabolism ; Phenotype ; },
abstract = {Beyond its well-established role in cancer, KRAS is also crucial for embryogenesis, as its absence leads to embryonic lethality. However, the precise mechanisms underlying the developmental functions of KRAS, as well as the respective roles of its two splicing isoforms, KRAS4A and KRAS4B, remain incompletely characterized. To address these issues, we generated Kras4A knock-out (Kras4A[-/-]) and Kras4B[-/-] mouse models using CRISPR/Cas9 technology, and compared their phenotypes to those of a Kras[-/-] model, in which both isoforms are simultaneously inactivated. We observed that Kras[-/-] and Kras4B[-/-] embryos show a lethality that starts around E13.5, while Kras4A[-/-] embryos develop normally, with no detectable abnormalities. In contrast, Kras[-/-] embryos displayed a dual phenotype affecting both the heart and placenta, whereas Kras4B[-/-] embryos exhibited only the placental phenotype. The cardiac phenotype was complex, combining ventricular non-compaction, ventricular septal defects, double outlet right ventricle, and overriding aorta, likely resulting from impaired cardiac precursor proliferation. The placental phenotype was characterized by reduced placental size, and a marked decrease in glycogen trophoblast cells, correlating with hypoglycemia and hypoxia in Kras[-/-] and Kras4B[-/-] embryos. Thus, our findings confirm the predominant role of KRAS4B in KRAS-mediated developmental functions, but also suggest hidden functions of KRAS4A. Importantly, this study is the first to identify KRAS as a key regulator of a specific cell differentiation process and to characterize the biological defects caused by its loss.},
}
@article {pmid40801599,
year = {2025},
author = {Yang, J and Guo, F and Chin, HS and Chen, GB and Zhang, Z and Williams, L and Kueh, AJ and Chow, PKH and Herold, MJ and Fu, NY},
title = {Rapid and Robust Generation of Homozygous Fluorescent Reporter Knock-In Cell Pools by CRISPR-Cas9.},
journal = {Cells},
volume = {14},
number = {15},
pages = {},
pmid = {40801599},
issn = {2073-4409},
support = {2020-Gilead-001//Gilead Research Scholars Liver Disease Program (Asia)/ ; MOH&#x2011;000935//NMRC OF&#x2011;YIRG/ ; MCRF22013//Victorian Cancer Agency Mid&#x2011;Career Research Fellowship/ ; 2037907//NHMRC 2024 Ideas Grants/ ; MOH&#x2011;000546//NMRC OF&#x2011;YIRG/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; *Gene Knock-In Techniques/methods ; Humans ; *Genes, Reporter/genetics ; HEK293 Cells ; *Homozygote ; Gene Editing/methods ; Lentivirus/genetics ; Genetic Vectors ; },
abstract = {Conventional methods for generating knock-out or knock-in mammalian cell models using CRISPR-Cas9 genome editing often require tedious single-cell clone selection and expansion. In this study, we develop and optimise rapid and robust strategies to engineer homozygous fluorescent reporter knock-in cell pools with precise genome editing, circumventing clonal variability inherent to traditional approaches. To reduce false-positive cells associated with random integration, we optimise the design of donor DNA by removing the start codon of the fluorescent reporter and incorporating a self-cleaving T2A peptide system. Using fluorescence-assisted cell sorting (FACS), we efficiently identify and isolate the desired homozygous fluorescent knock-in clones, establishing stable cell pools that preserve parental cell line heterogeneity and faithfully reflect endogenous transcriptional regulation of the target gene. We evaluate the knock-in efficiency and rate of undesired random integration in the electroporation method with either a dual-plasmid system (sgRNA and donor DNA in two separate vectors) or a single-plasmid system (sgRNA and donor DNA combined in one vector). We further demonstrate that coupling our single-plasmid construct with an integrase-deficient lentivirus vector (IDLV) packaging system efficiently generates fluorescent knock-in reporter cell pools, offering flexibility between electroporation and lentivirus transduction methods. Notably, compared to the electroporation methods, the IDLV system significantly minimises random integration. Moreover, the resulting reporter cell lines are compatible with most of the available genome-wide sgRNA libraries, enabling unbiased CRISPR screens to identify key transcriptional regulators of a gene of interest. Overall, our methodologies provide a powerful genetic tool for rapid and robust generation of fluorescent reporter knock-in cell pools with precise genome editing by CRISPR-Cas9 for various research purposes.},
}
@article {pmid40801569,
year = {2025},
author = {Rauf, MA and Rao, A and Sivasoorian, SS and Iyer, AK},
title = {Nanotechnology-Based Delivery of CRISPR/Cas9 for Cancer Treatment: A Comprehensive Review.},
journal = {Cells},
volume = {14},
number = {15},
pages = {},
pmid = {40801569},
issn = {2073-4409},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Neoplasms/therapy/genetics ; Gene Editing/methods ; *Nanotechnology/methods ; Animals ; Genetic Therapy/methods ; *Gene Transfer Techniques ; },
abstract = {CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats-associated protein 9)-mediated genome editing has emerged as a transformative tool in medicine, offering significant potential for cancer therapy because of its capacity to precisely target and alter the genetic modifications associated with the disease. However, a major challenge for its clinical translation is the safe and efficient in vivo delivery of CRISPR/Cas9 components to target cells. Nanotechnology is a promising solution to this problem. Nanocarriers, owing to their tunable physicochemical properties, can encapsulate and protect CRISPR/Cas9 components, enabling targeted delivery and enhanced cellular uptake. This review provides a comprehensive examination of the synergistic potential of CRISPR/Cas9 and nanotechnology in cancer therapy and explores their integrated therapeutic applications in gene editing and immunotherapy. A critical aspect of in vivo CRISPR/Cas9 application is to achieve effective localization at the tumor site while minimizing off-target effects. Nanocarriers can be engineered to overcome biological barriers, thereby augmenting tumor-specific delivery and facilitating intracellular uptake. Furthermore, their design allows for controlled release of the therapeutic payload, ensuring sustained efficacy and reduced systemic toxicity. The optimization of nanocarrier attributes, including size, shape, surface charge, and composition, is crucial for improving the cellular internalization, endosomal escape, and nuclear localization of CRISPR/Cas9. Moreover, surface functionalization with targeting ligands can enhance the specificity of cancer cells, leading to improved gene-editing accuracy. This review thoroughly discusses the challenges associated with in vivo CRISPR/Cas9 delivery and the innovative nanotechnological strategies employed to overcome them, highlighting their combined potential for advancing cancer treatment for clinical application.},
}
@article {pmid40795957,
year = {2025},
author = {Li, Z and Zhao, W and Li, S and Luo, X and Wei, Y and Zhang, Y and Ye, Z and Li, S and Wang, M and Tan, J and Bock, R},
title = {Engineering of high-precision C-to-G base editors with expanded site selectivity and target compatibility.},
journal = {Nucleic acids research},
volume = {53},
number = {15},
pages = {},
pmid = {40795957},
issn = {1362-4962},
support = {2023ZD04074//National Science and Technology Major Project/ ; NAUSY-ZZ01//Nanjing Agricultural University/ ; BE2023369//Jiangsu Province Key Research and Development Program/ ; ZSBBL-KY2023-04//Project of Zhongshan Biological Breeding Laboratory/ ; //Innovative and Entrepreneurial Talent/ ; },
mesh = {*Gene Editing/methods ; Humans ; CRISPR-Cas Systems ; Saccharomyces cerevisiae/genetics ; Saccharomyces cerevisiae Proteins/genetics/metabolism ; Base Sequence ; },
abstract = {Base editors (BEs) are powerful tools for single nucleotide substitutions without requiring DNA double-stranded breaks or donor templates. The development of C-to-G base editors (CGBEs) represents a significant advancement by enabling base transversions, thus expanding the range of genetic modifications beyond traditional transitions and facilitating a broader spectrum of (therapeutic) applications. However, current CGBEs suffer from limitations in their editing range, mostly modifying position 6 relative to the distal end of the PAM, and their editing efficiency depends on the sequence context. In this study, by systematic exploration of deaminases to construct CGBEs, we have identified PmCDA1-based CGBEs that preferentially edit position 3. Furthermore, we report that truncations of the CDA1 C-terminus significantly enhance C-to-G editing efficiency. Our CDA1Δ-CGBEs not only exhibit high precision but also display remarkable compatibility with diverse substrate sequence contexts. We also show that they can substantially reduce, or even eliminate, genome-wide off-target editing. Importantly, we demonstrate that the strategy of using truncated CDA1 variants to improve C-to-G editing is effective not only in yeast but also in human and rice cells. These enhanced C-to-G base editing tools hold great promise for a wide range of applications in gene therapy, precision breeding, and fundamental research.},
}
@article {pmid40780174,
year = {2025},
author = {Zhu, Y and Lai, J and Yang, X and Wang, S and Gu, D and Huang, Y and Liu, Y and Liu, C},
title = {CRISPR/Cas13a-driven lateral flow assay for preamplification-free and ultrasensitive miRNA-21 detection.},
journal = {Biosensors &amp; bioelectronics},
volume = {288},
number = {},
pages = {117850},
doi = {10.1016/j.bios.2025.117850},
pmid = {40780174},
issn = {1873-4235},
mesh = {*MicroRNAs/isolation &amp; purification/genetics/blood ; *Biosensing Techniques/methods ; *CRISPR-Cas Systems/genetics ; Humans ; Limit of Detection ; Spectrum Analysis, Raman/methods ; Colorimetry/methods ; },
abstract = {Developing a preamplification-free and sensitive clustered regularly interspaced short palindromic repeats (CRISPR)-based method is significant but still extremely challenging for microRNA (miRNA) detection. Here we present a combination of a CRISPR/Cas13a-based reaction with a lateral flow biosensor, which enables the quantitative and colorimetric readout of preamplification-free miRNA detection at room temperature. In this work, the reaction principle and the structure of the lateral flow strip are well-designed to achieve surface-enhanced Raman scattering (SERS)/colorimetric dual-signal "turn-on" response of target miRNA. The CRISPR/Cas13a Reporter is engineered with a DNA-RNA splicing structure to generate DNA cleavage products and reduce nonspecific collateral cleavage. Without the need for nucleic acid preamplification strategy, the developed CRISPR/Cas13a-driven lateral flow biosensor enables the microRNA-21 (miR-21) detection at room temperature with a readout time of 10 min and a total process time of less than 45 min, achieving an impressive limit of detection of 8.96 aM by SERS and 1 fM by visualization, respectively. Moreover, the platform demonstrated excellent recovery rates in spiked human serum samples. The proposed CRISPR/Cas13a-driven, dual-signal "turn-on"-responded lateral flow platform has the potential to simultaneously meet the requirements of convenient point-of-care visualization detection and more accurate and sensitive SERS detection of miR-21, offering a cost-effective, rapid, and reliable tool for early cancer diagnosis.},
}
@article {pmid40780172,
year = {2025},
author = {Feng, J and Wu, Z and Zhu, W and Jin, F and Zhao, M and Zhong, W and Dai, C and He, Y and Yan, L and Wu, S and Wang, Y and Rui, Y and Zheng, L and Fu, Q},
title = {A novel thermo-activated one-pot RPA-CRISPR-Cas12b assay for Mycoplasma pneumoniae POCT.},
journal = {Biosensors &amp; bioelectronics},
volume = {288},
number = {},
pages = {117839},
doi = {10.1016/j.bios.2025.117839},
pmid = {40780172},
issn = {1873-4235},
mesh = {Humans ; *Mycoplasma pneumoniae/isolation &amp; purification/genetics ; *CRISPR-Cas Systems/genetics ; *Biosensing Techniques/methods ; *Nucleic Acid Amplification Techniques/methods ; Point-of-Care Testing ; *Pneumonia, Mycoplasma/diagnosis/microbiology ; Limit of Detection ; Temperature ; },
abstract = {Mycoplasma pneumoniae (M. pneumoniae), a major human respiratory pathogen, necessitates the development of rapid point-of-care testing (POCT) platforms for clinical management. However, current two-step workflows suffer from operational complexity and aerosol contamination risks. This limitation stems from CRISPR-Cas12 mediated template degradation in single-reaction systems, which compromises amplification efficiency and detection sensitivity. Here, we combined RPA and CRISPR Cas12b by leveraging the difference in their optimal temperatures to construct a novel TRACER (Thermo-activated RPA Amplification for CRISPR-Cas12b Efficient Recognition) technology. Through precise temperature modulation, TRACER sequentially executes isothermal amplification and CRISPR-mediated detection while preventing premature template cleavage, thereby maintaining optimal reaction efficiency. The platform demonstrates exceptional analytical sensitivity with a detection limit of 1 copy/μL, representing a 100-fold improvement over conventional one-pot RPA-CRISPR-Cas12a systems. Clinical validation using 195 specimens revealed diagnostic performance metrics of 99.2 % sensitivity (119/120), 100.0 % specificity (75/75), and 99.5 % accuracy (194/195). This innovative combination of single-tube reaction, field-deployable instrumentation, and cost-effectiveness establishes TRACER as an ideal POCT solution for M. pneumoniae detection in diverse clinical settings.},
}
@article {pmid40770919,
year = {2025},
author = {Vega-Hernández, G and Duque, J and Klein, BJC and Soueid, DM and Rech, JC and Wang, H and Zhou, W and Garner, AL},
title = {CRISPR RiPCA for Investigating eIF4E-m[7]GpppX Capped mRNA Interactions.},
journal = {ACS chemical biology},
volume = {20},
number = {8},
pages = {2038-2048},
doi = {10.1021/acschembio.5c00471},
pmid = {40770919},
issn = {1554-8937},
support = {R01 GM135252/GM/NIGMS NIH HHS/United States ; R35 GM153185/GM/NIGMS NIH HHS/United States ; },
mesh = {*Eukaryotic Initiation Factor-4E/metabolism/genetics ; Humans ; *RNA, Messenger/metabolism/genetics ; *CRISPR-Cas Systems ; *RNA Caps/metabolism/genetics ; RNA-Binding Proteins/metabolism ; HEK293 Cells ; Protein Binding ; },
abstract = {Post-transcriptional modifications expand the information encoded by an mRNA. These dynamic and reversible modifications are specifically recognized by reader RNA-binding proteins (RBPs), which mediate the regulation of gene expression, RNA processing, localization, stability, and translation. Given their crucial functions, any disruptions in the normal activity of these readers can have significant implications for cellular health. Consequently, the dysregulation of these RBPs has been associated with neurodegenerative disorders, cancers, and viral infections. Therefore, there has been growing interest in targeting reader RBPs as a potential therapeutic strategy since developing molecules that restore proper RNA processing and function may offer a promising avenue for treating diseases. In this work, we coupled our previously established live-cell RNA-protein interaction (RPI) assay, RNA interaction with Protein-mediated Complementation Assay (RiPCA), with CRISPR technology to build a new platform, CRISPR RiPCA. As a model for development, we utilized the interaction of eukaryotic translation initiation factor 4E (eIF4E), a reader RBP that binds to the m[7]GpppX cap present at the 5' terminus of coding mRNAs, with an m[7]G capped RNA substrate. Using eIF4E CRISPR RiPCA, we demonstrate our technology's potential for measuring on-target activity of inhibitors of the eIF4E RPI of relevance to cancer drug discovery.},
}
@article {pmid40751732,
year = {2025},
author = {Ding, S and Li, H and Li, J and Lu, D and Yang, J and Tang, Z},
title = {One-Pot Detection of Biomarker Apurinic/Apyrimidinic Endonuclease 1 Based on the Modified-crRNA Regulated Trans-Cleavage Activity of CRISPR/Cas12a.},
journal = {ACS synthetic biology},
volume = {14},
number = {8},
pages = {3186-3195},
doi = {10.1021/acssynbio.5c00335},
pmid = {40751732},
issn = {2161-5063},
mesh = {*DNA-(Apurinic or Apyrimidinic Site) Lyase/genetics/metabolism/analysis ; *CRISPR-Cas Systems/genetics ; Humans ; Biomarkers/analysis/metabolism ; *Biosensing Techniques/methods ; *Endodeoxyribonucleases/metabolism/genetics ; DNA/genetics/metabolism ; *CRISPR-Associated Proteins/metabolism/genetics ; Bacterial Proteins ; },
abstract = {Apurinic/apyrimidinic endonuclease 1 (APE1), a critical protein in DNA repair, plays indispensable roles in the maintenance of cellular homeostasis, thereby garnering significant attention as a biomarker and therapeutic target for various disorders. Current APE1 sensing methods always require multiple enzymes or complex signal amplification. The high programmability of the CRISPR/Cas12-based signal amplifier provides a new chance for developing biosensors. In this study, we introduce a novel method for the detection of APE1 by leveraging the discovery that modulating the length of modified DNA within CRISPR RNA (crRNA) enables precise control over the trans-cleavage activity of CRISPR/Cas12a. By designing a specific crRNA, the APE1-mediated activity recovery of Cas12a (ARC) was developed for rapid, specific, and one-pot detection of APE1. ARC presented a detection limit of 1.74 × 10[-6] U/μL with high specificity in detecting APE1 in biological samples. Besides, this simple method was feasible for APE1 inhibition assays, highlighting its potential for inhibitor screening and evaluation. Collectively, our findings present an innovative approach for APE1 activity analysis and expand the CRISPR-based non-nucleic acid target sensing toolbox through a novel crRNA design.},
}
@article {pmid40677232,
year = {2025},
author = {Chen, Z and Zhu, D and Lai, KS and Chen, Y and Hu, Y and Fang, Y and Yan, Z and Hu, B and Zhang, Z and Zhang, M and Li, F},
title = {VEGFA Stop-Gained Variant Deteriorates Cardiac Remodeling in Myocardial Infarction.},
journal = {Circulation. Genomic and precision medicine},
volume = {18},
number = {4},
pages = {e004879},
doi = {10.1161/CIRCGEN.124.004879},
pmid = {40677232},
issn = {2574-8300},
mesh = {Animals ; *Myocardial Infarction/genetics/pathology/metabolism/physiopathology ; *Vascular Endothelial Growth Factor A/genetics/metabolism ; Mice ; *Ventricular Remodeling/genetics ; Humans ; Disease Models, Animal ; Male ; CRISPR-Cas Systems ; },
abstract = {BACKGROUND: A sustained dosage of VEGFA (vascular endothelial growth factor A) is crucial for angiogenesis in both homeostasis and cardiovascular diseases. Start codon CUG-initiated alternative translation is a conserved mechanism for producing mature VEGFA. Genetic surveys have identified stop-gained variants predicted to prematurely terminate CUG-initiated translation without affecting start codon ATG-initiated translation. However, the impacts of these variants on the vasculature in steady-state and disease conditions remain unknown.<br><br>METHODS: Using CRISPR/Cas9 genome editing, we established the Vegfa[Q150X/Q150] allele (Q150X), a mouse genetic model that mimics the human VEGFA stop-gained variant. The effects of this variant were tested in both adult homeostatic conditions and the acute myocardial infarction (MI) model. We analyzed and quantified cardiac vasculature structure using immunofluorescence and light-sheet imaging. Furthermore, we characterized cellular heterogeneity, cell-cell interactions, and gene regulation using single-nucleus RNA sequencing, as well as cell type-specific transcriptomics and epigenomics.<br><br>RESULTS: Homozygous mice carrying the stop-gained variant were viable. VEGFA dosage was reduced to 70% in the Q150X homeostatic heart, with no significant alteration in cardiac function or vasculature. In the MI model, VEGFA dosage in Q150X was reduced to about 40% within the first week post-infarction, leading to functional deterioration in the post-MI hearts. Significant changes in cellular composition were observed 3 days post-MI. In particular, endothelial cells in Q150X diverged into a state that showed a higher level of hypoxia stress, an elevated inflammatory response, and increased extracellular matrix secretion. In addition, we observed an increase in Nppb[+] stressed cardiomyocytes in both 3 days post-MI and homeostasis. Finally, proinflammatory macrophages, neutrophils, and Cd8[+]T cells were enriched in the ischemic zone of Q150X hearts.<br><br>CONCLUSIONS: CUG-initiated translation contributes significantly to the production of mature VEGFA in ischemic hearts. VEGFA dosage is critical in determining the cellular microenvironment during ischemic injury.},
}
@article {pmid40643567,
year = {2025},
author = {Majdi, M and Wahl, NJ and Li, L and King, G and Scott, H and Beal, J and Lenaghan, SC and Occhialini, A},
title = {Development of CRISPRi Orthogonal Repression Systems in Plant Cells Using Synthetic Variants of the Figwort Mosaic Virus 34S Promoter with Two Identical sgRNA Binding Sites.},
journal = {ACS synthetic biology},
volume = {14},
number = {8},
pages = {3219-3231},
doi = {10.1021/acssynbio.5c00356},
pmid = {40643567},
issn = {2161-5063},
mesh = {*Promoter Regions, Genetic/genetics ; Nicotiana/genetics ; Binding Sites/genetics ; *RNA, Guide, CRISPR-Cas Systems/genetics/metabolism ; Synthetic Biology/methods ; *Plant Cells/metabolism ; *CRISPR-Cas Systems/genetics ; Plants, Genetically Modified ; Plant Leaves/genetics ; },
abstract = {The plant synthetic biology toolbox is rapidly expanding; however, there are still limited options for engineering logic gates for the precise modulation of gene expression. CRISPR interference (CRISPRi) represents a promising strategy for engineering logic into plant cells; however, only a limited number of promoter modules have been characterized for CRISPRi-mediated repression. In this study, the transient transgene expression in agroinfiltrated Nicotiana benthamiana leaves was used to assess the repressibility of a number of promoters with different strengths, including the Figwort Mosaic Virus (FMV) 34S promoter, which showed high repression efficiency using CRISPRi. Using dCas9 fused to the SRDX repressor domain, we employed single and double (identical or heterogeneous) sgRNA strategies for evaluating the repressibility of a library of 33 variants of the 34S promoter. This investigation supported a previous computer simulation predicting that a promoter with identical sgRNA binding sites is more efficiently repressed than a counterpart with heterogeneous sites; however, the repression efficiency varied, depending on the binding site location within the target promoter. In a second step, the top-performing 34S mutant/sgRNA/dCas9-repressor was used in combination with a Cre/loxP RNA scaffold orthogonal system to design a genetic switch, providing a versatile tool for modulation of gene expression. These results provide valuable perspectives on the utilization of 34S promoter modules in plant synthetic biology and the design of valuable CRISPRi genetic tools for precise modulation of transgene expression.},
}
@article {pmid40854654,
year = {2025},
author = {Gelaye, Y and Li, J and Luo, H},
title = {Exploring the role of Peanut (Arachis hypogaea L.) root architecture in enhancing adaptation to climate change for sustainable agriculture and resilient crop production: A review.},
journal = {Journal, genetic engineering &amp; biotechnology},
volume = {23},
number = {3},
pages = {100535},
doi = {10.1016/j.jgeb.2025.100535},
pmid = {40854654},
issn = {2090-5920},
abstract = {Peanut (Arachis hypogaea L.) cultivation is increasingly vulnerable to climate change, with drought and heat stress emerging as major constraints to productivity and food security. This review explores the critical role of root architecture in enhancing peanut adaptation to environmental stressors, and evaluates current strategies and future directions for improving root traits through genetic, physiological, and agronomic approaches. Efficient root systems, characterized by deeper rooting and optimized xylem design, significantly improve water and nutrient acquisition under drought conditions. Key regulators such as abscisic acid (ABA), strigolactones, and specific root-related genes modulate root development and stress responses. Root exudates further enhance soil root interactions, while the peanut root microbiome contributes to nutrient cycling and resilience. Biotechnological tools, including quantitative trait loci (QTL) mapping and CRISPR/Cas-based genome editing, are being harnessed to manipulate root traits at the molecular level. Agronomic practices like mulching and cover cropping synergize with genetic improvements by enhancing soil structure and moisture retention. Strengthening peanut root architecture through the integration of modern breeding, biotechnological advances, and sustainable soil management offers a promising path toward climate-resilient peanut production. Future research should prioritize the convergence of these approaches, alongside microbiome exploration, to secure yield stability and food security in a changing climate.},
}
@article {pmid40854411,
year = {2025},
author = {Storz, U},
title = {The CRISPR Cas patent files, part 4: All back to zero, think again!.},
journal = {Journal of biotechnology},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.jbiotec.2025.08.001},
pmid = {40854411},
issn = {1873-4863},
abstract = {The epic patent disputes regarding CRISPR Cas9 have been keeping the Biotech community abuzz over the last 10 years. The unclear situation has created, and keeps creating, serious uncertainties among users of this groundbreaking technology. This article gives an overview of the dispute's history, and explains the actual state of the debate.},
}
@article {pmid40853412,
year = {2025},
author = {Iqbal, K and Mishra, A and Sreedharan, SM},
title = {The quintessence of algal biomass in bioplastic production: insightful advancement and sustainable use.},
journal = {Bioresources and bioprocessing},
volume = {12},
number = {1},
pages = {91},
pmid = {40853412},
issn = {2197-4365},
abstract = {Plastics are essential components of modern life, and their global demand is increasing daily. They are gaining recognition as a sustainable source for bioplastic production due to their rapid growth, carbon fixation ability, and capacity to utilize various waste streams. It seems that landfill, incineration, chemical treatment, and plastic recycling are not the best options for minimizing plastic pollution. A novel approach A new approach is needed to reduce this pollution. Bioplastics are biodegradable and come with less toxicity, a low carbon footprint, and are a better alternative to fossil-based plastics. This review explores recent advances in algal bioplastics, focusing on key polymers like polyhydroxyalkanoates (PHAs) and polylactic acid (PLA). Special attention is given to the use of genetic tools such as CRISPR-Cas systems to improve yield and carbon flux. Challenges related to downstream processing, low biomass productivity, and environmental variability are also discussed. This review highlights the importance of standardized life cycle assessments (LCAs) to evaluate environmental impact across the entire production chain. Additionally, regulatory frameworks from different countries are compared to identify gaps and promote progressive policy development. The review aims to provide an integrated perspective on the technical innovation, economic feasibility, and policy needed to support the future of algae-based bioplastics.},
}
@article {pmid40853001,
year = {2025},
author = {Ma, R and Zhong, H and Zhang, R},
title = {Prevalence, diversity, and parasitism of tailed prophages in Vibrio harveyi.},
journal = {mSphere},
volume = {},
number = {},
pages = {e0022825},
doi = {10.1128/msphere.00228-25},
pmid = {40853001},
issn = {2379-5042},
abstract = {Vibrio harveyi, a pathogenic bacterium, contains prophages that significantly influence its pathogenesis and evolutionary traits. Investigating the prevalence, evolution, and ecological roles of these prophages is of great importance as V. harveyi is responsible for luminous bacteriosis in aquatic organisms. In this study, 13 tailed prophages were identified from 55 globally sourced V. harveyi genomes, with prophage-bacterium junctions precisely annotated. These prophages exhibited distinct parasitic mechanisms, including Mu-type transposition, site-specific recombination, and a plasmid-like non-integrated state, reflecting their adaptive plasticity. Proteome-based phylogenetic analysis classified these prophages into eight subfamilies and nine genera, with half representing novel taxonomic singletons. Network analysis of V. harveyi prophages and a large set of prophages across Vibrio species revealed distinct prophage distribution patterns, including broad cross-species dissemination and clade-specific or strain-specific colonization. Further genomic analysis identified homologs of experimentally validated virulence factors associated with motility and biofilm formation, suggesting a potential role of these prophages in enhancing bacterial pathogenicity and adaptive fitness. CRISPR spacer matching provided the intra-species lytic history for 7 out of 13 identified prophages, underscoring their involvement in horizontal transfer of virulence traits. In summary, this study established a comprehensive genomic database of V. harveyi prophages, shedding light on their diversity, prevalence, and parasitic strategies.IMPORTANCEUnderstanding how prophages parasitize Vibrio harveyi holds significant commercial implications, given the pathogen's notoriety for inducing vibriosis across diverse aquatic species and causing substantial economic losses in the global aquaculture industry. We report here 13 well-curated prophage genomes identified from 55 globally collected V. harveyi genomes. Notably, these prophages exhibited previously unrecognized genomic diversity, along with distinct parasitic strategies and hierarchical distribution patterns. In-depth analysis of their genetic profiles identified multiple homologs of experimentally validated virulence determinants involved in regulating bacterial motility and biofilm formation. Lytic history was detected for over half of these prophages, suggesting their role in driving the dissemination of virulence traits within the species.},
}
@article {pmid40852733,
year = {2025},
author = {Brock, N and Kaur, N and Halford, NG},
title = {Advances in genome editing in plants within an evolving regulatory landscape, with a focus on its application in wheat breeding.},
journal = {Journal of plant biochemistry and biotechnology},
volume = {34},
number = {3},
pages = {599-614},
pmid = {40852733},
issn = {0971-7811},
abstract = {Population growth, diminishing resources and climate change are some of the many challenges that agriculture must address to satisfy the needs of the global population whilst ensuring the safety and nutritional value of our food. Wheat (Triticum aestivum) is tremendously important for human nutrition, providing starch (and, therefore, energy), fibre, protein, vitamins, and micronutrients. It is the second most widely grown crop behind maize (Zea mays), with 808 million tonnes of grain being produced in 2021-2022. In comparison, the production figure for 1961 was 222 million tonnes, and there have been similar increases for maize and rice (Oryza sativa). World population over the same period has increased from just over 3 billion to just over 8 billion, a stark reminder of just how important increased crop production has been in maintaining food security over that period, and for these cereals it has been achieved without additional land use. Plant breeding has played an important part in enabling crop production to keep increasing to meet demand and this will have to continue through the coming decades. Innovative technologies will play a part in that, and here we review how the new technology of genome editing is being applied in crop genetic improvement, with a focus on wheat. We cover oligonucleotide-directed mutagenesis and the use of site-directed nucleases, including meganucleases (MegNs), zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and clustered regularly interspaced short palindromic repeats (CRISPR)-associated (Cas) nucleases. We describe established genome editing strategies, mainly involving gene 'knockouts', and the new applications of base and prime editing using CRISPR/Cas. We also discuss how genome editing for crop improvement is developing in the context of an evolving regulatory landscape.},
}
@article {pmid40849498,
year = {2025},
author = {Feng, Y and Shi, J and Li, Z and Li, Y and Yang, J and Huang, S and Zheng, J and Han, W and Qiao, Y and Zhang, J and Liu, Q and Yang, Y and Hu, C and Wu, L and Zhang, X and Tang, J and Huang, X and Ma, P},
title = {Discovery of CRISPR-Cas12a clades using a large language model.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {7877},
pmid = {40849498},
issn = {2041-1723},
support = {23HC1400700//Science and Technology Commission of Shanghai Municipality (Shanghai Municipal Science and Technology Commission)/ ; 32161133022//National Natural Science Foundation of China (National Science Foundation of China)/ ; 22177073//National Natural Science Foundation of China (National Science Foundation of China)/ ; 24141901302//Shanghai Science and Technology Development Foundation (Shanghai Science and Technology Development Fund)/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; *CRISPR-Associated Proteins/genetics/metabolism/chemistry/classification ; *Bacterial Proteins/genetics/metabolism/chemistry ; Machine Learning ; *Endodeoxyribonucleases/genetics/metabolism/chemistry ; Polymorphism, Single Nucleotide ; Cryoelectron Microscopy ; Large Language Models ; },
abstract = {CRISPR-Cas systems revolutionize life science. Metagenomes contain millions of unknown Cas proteins. Traditional mining relies on protein sequence alignments. In this work, we employ an evolutionary scale language model (ESM) to learn the information beyond sequences. Trained with CRISPR-Cas data, ESM accurately identifies Cas proteins without alignment. Limited experimental data restricts feature prediction, but integrating with machine learning enables trans-cleavage activity prediction of uncharacterized Cas12a. We discover 7 undocumented Cas12a subtypes with unique CRISPR loci. Structural analyses reveal 8 subtypes of Cas1, Cas2, and Cas4. Cas12a subtypes display distinct 3D-folds. CryoEM analyses unveil unique RNA interactions with the uncharacterized Cas12a. These proteins show distinct double-strand and single-strand DNA cleavage preferences and broad PAM recognition. Finally, we establish a specific detection strategy for the oncogene SNP without traditional Cas12a PAM. This study highlights the potential of language models in exploring undocumented Cas protein function via gene cluster classification.},
}
@article {pmid40849322,
year = {2025},
author = {Lampe, GD and Liang, AR and Zhang, DJ and Fernández, IS and Sternberg, SH},
title = {Structure-guided engineering of type I-F CASTs for targeted gene insertion in human cells.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {7891},
pmid = {40849322},
issn = {2041-1723},
support = {DP2HG011650//U.S. Department of Health &amp; Human Services | NIH | National Human Genome Research Institute (NHGRI)/ ; },
mesh = {Humans ; *Transposases/genetics/metabolism/chemistry ; *Gene Editing/methods ; CRISPR-Cas Systems/genetics ; DNA/metabolism/genetics/chemistry ; Cryoelectron Microscopy ; HEK293 Cells ; RNA, Guide, CRISPR-Cas Systems/genetics/metabolism ; Protein Engineering/methods ; },
abstract = {Conventional genome editing tools rely on DNA double-strand breaks (DSBs) and host recombination proteins to achieve large insertions, resulting in heterogeneous mixtures of undesirable outcomes. We recently leveraged a type I-F CRISPR-associated transposase, PseCAST, for DSB-free DNA integration in human cells, albeit at low efficiencies; multiple lines of evidence suggest DNA binding may be a bottleneck for higher efficiencies. Here we report structural determinants of DNA recognition by the PseCAST QCascade complex using single-particle cryogenic electron microscopy (cryoEM), revealing subtype-specific interactions and RNA-DNA heteroduplex features. By combining structural data, library screens, and rationally engineered mutants, we uncover variants with increased integration efficiencies and modified PAM stringencies. We further leverage transpososome structural predictions to build hybrid CASTs that combine orthogonal DNA binding and integration modules. Our work provides unique structural insights into type I-F CASTs and showcases diverse strategies to investigate and engineer RNA-guided transposase architectures for human genome editing applications.},
}
@article {pmid40828659,
year = {2025},
author = {Wang, X and Chen, Z and Liu, C and Zhang, Z and Deng, Y and Tao, L and Tiedje, JM and Deng, J},
title = {Type I-F CRISPR-associated transposons contribute to genomic plasticity in Shewanella and mediate efficient programmable DNA integration.},
journal = {Microbial genomics},
volume = {11},
number = {8},
pages = {},
doi = {10.1099/mgen.0.001476},
pmid = {40828659},
issn = {2057-5858},
mesh = {*Shewanella/genetics ; *DNA Transposable Elements/genetics ; Genome, Bacterial ; *Clustered Regularly Interspaced Short Palindromic Repeats ; CRISPR-Cas Systems ; },
abstract = {The genome plasticity of species and strains in the genus Shewanella is closely associated with the diverse mobile genetic elements embedded in its genomes. One mobile element with potential for accurate and efficient DNA insertion in Shewanella is the type I-F3 CRISPR-associated transposon (I-F3 CAST). However, relatively little is known about the distribution and ecological significance of I-F3 CASTs and whether they could be suitable as a tool for targeted genetic manipulation in situ. To better understand the distribution of I-F3 CASTs in Shewanella, we analysed 602 Shewanella genomes. We found that I-F3 CASTs were present in 12% of all genomes, although differences in both gene arrangement and integration locus were observed. These Shewanella I-F3 CASTs carried up to 89 cargo genes, which were associated with diverse functions, including defence, resistance and electron transfer, demonstrating an important role in genomic diversification and ecological adaptation. We tested whether the I-F3 CAST present in Shewanella sp. ANA-3 enhanced gene insertion, both in situ and in a heterologous host. We observed I-F3 CAST-mediated crRNA-targeted integration of the supplied genes into the pyrF locus in Shewanella sp. ANA-3. Heterologous gene insertion with high integration efficiency in Escherichia coli was also demonstrated using a simplified version of ANA-3 I-F3 CAST. Altogether, this work highlights the important role of I-F3 CASTs in promoting genomic plasticity of the Shewanella genus and demonstrates the gene-editing capability of ANA-3-CAST both endogenously and heterologously.},
}
@article {pmid40828287,
year = {2025},
author = {Dave, S and Patel, C and Heu, C and Macias, VM},
title = {Adapting and Testing ReMOT Control for Expanded CRISPR-Era Genome Functions in Non-model Insects.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2935},
number = {},
pages = {385-413},
pmid = {40828287},
issn = {1940-6029},
mesh = {Animals ; *CRISPR-Cas Systems ; Female ; Oocytes/metabolism ; RNA, Guide, CRISPR-Cas Systems/genetics ; Gene Editing/methods ; Drosophila Proteins/genetics/metabolism ; Egg Proteins/genetics/metabolism ; Gene Knockdown Techniques/methods ; *Genome, Insect ; Microinjections ; },
abstract = {The movement of the Drosophila yolk protein (DmYP) across the mosquito oocyte membrane was both fortuitous and puzzling; the cells that become future offspring--oocytes--are closed off to molecules that are not specifically recognized by a receptor, but there is no obvious ortholog of the yolk protein/receptor for DmYP in mosquitoes. Nonetheless, a small fragment of DmYP was sufficient to move the massive ribonucleoprotein complex of Cas9 and a guide RNA from the open circulatory system of a female mosquito into the mosquito oocyte for targeting of the germline DNA and heritable mutation. This procedure, known as ReMOT Control, is a robust method for CRISPR/Cas9-mediated gene knockdown that has been adapted for many orders of insects, for ticks, and even for several species of crustacean by first identifying a suitable peptide for oocyte uptake, then expressing Cas9 as a fusion protein with the peptide and finally performing adult injections with expressed, purified protein and guide RNA against a gene with a visible marker phenotype. In order to support the adaptation of this procedure widely among entomologists, herein, we provide the protocols to: (a) Identify a suitable peptide for any insect by identifying the receptor-binding region of vitellogenin. (b) Clone a nucleotide coding the peptide and a fluorescent protein into the commercially available Addgene plasmid pET28a/Cas9-cys to generate a fusion-protein encoding gene. (c) Express a fusion protein for specific delivery of ReMOT Cas9 to the ovaries of an insect of interest. (d) Adapt a generalized procedure for adult injection of insects targeting the hemolymph and detecting ovary translocation and heritable gene editing.},
}
@article {pmid40824497,
year = {2025},
author = {Ji, W and Yuan, L and Hu, T and Jiang, M and Shen, J},
title = {Establishment of a rapid Bordetella pertussis detection method based on RPA-CRISPR-Cas12a technology.},
journal = {Archives of microbiology},
volume = {207},
number = {9},
pages = {226},
pmid = {40824497},
issn = {1432-072X},
support = {2024zh-06//Anhui Institute of Translational Medicine/ ; 2021xkj061//the Youth Science Foundation of Anhui Medical University/ ; 2023AH050568//he Key Project of Natural Science in Colleges and Universities of Anhui Province/ ; },
mesh = {*Bordetella pertussis/genetics/isolation &amp; purification ; Humans ; *Whooping Cough/diagnosis/microbiology ; *CRISPR-Cas Systems ; Sensitivity and Specificity ; *Nucleic Acid Amplification Techniques/methods ; Recombinases/genetics/metabolism ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {Whooping cough (pertussis) is an acute respiratory infectious disease caused by Bordetella pertussis (BP). It poses a risk to infants and young children. This investigation aimed to construct a simple, rapid, and accurate diagnostic protocol for BP detection that does not depend on complex equipment or large-scale instruments. This study combines Recombinase Polymerase Amplification (RPA) technology with the CRISPR/Cas12a system, utilizing immunochromatographic lateral flow strips (ILFS) test and fluorescence curves to observe data. This diagnostic strategy does not require complex equipment used in traditional diagnostic approaches (such as bacterial culture, pathogen detection, and molecular biology techniques), which has increased its accessibility and ease of use. The validation data indicate that the RPA-CRISPR/Cas12a-ILFS and RPA-CRISPR/Cas12a fluorescence detection analyses had a lower detection threshold of 10[2] copies/µL and did not cross-react with other prevalent infections. Furthermore, 40 clinical samples were evaluated and compared via qPCR, which revealed that the RPA-CRISPR/Cas12a method has 100% sensitivity and specificity. In addition, the RPA-CRISPR/Cas12a diagnostic platform showed significant potential for clinical application, specifically when resources are limited, enabling point-of-care testing. This platform's simplicity, accuracy, and efficiency make it a powerful tool for pertussis diagnosis, which can improve patient care and public health outcomes.},
}
@article {pmid40814228,
year = {2025},
author = {Jiang, Y and Li, B and Xiong, J and Liu, X},
title = {Graph-CRISPR: a gene editing efficiency prediction model based on graph neural network with integrated sequence and secondary structure feature extraction.},
journal = {Briefings in bioinformatics},
volume = {26},
number = {4},
pages = {},
pmid = {40814228},
issn = {1477-4054},
support = {12472248//National Natural Science Foundation of China/ ; FRF-IDRY-24-024//The Fundamental Research Funds for the Central Universities/ ; GJJ2022-18//The Beijing Advanced Innovation Center for Materials Genome Engineering/ ; },
mesh = {*Gene Editing/methods ; *Neural Networks, Computer ; *CRISPR-Cas Systems ; Nucleic Acid Conformation ; Computational Biology/methods ; RNA, Guide, CRISPR-Cas Systems/genetics/chemistry ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Humans ; Graph Neural Networks ; },
abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR) gene-editing technology has transformed molecular biology. Predicting editing efficiency is crucial for optimization, and numerous computational models have been created. However, many current models struggle to generalize across diverse editing systems, often experiencing performance drops with varying conditions or systems. Additionally, most models focus on ribonucleic acid (RNA) sequence and thermodynamic features, overlooking the importance of secondary structure information. Here, we present the first graph-based model (Graph-CRISPR) that integrates both sequence and secondary structure features of single guide RNA enhancing editing efficiency prediction. Tests show Graph-CRISPR consistently surpasses baseline models across systems like CRISPR-Cas9, prime editing, and base editing. It also demonstrates strong resilience, maintaining robust performance under varying experimental conditions. This work highlights the potential of integrating sequence and structural information through graph-based modeling to enhance predictive accuracy and adaptability in gene editing applications. The datasets and source codes are publicly available at: https://github.com/MoonLBH/Graph-CRISPR.},
}
@article {pmid40811470,
year = {2025},
author = {Ogo, Y and Kawauchi, T and Mimura, M and Naito, K and Itoh, H and Izawa, T},
title = {A 65-kb deletion survey identifies a distal cis-regulatory region for red-light induction of Ghd7, a key rice floral repressor.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {122},
number = {33},
pages = {e2423119122},
pmid = {40811470},
issn = {1091-6490},
support = {JP17H06246//MEXT | Japan Society for the Promotion of Science (JSPS)/ ; JP20K22585//MEXT | Japan Society for the Promotion of Science (JSPS)/ ; JP22H05180//MEXT | Japan Society for the Promotion of Science (JSPS)/ ; JP22H05172//MEXT | Japan Society for the Promotion of Science (JSPS)/ ; JP22H00367//MEXT | Japan Society for the Promotion of Science (JSPS)/ ; RGP0011/2019//Human Frontier Science Program (HFSP)/ ; },
mesh = {*Oryza/genetics/metabolism/radiation effects/growth &amp; development ; *Gene Expression Regulation, Plant/radiation effects ; *Flowers/genetics/metabolism/radiation effects/growth &amp; development ; Light ; *Plant Proteins/genetics/metabolism ; Circadian Rhythm/genetics ; Sequence Deletion ; CRISPR-Cas Systems ; Regulatory Sequences, Nucleic Acid ; },
abstract = {The Ghd7 (Grain number, plant height, and heading date 7) gene integrates red light signals and circadian rhythms to control floral repression under long-day conditions in rice. CRISPR/Cas9 systems were employed to create a series of deletion mutant lines in the upstream regions of Ghd7, covering a 65-kb genomic region from its transcription start site (TSS). These deletions ranged from 2 to 25 kb in size. Three deletion lines, those from 0 to -3 kb (0/-3 K), -20 to -40 kb (-20/-40 K), and -26 to -30 kb (-26/-30 K) from the TSS, resulted in early flowering, similar to Ghd7 knockout lines. The -20/-40 and -26/-30 K lines exhibited a loss of acute Ghd7 morning induction. Night-break experiments consistently supported these findings, suggesting that the key cis-regulatory region for red light responses was located within the 3.7-kb region in -26/-30 K. In seedlings of the 0/-3 K deletion line, which retains the -29 to -86 bp region, Ghd7 showed a diurnal pattern similar to wild type. This suggests that the deleted region in 0/-3 K is dispensable for both circadian rhythms and red-light responses. Further analyses of two deletion lines within the -26/-30 K region allowed us to narrow down the core cis-regulatory elements, responsive to morning-light signals, within a 228-bp segment located at 28-kb upstream of the TSS in Ghd7.},
}
@article {pmid40809520,
year = {2025},
author = {Ji, T and Fang, X and Gao, Y and Yu, K and Gao, X},
title = {Research progress on the application of RPA-CRISPR/Cas12a in the rapid visual detection of pathogenic microorganisms.},
journal = {Frontiers in cellular and infection microbiology},
volume = {15},
number = {},
pages = {1640938},
pmid = {40809520},
issn = {2235-2988},
mesh = {*CRISPR-Cas Systems ; Humans ; *Nucleic Acid Amplification Techniques/methods ; *Molecular Diagnostic Techniques/methods ; *Recombinases/metabolism ; *Bacteria/genetics/isolation &amp; purification ; Point-of-Care Testing ; Sensitivity and Specificity ; Point-of-Care Systems ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {In an increasingly complex global public health landscape, the continuous emergence of novel pathogens and the growing problem of antibiotic resistance highlight the urgent need for rapid, efficient, and precise detection technologies for pathogenic microorganisms. The innovative combination of Recombinase Polymerase Amplification (RPA) and CRISPR/Cas12a enables the rapid amplification of target gene fragments under isothermal conditions and the precise recognition and cleavage of specific nucleic acid sequences. The integration of RPA and CRISPR/Cas12a significantly enhances the sensitivity and accuracy of detection simplifies operational procedures, and reduces the dependence on specialized equipment for testing personnel. This combination demonstrates great potential for application in clinical diagnostics and point-of-care testing. This article provides a detailed overview of the principles of RPA-CRISPR/Cas12a and its latest research progress in the field of pathogen detection, aiming to promote the widespread application of RPA-CRISPR/Cas12a technology in clinical medicine and public health and to offer theoretical support for its further optimization.},
}
@article {pmid40806519,
year = {2025},
author = {Gao, G and Zhang, L and Tong, P and Yan, G and Wu, X},
title = {Enhancing Oil Content in Oilseed Crops: Genetic Insights, Molecular Mechanisms, and Breeding Approaches.},
journal = {International journal of molecular sciences},
volume = {26},
number = {15},
pages = {},
pmid = {40806519},
issn = {1422-0067},
mesh = {*Plant Breeding/methods ; Quantitative Trait Loci ; *Crops, Agricultural/genetics/metabolism ; *Plant Oils/metabolism ; Gene Editing ; *Seeds/genetics/metabolism ; Genome-Wide Association Study ; CRISPR-Cas Systems ; },
abstract = {Vegetable oils are essential for human nutrition and industrial applications. With growing global demand, increasing oil content in oilseed crops has become a top priority. This review synthesizes recent progress in understanding the genetic, environmental, and molecular mechanisms regulating oil content, and presents biotechnological strategies to enhance oil accumulation in major oilseed crops. Oil biosynthesis is governed by intricate genetic-environmental interactions. Environmental factors and agronomic practices significantly impact oil accumulation dynamics. Quantitative trait loci (QTL) mapping and genome-wide association studies (GWAS) have identified key loci and candidate genes involved in lipid biosynthesis pathways. Transcription factors and epigenetic regulators further fine-tune oil accumulation. Biotechnological approaches, including marker-assisted selection (MAS) and CRISPR/Cas9-mediated genome editing, have successfully generated high-oil-content variants. Future research should integrate multi-omics data, leverage AI-based predictive breeding, and apply precision genome editing to optimize oil yield while maintaining seed quality. This review provides critical references for the genetic improvement and breeding of high- and ultra-high-oil-content varieties in oilseed crops.},
}
@article {pmid40806492,
year = {2025},
author = {Șerban, M and Toader, C and Covache-Busuioc, RA},
title = {Precision Neuro-Oncology in Glioblastoma: AI-Guided CRISPR Editing and Real-Time Multi-Omics for Genomic Brain Surgery.},
journal = {International journal of molecular sciences},
volume = {26},
number = {15},
pages = {},
pmid = {40806492},
issn = {1422-0067},
mesh = {Humans ; *Glioblastoma/genetics/surgery ; *Brain Neoplasms/genetics/surgery ; *Precision Medicine/methods ; *Genomics/methods ; *CRISPR-Cas Systems ; *Artificial Intelligence ; *Gene Editing/methods ; Proteomics/methods ; Metabolomics/methods ; Multiomics ; },
abstract = {Precision neurosurgery is rapidly evolving as a medical specialty by merging genomic medicine, multi-omics technologies, and artificial intelligence (AI) technology, while at the same time, society is shifting away from the traditional, anatomic model of care to consider a more precise, molecular model of care. The general purpose of this review is to contemporaneously reflect on how these advances will impact neurosurgical care by providing us with more precise diagnostic and treatment pathways. We hope to provide a relevant review of the recent advances in genomics and multi-omics in the context of clinical practice and highlight their transformational opportunities in the existing models of care, where improved molecular insights can support improvements in clinical care. More specifically, we will highlight how genomic profiling, CRISPR-Cas9, and multi-omics platforms (genomics, transcriptomics, proteomics, and metabolomics) are increasing our understanding of central nervous system (CNS) disorders. Achievements obtained with transformational technologies such as single-cell RNA sequencing and intraoperative mass spectrometry are exemplary of the molecular diagnostic possibilities in real-time molecular diagnostics to enable a more directed approach in surgical options. We will also explore how identifying specific biomarkers (e.g., IDH mutations and MGMT promoter methylation) became a tipping point in the care of glioblastoma and allowed for the establishment of a new taxonomy of tumors that became applicable for surgeons, where a change in practice enjoined a different surgical resection approach and subsequently stratified the adjuvant therapies undertaken after surgery. Furthermore, we reflect on how the novel genomic characterization of mutations like DEPDC5 and SCN1A transformed the pre-surgery selection of surgical candidates for refractory epilepsy when conventional imaging did not define an epileptogenic zone, thus reducing resective surgery occurring in clinical practice. While we are atop the crest of an exciting wave of advances, we recognize that we also must be diligent about the challenges we must navigate to implement genomic medicine in neurosurgery-including ethical and technical challenges that could arise when genomic mutation-based therapies require the concurrent application of multi-omics data collection to be realized in practice for the benefit of patients, as well as the constraints from the blood-brain barrier. The primary challenges also relate to the possible gene privacy implications around genomic medicine and equitable access to technology-based alternative practice disrupting interventions. We hope the contribution from this review will not just be situational consolidation and integration of knowledge but also a stimulus for new lines of research and clinical practice. We also hope to stimulate mindful discussions about future possibilities for conscientious and sustainable progress in our evolution toward a genomic model of precision neurosurgery. In the spirit of providing a critical perspective, we hope that we are also adding to the larger opportunity to embed molecular precision into neuroscience care, striving to promote better practice and better outcomes for patients in a global sense.},
}
@article {pmid40806337,
year = {2025},
author = {Carullo, N and Haellman, V and Gutbier, S and Schlicht, S and Nguyen, TT and Blum Marti, R and Hartz, P and Lindemann, L and Schukur, L},
title = {Development and Characterization of a Novel α-Synuclein-PEST H4 Cell Line for Enhanced Drug Screening in α-Synucleinopathies.},
journal = {International journal of molecular sciences},
volume = {26},
number = {15},
pages = {},
pmid = {40806337},
issn = {1422-0067},
support = {71095//Roche Pharma AG (Germany)/ ; },
mesh = {*alpha-Synuclein/metabolism/genetics ; Humans ; Drug Evaluation, Preclinical/methods ; *Synucleinopathies/metabolism/drug therapy/genetics ; Cell Line ; Proteolysis ; CRISPR-Cas Systems ; Parkinson Disease/metabolism/drug therapy ; Half-Life ; },
abstract = {Alpha-Synuclein (α-Syn) is a presynaptic neuronal protein implicated in the pathogenesis of Parkinson's disease (PD) and other synucleinopathies, primarily through its aggregation into insoluble fibrils. The extended α-Syn half-life necessitates treatment durations that are incompatible with efficient high-throughput drug screening, can risk compound stability or cause cellular toxicity. To address this, we inserted a PEST sequence, a motif known to promote rapid protein degradation, at the C-terminus of the SNCA gene using CRISPR/Cas9 to create a novel cell line with reduced α-Syn half-life. This modification accelerates α-Syn turnover, providing a robust model for studying α-Syn dynamics and offering a platform that is applicable to other long-lived proteins. Our results demonstrate a six-fold reduction in α-Syn half-life, enabling the rapid detection of changes in protein levels and facilitating the identification of molecules that modulate α-Syn production and degradation pathways. Using inhibitors of the proteasome, transcription, and translation further validated the model's utility in examining various mechanisms that impact protein levels. This novel cell line represents a significant advancement for studying α-Syn dynamics and offers promising avenues to develop therapeutics for α-synucleinopathies. Future research should focus on validating this model in diverse experimental settings and exploring its potential in high-throughput screening applications.},
}
@article {pmid40806299,
year = {2025},
author = {Citterio, CE and Morales-Rodriguez, B and Liao, XH and Vu, C and Nguyen, R and Tsai, J and Le, J and Metawea, I and Liu, M and Olson, DP and Refetoff, S and Arvan, P},
title = {A Genetically-Engineered Thyroid Gland Built for Selective Triiodothyronine Secretion.},
journal = {International journal of molecular sciences},
volume = {26},
number = {15},
pages = {},
pmid = {40806299},
issn = {1422-0067},
support = {K01 DK125448/DK/NIDDK NIH HHS/United States ; R01 DK132017/DK/NIDDK NIH HHS/United States ; DK125448 (to C.E.C.)/NH/NIH HHS/United States ; R01-DK132017 (to P. A.)/NH/NIH HHS/United States ; },
mesh = {Animals ; *Triiodothyronine/metabolism ; Mice ; *Thyroid Gland/metabolism ; CRISPR-Cas Systems ; Gene Knock-In Techniques ; Thyroglobulin/genetics/metabolism ; Thyroxine/metabolism ; Hypothyroidism/genetics/metabolism ; Thyrotropin/metabolism ; *Genetic Engineering/methods ; Humans ; },
abstract = {Thyroid hormones (thyroxine, T4, and triiodothyronine, T3) are indispensable for sustaining vertebrate life, and their deficiency gives rise to a wide range of symptoms characteristic of hypothyroidism, affecting 5-10% of the world's population. The precursor for thyroid hormone synthesis is thyroglobulin (Tg), a large iodoglycoprotein consisting of upstream regions I-II-III (responsible for synthesis of most T4) and the C-terminal CholinEsterase-Like (ChEL) domain (responsible for synthesis of most T3, which can also be generated extrathyroidally by T4 deiodination). Using CRISPR/Cas9-mediated mutagenesis, we engineered a knock-in of secretory ChEL into the endogenous TG locus. Secretory ChEL acquires Golgi-type glycans and is properly delivered to the thyroid follicle lumen, where T3 is first formed. Homozygous knock-in mice are capable of thyroidal T3 synthesis but largely incompetent for T4 synthesis such that T4-to-T3 conversion contributes little. Instead, T3 production is regulated thyroidally by thyrotropin (TSH). Compared to cog/cog mice with conventional hypothyroidism (low serum T4 and T3), the body size of ChEL-knock-in mice is larger; although, these animals with profound T4 deficiency did exhibit a marked elevation of serum TSH and a large goiter, despite normal circulating T3 levels. ChEL knock-in mice exhibited a normal expression of hepatic markers of thyroid hormone action but impaired locomotor activities and increased anxiety-like behavior, highlighting tissue-specific differences in T3 versus T4 action, reflecting key considerations in patients receiving thyroid hormone replacement therapy.},
}
@article {pmid40804581,
year = {2025},
author = {Li, H and Wang, G and Wu, X and Ying, X and Luo, Z and Lu, C and Zhang, P},
title = {Rapid and Specific Visual Detection of Amanita fuliginea by Combining Recombinase Polymerase Amplification with CRISPR/Cas12b and Lateral Flow Strip.},
journal = {Current microbiology},
volume = {82},
number = {10},
pages = {450},
pmid = {40804581},
issn = {1432-0991},
support = {No. 31670015//National Natural Science Foundation of China/ ; No. 31750001//National Natural Science Foundation of China/ ; },
mesh = {*CRISPR-Cas Systems ; *Nucleic Acid Amplification Techniques/methods ; Recombinases/metabolism/genetics ; *Amanita/genetics/isolation &amp; purification/classification ; Sensitivity and Specificity ; },
abstract = {Amanita fuliginea is a common lethal mushroom with high mortality rates due to its ease of ingestion and concealed early symptoms. Rapid and specific identification of this species is crucial for clinical diagnosis and treatment. However, the existing rapid detection methods for nucleic acids of A. fuliginea were still plagued by specificity issues. Herein, we developed a recombinase polymerase amplification (RPA) coupled with CRISPR/Cas12b and lateral flow strip (LFS) assay for the rapid and specific detection of A. fuliginea. The RPA-CRISPR/Cas12b-LFS assay provides visual results within 35 min and does not rely on expensive equipment. High specificity was demonstrated against other Amanita species and non-Amanita species, with a sensitivity of 31 pg of genomic DNA per reaction. In conclusion, the RPA-CRISPR/Cas12b-LFS assay presents a rapid, specific, sensitive, and convenient tool for identifying A. fuliginea, particularly suitable for primary healthcare institutions or remote areas.},
}
@article {pmid40796566,
year = {2025},
author = {Saha, S and Haynes, WJ and Seo, J and Del Rio, NM and Young, EE and Zhang, J and Holm, AM and Pimentel, M and Flannagan, L and Huang, L and Blashka, W and Murphy, L and Scholz, MJ and Henrichs, A and Suresh Babu, J and Steill, J and Kratz, J and Stewart, R and Kamp, TJ and Brown, ME},
title = {Diminished immune cell adhesion in hypoimmune ICAM-1 knockout human pluripotent stem cells.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {7415},
pmid = {40796566},
issn = {2041-1723},
support = {T32 GM081061/GM/NIGMS NIH HHS/United States ; T32 DC009401/DC/NIDCD NIH HHS/United States ; U01 HL134764/HL/NHLBI NIH HHS/United States ; P30 CA014520/CA/NCI NIH HHS/United States ; U01HL134764//U.S. Department of Health &amp; Human Services | NIH | National Heart, Lung, and Blood Institute (NHLBI)/ ; R56HL165189//U.S. Department of Health &amp; Human Services | NIH | National Heart, Lung, and Blood Institute (NHLBI)/ ; 75N93021C00004/AI/NIAID NIH HHS/United States ; P50 HD105353/HD/NICHD NIH HHS/United States ; R56 HL165189/HL/NHLBI NIH HHS/United States ; },
mesh = {*Intercellular Adhesion Molecule-1/genetics/immunology/metabolism ; Humans ; Animals ; Mice ; Cell Adhesion/immunology/genetics ; *Pluripotent Stem Cells/immunology/metabolism ; CRISPR-Cas Systems ; Gene Editing ; Gene Knockout Techniques ; T-Lymphocytes/immunology ; Immunity, Innate ; Graft Rejection/immunology ; Cell Proliferation ; },
abstract = {Gene edited human pluripotent stem cells are a promising platform for developing reparative cellular therapies that evade immune rejection. Existing first-generation hypoimmune strategies have used CRISPR/Cas9 editing to modulate genes associated with adaptive immune responses, but have largely not addressed the innate immune cells, such as neutrophils, that mediate inflammation and rejection processes occurring early after graft transplantation. We identify the adhesion molecule ICAM-1 as a hypoimmune target that plays multiple critical roles in both adaptive and innate immune responses post-transplantation. In our experiments, we find that ICAM-1 blocking or knockout in human pluripotent stem cell-derived cardiovascular therapies imparts significantly diminished binding of multiple immune cell types. ICAM-1 knockout results in diminished T cell proliferation and activation responses in vitro and in longer in vivo retention/protection of knockout grafts following immune cell encounter in NeoThy humanized mice. We also introduce the ICAM-1 knockout edit into existing first-generation hypoimmune human pluripotent stem cells and prevent immune cell binding. This promising hypoimmune editing strategy has the potential to improve transplantation outcomes for regenerative therapies in the setting of cardiovascular pathologies and several other diseases.},
}
@article {pmid40778406,
year = {2025},
author = {Yuan, X and Xu, Y and Zhang, G and Wang, Y and Jin, X},
title = {CRISPR/Cas12a-Mediated Electrochemical Aptasensor for Simultaneous Determination of Alzheimer's Disease Biomarkers in Human Blood.},
journal = {Analytical chemistry},
volume = {97},
number = {32},
pages = {17715-17724},
doi = {10.1021/acs.analchem.5c03015},
pmid = {40778406},
issn = {1520-6882},
mesh = {Humans ; *Alzheimer Disease/blood/diagnosis ; *Amyloid beta-Peptides/blood ; *Electrochemical Techniques/methods ; *Aptamers, Nucleotide/chemistry/genetics ; Biomarkers/blood ; *Biosensing Techniques/methods ; *CRISPR-Cas Systems ; *Peptide Fragments/blood ; Limit of Detection ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {Alzheimer's disease (AD) is an irreversible and progressive neurodegenerative disease, and the ratio of 40 and 42 residue amyloid beta peptides (Aβ40 and Aβ42) plays a crucial role as a biomarker for the early diagnosis of AD. Conventional laboratory-based assays for Aβ proteins analysis typically relies on the collection of cerebrospinal fluid (CSF). In contrast, blood samples offer a less invasive alternative but present challenges due to lower concentrations of Aβ. This study presents a novel strategy that combines aptamer-specific recognition with CRISPR/Cas12a-mediated signal amplification in an electrochemical sensing platform. The proposed electrochemical sensing platform achieved reliable and accurate results when applied to clinical serum samples, validating its effectiveness in practical scenarios. This approach enables highly specific detection of Aβ40 and Aβ42 with detection limits as low as 1.1 and 0.8 pg/mL, respectively. Moreover, the platform demonstrated outstanding repeatability and operational stability, underscoring its potential as a robust and highly sensitive diagnostic tool for the early detection of AD.},
}
@article {pmid40774925,
year = {2025},
author = {Zhu, L and Hu, Q and Wang, Z and Lin, JM and Zhao, RS},
title = {CRISPR/Cas12a-Mediated Rolling Circle Amplification for the Development of Liquid Crystal-Based Sensors.},
journal = {Analytical chemistry},
volume = {97},
number = {32},
pages = {17825-17832},
doi = {10.1021/acs.analchem.5c03500},
pmid = {40774925},
issn = {1520-6882},
mesh = {*CRISPR-Cas Systems ; *Liquid Crystals/chemistry ; *Nucleic Acid Amplification Techniques/methods ; *Aflatoxin B1/analysis ; *Biosensing Techniques/methods ; Aptamers, Nucleotide/chemistry ; Limit of Detection ; Food Contamination/analysis ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {The development of high-performance liquid crystal (LC)-based sensors with remarkable sensitivity and excellent selectivity is of great importance. Herein, a CRISPR/Cas12a-based LC sensor for detecting mycotoxins in food is first reported, and the detection of aflatoxin B1 (AFB1) is chosen as a model. AFB1 is added to magnetic beads (MBs) functionalized with double-stranded DNA (dsDNA) consisting of AFB1 aptamer and cDNA. As the AFB1 aptamer specifically recognizes AFB1, cDNA is released and activates the CRISPR/Cas12a system to cut ligation DNA, thereby preventing the initiation of the rolling circle amplification (RCA) reaction. As the long-chain single-stranded DNA (ssDNA) cannot be produced to capture myristoylcholine (Myr) in the aqueous solution, a dark LC image is obtained because a Myr monolayer forms at the aqueous/LC interface. In contrast, when the RCA reaction is initiated in the absence of AFB1, Myr in the aqueous solution is captured by long-chain ssDNA generated from the RCA reaction, causing a bright LC image. Notably, the RCA reaction on MBs exponentially amplifies the CRISPR/Cas12a-generated signals, resulting in enhanced sensitivity. The limit of detection (LOD) is about 0.312 ng/mL. Furthermore, the selectivity is greatly enhanced due to the introduction of the AFB1 aptamer and MBs. Furthermore, a portable device is developed for rapid onsite detection. Therefore, the study provides a sensitive, selective, convenient, and promising assay for detecting mycotoxins in food.},
}
@article {pmid40749399,
year = {2025},
author = {Yan, B and Wei, C and Lei, X and Ding, L and Yu, S},
title = {cliCRISPR: crRNA-limited CRISPR/Cas12a system for multiplexed detection.},
journal = {Biosensors &amp; bioelectronics},
volume = {288},
number = {},
pages = {117834},
doi = {10.1016/j.bios.2025.117834},
pmid = {40749399},
issn = {1873-4235},
mesh = {*CRISPR-Cas Systems/genetics ; *Biosensing Techniques/methods ; Humans ; *RNA/genetics/analysis ; Genotype ; Genotyping Techniques ; Spectrometry, Fluorescence ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {The CRISPR/Cas12a system is extensively employed for nucleic acid detection, where crRNA is typically administered in excess to quantify target levels. However, its non-specific trans-cleavage activity poses challenges for achieving multiplexed detection within a single-pot CRISPR/Cas12a assay. In this study, we introduce a crRNA-limited strategy for multiplexed detection based on the CRISPR/Cas12a system, termed cliCRISPR. This approach correlates fluorescence intensity with crRNA concentration rather than target concentration. By precisely controlling crRNA concentrations, distinct low, medium, and high fluorescence intensities can be observed for multiple targets. As proof of concept, distinguishable fluorescence intensities for wild-type, mutant, and heterozygous genotypes of rs4646536 which is associated with vitamin D deficiency could be obtained with 10 nM crRNA1 and 3 nM crRNA2. Subsequently, a logic-gate strategy is utilized for rs4646536 genotyping by comparing fluorescence intensity slope with the cut-off values (V0 = 25.96, V1 = 40.16 and V2 = 1815.56). To validate the practicability, the proposed cliCRISPR method was applied to genotype rs4646536 for 10 samples from 2 families. The results were consistent with those obtained using TaqMan qPCR and adhered to Mendel's laws of inheritance. Therefore, cliCRISPR demonstrates potential for developing multiplexed biosensors based on the CRISPR/Cas12a system.},
}
@article {pmid40749158,
year = {2025},
author = {Baek, M and Kweon, S and Kim, Y and Lewis, NE and Lee, JS and Lee, GM},
title = {Recombinase-Mediated Cassette Exchange-Based CRISPR Activation Screening Identifies Hyperosmotic Stress-Resistant Genes in Chinese Hamster Ovary Cells.},
journal = {ACS synthetic biology},
volume = {14},
number = {8},
pages = {3116-3126},
doi = {10.1021/acssynbio.5c00268},
pmid = {40749158},
issn = {2161-5063},
mesh = {Animals ; CHO Cells ; Cricetulus ; *CRISPR-Cas Systems/genetics ; *Osmotic Pressure ; *Recombinases/metabolism/genetics ; Cell Proliferation/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Cricetinae ; },
abstract = {Chinese hamster ovary (CHO) cells are ubiquitously used for therapeutic protein production. However, fed-batch culture, typically used for large-scale production, often induces hyperosmotic stress, negatively impacting cell growth and productivity. To identify genes conferring resistance to hyperosmotic stress, we performed genome-wide CRISPRa screening in bispecific antibody (bsAb)-producing CHO (CHO-bsAb) cells. Using a virus-free recombinase-mediated cassette exchange (RMCE) system, we established a CRISPRa library and cultured cells in standard and hyperosmolar media. Next-generation sequencing identified 122 significantly enriched and 171 significantly depleted genes under hyperosmolar conditions, with functional enrichment analysis highlighting pathways related to cell proliferation and transcriptional regulation. Among the enriched genes, CRISPRa-based activation of 24 candidates demonstrated that 23 improved cell growth under hyperosmolar conditions. Notably, stable expression of Siah2 or C2cd4a significantly enhanced cell growth, and optimizing their expression levels increased bsAb production by up to 1.3-fold. Additional knockout of Zfr, previously identified in CRISPR knockout screening, further improved cell growth and bsAb production, demonstrating the synergistic benefits of integrating CRISPR knockout and CRISPRa approaches. Thus, CRISPRa screening is a powerful tool for identifying novel engineering targets, facilitating the development of stress-resistant CHO cell lines, and enhancing therapeutic protein production.},
}
@article {pmid40743400,
year = {2025},
author = {Lu, B and Lin, S and Lang, Z and Yin, Q and Cao, H},
title = {Aptamer Probe-Assisted Strand Displacement-CRISPR/Cas12a Biosensor for Ultrasensitive Detection of AFB1.},
journal = {Journal of agricultural and food chemistry},
volume = {73},
number = {32},
pages = {20500-20507},
doi = {10.1021/acs.jafc.5c05775},
pmid = {40743400},
issn = {1520-5118},
mesh = {*Biosensing Techniques/methods/instrumentation ; *Aflatoxin B1/analysis ; *Aptamers, Nucleotide/chemistry/genetics ; Food Contamination/analysis ; Limit of Detection ; CRISPR-Cas Systems ; *CRISPR-Associated Proteins/genetics ; Bacterial Proteins/genetics ; Endodeoxyribonucleases ; },
abstract = {The sensitive and accurate detection of aflatoxin B1 (AFB1) is crucial for public health. Herein, the aptamer (Apt)-lock-key-structure (A-LKS), composed of Apt capable of spontaneous amplification and its complementary ssDNA (cDNA), was designed. Based on the identification of AFB1 in A-LKS, an A-LKS-mediated-SDA-Cas12a signal cascade (ASCC) biosensor was developed for ultrasensitive AFB1 detection. In the absence of AFB1, the Apt initiates amplification using DNA hanging from the 5' end of cDNA as a template, thereby enhancing the stability of A-LKS and reducing nonspecific amplification. When AFB1 is present, Apt binds to it, initiating the SDA reaction and activating Cas12a to generate strong fluorescence signals. The proposed biosensor demonstrates excellent selectivity and high sensitivity, with a low LOD of 3.6 pg/mL and a linear range of 0.01-100 ng/mL. This biosensor was successfully applied in real samples with satisfactory recoveries (88.69-105.48%), indicating its potential application in real samples.},
}
@article {pmid40736916,
year = {2025},
author = {Huang, Y and Yang, X and Liu, H and Xie, S and Chen, W and Dai, C and Zhao, C},
title = {Enhanced Paramylon Production in Euglena gracilis through CRISPR/Cas9 and Its Antiaging Effects.},
journal = {Journal of agricultural and food chemistry},
volume = {73},
number = {32},
pages = {20163-20171},
doi = {10.1021/acs.jafc.5c03014},
pmid = {40736916},
issn = {1520-5118},
mesh = {*Euglena gracilis/metabolism/genetics ; Animals ; Caenorhabditis elegans/drug effects/metabolism/genetics ; *CRISPR-Cas Systems ; *Glucans/pharmacology/metabolism/biosynthesis ; Reactive Oxygen Species/metabolism ; Oxidative Stress/drug effects ; Mitochondria/metabolism ; Longevity/drug effects ; },
abstract = {Euglena gracilis is a photosynthetic microalga that can synthesize paramylon. This study utilized CRISPR/Cas9 to knock out β-1,3-glucan phosphorylase, resulting in engineered strains to increase paramylon production. The ldp1 mutant strain produced paramylon constituting 68.20% of the cellular dry weight and a yield of 1.49 g/L. Replacing the carbon source in AF-6 medium with glucose resulted in an increase in the paramylon content to 72.92% of the dry cell weight and a yield of 1.51 g/L. The observation of a single distinct peak in the light scattering spectrum suggests a high degree of purity (approaching 100%). Paramylon significantly reduces reactive oxygen species in Caenorhabditis elegans, enhances antioxidant enzyme activity, and improves resistance to oxidative stress and high temperatures. It also reduces DNA damage and extends the lifespan. The antiaging effects of paramylon may be mediated through the regulation of the insulin/insulin-like growth factor signaling pathway. In C. elegans (myo-3:GFP(mit)), paramylon treatment increased mitochondrial signaling (p < 0.01), ATP production, membrane potential, and the expression of longevity-related genes, suggesting enhanced mitochondrial function. This research not only optimizes paramylon production but also highlights its potential as an antioxidative and antiaging agent.},
}
@article {pmid40735939,
year = {2025},
author = {Hulleman, JD and Jeon, S and Bali, S and DiCesare, SM and Abbas, A and Daniel, S and Ortega, AJ and Collier, GE and Yang, J and Bhattacharyaa, A and McCoy, MK and Joachimiak, LA and Posner, BA},
title = {Select Azo Compounds Post-translationally Modulate HTRA1 Abundance and Activity Potentially through Interactions at the Trimer Interface.},
journal = {ACS chemical biology},
volume = {20},
number = {8},
pages = {1849-1862},
doi = {10.1021/acschembio.4c00818},
pmid = {40735939},
issn = {1554-8937},
mesh = {*High-Temperature Requirement A Serine Peptidase 1/metabolism/genetics/chemistry ; Humans ; *Azo Compounds/pharmacology/chemistry ; *Protein Processing, Post-Translational/drug effects ; Macular Degeneration/genetics ; Retinal Pigment Epithelium/metabolism ; CRISPR-Cas Systems ; },
abstract = {High-temperature requirement protein A1 (HTRA1) is a secreted serine protease with diverse substrates, including extracellular matrix proteins, proteins involved in amyloid deposition, and growth factors. Accordingly, HTRA1 has been implicated in a variety of neurodegenerative diseases including a leading cause of blindness in the elderly, age-related macular degeneration (AMD). In fact, genomewide association studies have identified that the 10q26 locus that contains HTRA1 confers the strongest genetic risk factor for AMD. A recent study has suggested that AMD-associated risk alleles located in the HTRA1 gene correlate with a significant age-related defect in HTRA1 synthesis in the retinal pigmented epithelium (RPE) within the eye, possibly accounting for AMD susceptibility. Thus, we sought to identify small molecule enhancers of HTRA1 transcription and/or protein abundance using an unbiased high-throughput screening approach. To accomplish this goal, we used CRISPR/Sp.Cas9 engineering to introduce an 11-amino-acid luminescent peptide tag (HiBiT) onto the C-terminus of HTRA1 in immortalized ARPE-19 cells. Editing was very efficient (∼88%), verified by genomic DNA analysis, short interfering RNA (siRNA), and HiBiT blotting. A total of 1920 compounds from two libraries were screened. An azo compound with reported antiamyloidogenic and cardioprotective activity, Chicago Sky Blue 6B (CSB), was identified as an enhancer of endogenous HTRA1 secretion (2.0 ± 0.3 fold) and intracellular levels (1.7 ± 0.2 fold). These results were counter-screened using HiBiT complement factor H (CFH) edited ARPE-19 cells, verified using HiBiT blotting, and were not due to HTRA1 transcriptional changes. Importantly, serine hydrolase activity-based protein profiling (SH-ABPP) demonstrated that CSB does not affect HTRA1's specific activity. However, interestingly, in follow-up studies, Congo Red, another azo compound structurally similar to CSB, also substantially increased intracellular HTRA1 levels (up to 3.6 ± 0.3 fold) but was found to significantly impair HTRA1 enzymatic reactivity (0.45 ± 0.07 fold). Computational modeling of potential azo dye interaction with HTRA1 suggests that CSB and Congo Red can bind to the noncatalytic face of the trimer interface but with different orientation tolerances and interaction energies. These studies identify select azo dyes as HTRA1 chemical probes that may serve as starting points for future HTRA1-centered small molecule therapeutics.},
}
@article {pmid40729556,
year = {2025},
author = {Laforge, N and Calabre, M and Jules, M and Planson, AG},
title = {Multiplex Genome Editing and Regulation in Bacillus subtilis with CRISPR-MAD7.},
journal = {ACS synthetic biology},
volume = {14},
number = {8},
pages = {3142-3153},
doi = {10.1021/acssynbio.5c00274},
pmid = {40729556},
issn = {2161-5063},
mesh = {*Bacillus subtilis/genetics/metabolism ; *Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics/metabolism ; Bacterial Proteins/genetics/metabolism ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Genome, Bacterial/genetics ; },
abstract = {With the advent of MAD7, a Cpf1-like nuclease, there has been a renewed focus on the development of CRISPR-based genome engineering tools in recent years. To improve genome engineering methodologies in B. subtilis, we revisited the potential of MAD7 for gene modification and expression interference. A key challenge in these endeavors is the limited transformation efficiency. To overcome this, we developed an efficient transformation protocol using strains overexpressing competence genes. Our results showed that although MAD7 together with a B. subtilis chromosome-targeting gRNA is lethal, enabling robust counterselection, we successfully engineered a strain carrying the MAD7-gRNA machinery in a reversibly inactivated state, marking a significant advance in the field. We demonstrated that both MAD7 and its catalytically inactive variant (dMAD7) can be conditionally regulated by inactivation at elevated temperatures. In addition, the MAD7-gRNA complex is effective for multiplex genome editing, allowing for the simultaneous deletion, mutation, or insertion of up to four loci, and enabling the combination of gene deletion, gene insertion, and point mutations. Furthermore, we established a strategy that achieves the simultaneous removal of MAD7 and the gRNA along with the desired genome edits. Altogether, this comprehensive study underscores the versatility of MAD7 for complex, scarless genome engineering and lays a strong foundation for further advancing genetic manipulation in B. subtilis.},
}
@article {pmid40721107,
year = {2025},
author = {Oh, SJ and Lim, G and Han, Y and Kim, H and Kim, YG and Bhatia, SK and Yang, YH},
title = {Development of a DNA endonuclease I-SceI-based scarless genome editing system for Cupriavidus necator.},
journal = {Journal of biotechnology},
volume = {406},
number = {},
pages = {285-295},
doi = {10.1016/j.jbiotec.2025.07.020},
pmid = {40721107},
issn = {1873-4863},
mesh = {*Cupriavidus necator/genetics/metabolism ; *Gene Editing/methods ; Plasmids/genetics ; *Deoxyribonuclease I/genetics/metabolism ; CRISPR-Cas Systems ; Homologous Recombination ; Genome, Bacterial ; *Deoxyribonucleases, Type II Site-Specific/genetics/metabolism ; },
abstract = {Cupriavidus necator is a promising microbial chassis capable of fixing CO2 and producing high polyhydroxyalkanoate yields. Consequently, various genetic engineering methods have been explored. While sacB-based homologous recombination (HR) and CRISPR-Cas9 have shown both advantages and disadvantages in C. necator, alternative tools, including the DNA endonuclease I-SceI-mediated HR system could enable precise, scarless genome editing without requiring a large database. We developed a two-plasmid-based I-SceI HR system for efficient gene deletion and insertion in C. necator by altering origin replication and induction systems. The pOUO-1 plasmid was designed for conjugation-based genome integration via first HR, whereas the pOH-4 plasmid was constructed to express I-SceI, inducing second HR. Unlike conventional I-SceI expression strategies, which fail to trigger second HR in C. necator, transformation with pOH-4 alone was sufficient for recombination. A plasmid-curing strategy was optimized to eliminate the highly stable pOH-4 by increasing the incubation temperature to 37°C. Using this optimized system, the phaC1 gene was successfully knocked out; the phaCBP-M-CPF4 was inserted at the same site, resulting in a novel poly(3-hydroxybutyrate-co-5-hydroxyvalerate)-producing strain. This newly established I-SceI HR technique significantly simplifies genome engineering in C. necator, reducing the timeframe to a few weeks and facilitating its further applications in synthetic biology.},
}
@article {pmid40711424,
year = {2025},
author = {Zou, X and Gu, T and Li, X and Deng, L and Zhu, S and Dong, J and Deng, F and Hou, C and Huo, D},
title = {PAM-free activation of CRISPR/Cas12a via semi-nested asymmetric RPA: highly specific detection of HPV16 dsDNA.},
journal = {The Analyst},
volume = {150},
number = {17},
pages = {3891-3898},
doi = {10.1039/d5an00491h},
pmid = {40711424},
issn = {1364-5528},
mesh = {*Human papillomavirus 16/genetics/isolation &amp; purification ; *CRISPR-Cas Systems/genetics ; Humans ; *DNA, Viral/analysis/genetics ; *Nucleic Acid Amplification Techniques/methods ; Limit of Detection ; DNA, Single-Stranded/genetics ; *Bacterial Proteins/metabolism/genetics ; *Endodeoxyribonucleases/metabolism/genetics ; Uterine Cervical Neoplasms/virology/diagnosis ; CRISPR-Associated Proteins ; },
abstract = {Early and accurate detection of HPV16 nucleic acids is therefore critical for the effective screening, diagnosis, and prevention of cervical cancer. Although CRISPR/Cas12a-based molecular diagnostics offer a rapid and sensitive approach for HPV16 detection, their application to double-stranded DNA (dsDNA) targets remains constrained by two major limitations: the strict requirement for a protospacer adjacent motif (PAM) site and the insufficient specificity of current amplification strategies, which can lead to off-target amplification and false-positive results. To address these challenges, we developed a semi-nested asymmetric recombinase polymerase amplification (SNA-RPA) method combined with CRISPR/Cas12a for the detection of HPV16 dsDNA. This strategy employs a semi-nested primer design to significantly enhance target sequence specificity during amplification, while asymmetric primer ratios promote the efficient generation of single-stranded DNA (ssDNA) that directly activates Cas12a without the need for a PAM site. Using this approach, we achieved rapid and highly specific detection of HPV16 dsDNA, with a limit of detection as low as 18 aM. Beyond achieving PAM-free detection, our method also substantially improves amplification fidelity, offering a promising solution for precise and reliable HPV diagnostics and cervical cancer screening.},
}
@article {pmid40704803,
year = {2025},
author = {Mao, Y and Lv, R and Shao, H and Zhao, Y and Wang, J and Chen, Q and Yi, H and Ge, Y and Wang, H and Li, Y and Qi, Y},
title = {An ELISA-like sensitive and visual detection system targeting Yersinia pestis based on CRISPR/Cas12a and DNAzyme.},
journal = {Journal of clinical microbiology},
volume = {63},
number = {8},
pages = {e0027425},
pmid = {40704803},
issn = {1098-660X},
support = {JK2023gk002//Medical Science and Technology Project/ ; },
mesh = {*Yersinia pestis/genetics/isolation &amp; purification ; *DNA, Catalytic/genetics ; Sensitivity and Specificity ; Humans ; Enzyme-Linked Immunosorbent Assay/methods ; *CRISPR-Cas Systems ; Bacterial Proteins/genetics ; *Plague/diagnosis/microbiology ; Clustered Regularly Interspaced Short Palindromic Repeats ; *CRISPR-Associated Proteins/genetics ; *Endodeoxyribonucleases/genetics ; G-Quadruplexes ; Nucleic Acid Amplification Techniques/methods ; },
abstract = {Yersinia pestis is the causative agent of plague, a human disease with potentially devastating consequences. Here, we developed an enzyme-linked immunosorbent assay-like visual detection method based on clustered regularly interspaced short palindromic repeats (CRISPR) detection and DNAzyme for the cost-effective and highly sensitive detection of Y. pestis. A novel specific gene sequence (CH57_3927) was screened for the detection target of Y. pestis. The recombinase-aided amplification (RAA) assay, CRISPR/Cas12a detection assay, and G-quadruplex (G4) DNAzyme-based color development assay were separately established and optimized. These three optimized assays were integrated into an advanced ELISA-like visual detection method-RAA-CRISPR/Cas12a-DNAzyme (RCCD)-by further optimization of their components to improve the compatibility between them. The amplified target sequence binds to crRNA and activates the Cas12a nucleases for trans-cleave G4. As a result, the cleaved G4 is unable to bind with hemin to exert peroxidase activity, thus impeding the catalysis of the 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS[2-]) colorimetric reaction. Consequently, negative samples exhibit a dark green coloration, while the positive products appear nearly colorless, facilitating visual differentiation with the naked eye. In addition, the RCCD detection platform effectively distinguished Y. pestis from all other closely related species, with a detection limit of 1 copy/reaction. Evaluated using Y. pestis DNA-spiked blood samples and uninfected samples, both sensitivity and specificity were 100%. The method shows significant potential for detecting targets in clinical samples and is well-suited for use in resource-limited environments. It offers advantages such as visual detection, batch detection, and low cost.IMPORTANCEWe utilized Mauve software to screen Yersinia pestis specific genes and integrated CRISPR-Cas12a, RAA amplification, and G-quadruplex DNAzyme technology to establish an advanced ELISA-like visual detection method. The visual detection method offers a more cost-effective alternative compared to the conventional CRISPR detection method that relies on fluorescence-labeled ssDNA reporter or lateral flow (LF) test strips. With only one thermostatic device required, it enhances the convenience of rapid on-site screening of Y. pestis outbreaks, providing effective support for plague detection, prevention, and control within primary medical and health institutions.},
}
@article {pmid40701479,
year = {2025},
author = {Duan, X and Wu, S and Song, J and Xiong, G and Luo, J and Lu, Y and Tan, D and Lou, J and Hu, H and Tong, X and Ding, X and Dai, F},
title = {Bicaudal-C plays a critical role in regulating oogenesis in the lepidopteran insect Bombyx mori.},
journal = {International journal of biological macromolecules},
volume = {321},
number = {Pt 2},
pages = {145939},
doi = {10.1016/j.ijbiomac.2025.145939},
pmid = {40701479},
issn = {1879-0003},
mesh = {Animals ; *Bombyx/genetics/metabolism/physiology ; *Oogenesis/genetics ; Female ; *Insect Proteins/genetics/metabolism ; Oocytes/metabolism ; CRISPR-Cas Systems ; },
abstract = {Oogenesis is an intricate cellular specialization process that requires the precise expression of multiple genes. However, the functional genes and mechanisms contributing to lepidopteran oogenesis remain incompletely understood. In silkworm, the eggless mutant (sm[n]) has a typical oogenesis disorder phenotype. In sm[n] females, oocyte development is delayed, causing follicle degeneration and the absence of mature eggs in virgin ovarioles. Here, we revealed that BmBic-C, an ortholog of Drosophila melanogaster Bicaudal-C (Bic-C), is responsible for sm[n]. BmBic-C expression was significantly downregulated in sm[n], likely due to an Organdy transposon insertion upstream of BmBic-C. CRISPR/Cas9-mediated knockout of BmBic-C results in a no-egg phenotype consistent with sm[n]. BmBic-C deficiency reduces the absorption efficiency of vitellogenin, a key factor in egg development. A comparative proteomic analysis between the ΔBmBic-C and control strains revealed 633 differentially expressed proteins (DEPs). Gene Ontology (GO) enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses of the DEPs suggested that BmBic-C might contribute to ovarian cell development and survival through signal transduction, autophagy, and other pathways. Moreover, BmBic-C dysfunction triggers the apoptosis cascade, highlighting the essential role of BmBic-C in regulating ovarian homeostasis and cell fate. Phylogenetic and selection pressure analyses revealed that Bic-C is highly conserved across lepidopteran species and has undergone purifying selection throughout evolution, suggesting that Bic-C is a key component in organism survival and reproductive regulation. This study provides novel insights into oogenesis and lays a foundation for precision lepidopteran pest control against species-specific genomic/coding variable regions.},
}
@article {pmid40693287,
year = {2025},
author = {Kiattisewee, C and Karanjia, AV and Cardiff, RAL and Olander, KE and Leejareon, P and Alvi, SS and Carothers, JM and Zalatan, JG},
title = {Systematic Mapping of Bacterial CRISPRa Systems for Synergistic Gene Activation Reveals Antagonistic Effects.},
journal = {ACS synthetic biology},
volume = {14},
number = {8},
pages = {3232-3244},
doi = {10.1021/acssynbio.5c00358},
pmid = {40693287},
issn = {2161-5063},
mesh = {Promoter Regions, Genetic/genetics ; *Transcriptional Activation/genetics ; *CRISPR-Cas Systems/genetics ; Escherichia coli/genetics ; Bacterial Proteins/genetics/metabolism ; Gene Expression Regulation, Bacterial ; Transcription Initiation Site ; },
abstract = {CRISPR gene activation (CRISPRa) tools have shown great promise for bacterial strain engineering but often require customization for each intended application. Our goal is to create generalizable CRISPRa tools that can overcome previous limitations of gene activation in bacteria. In eukaryotic cells, multiple activators can be combined for synergistic gene activation. To identify potential effectors for synergistic activation in bacteria, we systematically characterized bacterial activator proteins with a set of engineered synthetic promoters. We found that optimal target sites for different activators could vary by up to 200 bases in the region upstream of the transcription start site (TSS). These optimal target sites qualitatively matched previous reports for each activator, but the precise targeting rules varied between different promoters. By characterizing targeting rules in the same promoter context, we were able to test activator combinations with each effector positioned at its optimal target site. We did not find any activator combinations that produced synergistic activation, and we found that many combinations were antagonistic. This systematic investigation highlights fundamental mechanistic differences between bacterial and eukaryotic transcriptional activation systems and suggests that alternative strategies will be necessary for strong bacterial gene activation at arbitrary endogenous targets.},
}
@article {pmid40684992,
year = {2025},
author = {Tafech, B and Carlaw, T and Sadhnani, G and Schmidt, K and Morin, T and Leung, J and Weiner, J and An, K and Balázs, A and Ross, C and Beule, D and Mall, MA and Fuchs, H and Kulkarni, J and Cullis, PR and Hedtrich, S},
title = {Lung tissue-optimized gene editing in human cystic fibrosis models following topical application of lipid nanoparticles.},
journal = {Journal of controlled release : official journal of the Controlled Release Society},
volume = {385},
number = {},
pages = {114053},
doi = {10.1016/j.jconrel.2025.114053},
pmid = {40684992},
issn = {1873-4995},
mesh = {Humans ; *Cystic Fibrosis/genetics/therapy ; *Gene Editing/methods ; *Nanoparticles/administration &amp; dosage/chemistry ; Cystic Fibrosis Transmembrane Conductance Regulator/genetics ; *Lung/metabolism ; *Lipids/administration &amp; dosage/chemistry ; Administration, Topical ; Deoxyribonuclease I/administration &amp; dosage ; CRISPR-Cas Systems ; Liposomes ; Recombinant Proteins ; },
abstract = {Cystic fibrosis (CF) is a severe monogenic disease characterized by debilitating lung dysfunction caused by loss-of-function mutations in the CFTR gene. While CRISPR-based gene editing holds promise for correcting these mutations and potentially curing CF, efficient delivery of gene editors to the lung epithelium through the mucosal barrier remains a major challenge. In this study, we developed a lung-optimized gene editing strategy using lipid nanoparticles (LNPs) and evaluated it in increasingly complex, biomimetic human-based and patient-derived models. Systematic optimization of helper lipids, genetic cargo, guide RNA modifications, and gene editor ratios, alongside analysis of innate immune responses, achieved ∼50 % editing efficiency in the model gene HPRT in two-dimensional models. Editing efficiency significantly dropped to ∼5 % in biomimetic three-dimensional CF bronchial epithelial tissue models following topical LNP application. Pretreatment with the approved mucolytic agent dornase alpha increased editing efficiency to ∼12.7 %. Finally, in CF patient-derived cells harboring the CFTR[R1162X] mutation, our optimized LNP formulation achieved ∼12 % correction on gene level, offering a potential treatment avenue for this yet untreatable mutation. Taken together, this study demonstrates that optimizing the genetic cargo as well as the delivery vehicle is key when striving for clinically applicable treatment approaches. It further provides insights into gene editing rates in human-based normal and CF patient-derived bronchial tissue models which express all relevant biological barriers and, thus, can pave the way for topically applicable treatment options for patients with CF and other genetic lung diseases.},
}
@article {pmid40680952,
year = {2025},
author = {Gao, X and Dong, X and Song, H and Fu, Y and Li, J and Fan, G and Wang, T and Sun, Y and Wang, Y and Qiu, HJ and Luo, Y},
title = {A one-pot CRISPR-Cas12a-based assay for rapid, on-site detection of African swine fever virus.},
journal = {International journal of biological macromolecules},
volume = {321},
number = {Pt 2},
pages = {146109},
doi = {10.1016/j.ijbiomac.2025.146109},
pmid = {40680952},
issn = {1879-0003},
mesh = {*African Swine Fever Virus/genetics/isolation &amp; purification ; *CRISPR-Cas Systems/genetics ; Animals ; Swine ; *African Swine Fever/diagnosis/virology ; DNA, Viral/genetics ; Nucleic Acid Amplification Techniques/methods ; Sensitivity and Specificity ; Recombinases ; },
abstract = {African swine fever (ASF), caused by African swine fever virus (ASFV), is a highly contagious and devastating disease threatening global swine production. The disease has caused substantial economic losses, driving the need for efficient diagnostic tools to enhance surveillance and control. Despite various available assays for ASF, field-deployable tools enabling rapid, accurate, and user-friendly detection remain urgently needed. Here, we developed and validated a novel one-pot recombinase polymerase amplification (RPA)-CRISPR-Cas12a assay for rapid and sensitive detection of ASFV by integrating all components into a single sealed tube, which requires only isothermal heating and ultraviolet visualization. The assay demonstrated a detection limit of 56 TCID50/mL and could be completed within 35 min, and without cross-reactivity with non-ASFV porcine viruses. In comparative testing of 150 clinical samples, the one-pot RPA-CRISPR-Cas12a assay exhibited 100 % agreement with gold standard quantitative PCR (qPCR). Notably, the assay identified ASFV genomic DNA in whole blood as early as 3 days post-infection with sensitivity comparable to the qPCR. This early detection capability, combined with a field-deployable format, provides a robust tool for implementing timely containment measures against ASF, especially in resource-limited setting.},
}
@article {pmid40674851,
year = {2025},
author = {Li, D and Hu, J and Hu, J and Zhang, CY},
title = {Integration of AND logic circuit with CRISPR/Cas12a system for sensitive detection of biomarkers and accurate discrimination of breast cancer cells.},
journal = {Biosensors &amp; bioelectronics},
volume = {288},
number = {},
pages = {117790},
doi = {10.1016/j.bios.2025.117790},
pmid = {40674851},
issn = {1873-4235},
mesh = {Humans ; *Breast Neoplasms/diagnosis/genetics/pathology ; *CRISPR-Cas Systems/genetics ; *Biosensing Techniques/methods ; *Biomarkers, Tumor/genetics/analysis/isolation &amp; purification ; Female ; *Flap Endonucleases/genetics/isolation &amp; purification/analysis ; Cell Line, Tumor ; Limit of Detection ; },
abstract = {Flap endonuclease 1 (FEN1) is a structure-selective nuclease that is of great significance in maintaining genomic stability. FEN1 is up-regulation in various cancers and is regarded as a new biomarker for cancer diagnosis. Herein, we integrate DNA logic circuit with clustered regularly interspaced short palindromic repeats (CRISPR)/Cas12a system for sensitive detection of FEN1 at the cellular level and accurate discrimination of different cell types. In this design, FEN1 cleaves the flap-containing synthetic substrates, and subsequently magnetic separation separates the primers from the cleaved substrate fragments, inducing the release of the primers. The released primers can initiate strand displacement amplification (SDA) reaction to generate abundant activators that can activate the trans-cleavage activity of the CRISPR/Cas12a system to cleave the signal probes for the recovery of Cy5 fluorescence signal. This circuit displays superior sensitivity with a detection limit of 5.19 × 10[-5] U/μL, and it is capable of screening the FEN1 inhibitors, quantifying the activity of FEN1 with sensitivity at the single-cell level, and discriminating diverse FEN1 levels in clinical breast cancer tissues. We further construct an AND logic circuit based on miR-31 and FEN1 to simultaneously monitor the biomarkers at the cellular level and achieves accurate discrimination of different breast cancer cell types with a P-value of less than 0.05. The integration of DNA logic circuit with CRISPR/Cas12a system provides a new approach for biomarker-related biomedical research and clinical diagnostics.},
}
@article {pmid40619069,
year = {2025},
author = {Gao, S and Wang, X and Yang, R and Bian, Z and Yong, D and Xu, S and Huo, L and Tu, Q and Zhang, Y and Fu, J and Li, R},
title = {Efficient genome engineering in Agrobacterium tumefaciens C58 using recombineering assisted by CRISPR/Cas9.},
journal = {Journal of biotechnology},
volume = {406},
number = {},
pages = {99-104},
doi = {10.1016/j.jbiotec.2025.07.005},
pmid = {40619069},
issn = {1873-4863},
mesh = {*Agrobacterium tumefaciens/genetics ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; *Genome, Bacterial/genetics ; *Genetic Engineering/methods ; Homologous Recombination ; },
abstract = {Recombineering, a technique derived from phage-encoded homologous recombination, has emerged as a vital approach for bacterial genome engineering. Agrobacterium tumefaciens is extensively utilized to transfer DNA into the host plant genomes. To facilitate the transformation of various plant species, particularly those of considerable economic value, genetic modifications of Agrobacterium strains are essential. Our previous studies established an Agrobacterium-specific phage-encoded homologous recombination system for Agrobacterium species. Yet, recent investigations have indicated that there is a substantial variability in the recombination efficiency of these recombineering systems for gene editing across different genome loci in A. tumefaciens. In this work, we present the development of an efficient genome engineering tool for A. tumefaciens by integrating recombineering with CRISPR/Cas9 technology. Initially, we found that lengthening the homology arms significantly enhanced genome editing efficiency. Nevertheless, at certain genomic sites, even when the length of the homology arms was increased, the editing efficiency remained suboptimal. Subsequently, combination of the Agrobacterium-specific recombineering system with the CRISPR/Cas9 system markedly enhanced the genome engineering efficiency. This study offers an enhanced and efficient genome engineering tool for A. tumefaciens, which could potentially be applied to other species within the Agrobacterium genus.},
}
@article {pmid40607797,
year = {2025},
author = {Kuzmina, A and Wattad, S and Murugavelu, P and Amir, N and Tickotsky, N and Levin, L and Taube, R},
title = {Genome-wide CRISPR knockout screen identifies activating transcription factor (ATF1) as an activator of HIV gene expression.},
journal = {mBio},
volume = {16},
number = {8},
pages = {e0055725},
pmid = {40607797},
issn = {2150-7511},
support = {2021273//United States-Israel Binational Science Foundation/ ; 1884/24//Israel Science Foundation/ ; 508136175//Deutsche Forschungsgemeinschaft/ ; 5R21AI270197//All of Us Research Program (All of Us NIH)/ ; },
mesh = {Humans ; CD4-Positive T-Lymphocytes/virology ; *Gene Expression Regulation, Viral ; Virus Latency/genetics ; *HIV-1/genetics/physiology ; Promoter Regions, Genetic ; *Activating Transcription Factor 1/genetics/metabolism ; CRISPR-Cas Systems ; HIV Infections/virology/genetics ; Gene Knockout Techniques ; Receptors, CCR5/genetics/metabolism ; Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {UNLABELLED: Antiretroviral therapy against the human immunodeficiency virus (HIV) has significantly prolonged the life span of people living with HIV, transforming viral infection into a latent condition that is characterized with undetectable viral loads. Yet, a complete cure of infection is out of reach, as transcriptionally silent but replication-competent proviruses persist in a long-lived reservoir that is resistant to therapy. The current work follows a genome-wide CRISPR knockout screen in human CD4[+] T cells and defines the activating transcription factor 1 (ATF1) as an activator of HIV gene transcription with elevated expression levels in cells that carry transcriptionally active provirus. Additional gain and loss-of-function experiments show that depletion of ATF1 promotes latency. ATF1 directly occupies the HIV promoter, where it regulates the recruitment of RNA Polymerase II and the levels of H3K9me3 histone repression mark. Genome-wide, ATF1 binds cellular gene promoters. Among its targets, ATF1 modulates the levels of CCR5 antisense lncRNA, thereby regulating the protein expression of the CCR5 HIV co-receptor. We conclude that ATF1 is an activator of gene transcription that dictates HIV gene expression via both direct and indirect mechanisms.<br><br>IMPORTANCE: HIV persists in resting CD4[+] primary infected cells, forming a reservoir that is resistant to therapy, and thus a main barrier toward elimination of viral infection. An understanding of the mechanisms that control HIV gene expression and drive viral latency is therefore of high clinical importance. This study identifies activating transcription factor 1 (ATF1) as an activator of HIV gene expression. ATF1 binds the HIV promoter, where it modulates the occupancy of RNA Polymerase II and the levels of H3K9me3 histone repression mark. Genome-wide, ATF1 also occupies cellular promoters. One target of ATF1 is the antisense (AS) lncRNA. Through binding to CCR5-AS lncRNA, ATF1 induces CCR5 mRNA stability, thereby indirectly controlling HIV infection. Overall, we provide an additional understanding of the host transcription pathways that regulate HIV gene expression and potentially open new ways to manipulate its reservoir size.},
}
@article {pmid40592435,
year = {2025},
author = {Liu, Y and Li, Z and Zhou, L and Jia, W and Zhou, Z and Yang, Y and Sun, Y and Lin, P and Shen, G and Liu, Q and Hua, X and Zhao, P},
title = {BmPriS promotes silk gland growth by regulating endoreplication in silkworm.},
journal = {International journal of biological macromolecules},
volume = {320},
number = {Pt 1},
pages = {145640},
doi = {10.1016/j.ijbiomac.2025.145640},
pmid = {40592435},
issn = {1879-0003},
mesh = {Animals ; *Bombyx/genetics/growth &amp; development/metabolism ; *Silk/metabolism/biosynthesis/genetics ; *DNA Replication ; *Insect Proteins/genetics/metabolism ; Cell Cycle/genetics ; CRISPR-Cas Systems ; },
abstract = {Endoreplication, also known as the endocycle, is a variant of the cell cycle that occurs in the silk glands of silkworms. Although the primase α subunit (PriS) has been reported to be involved in the initiation of DNA replication, its role in silk gland cell endoreplication remains unclear. In our study, we observed that BmPriS expression in the posterior silk gland (PSG) progressively increased during the late fifth instar. Using the CRISPR/Cas9 system, we specifically mutated BmPriS in the PSG, resulting in a decrease in cocoon shell weight and thinner silk fibers. Comparison with the wild-type revealed that the PSG was completely absent in ∆BmPriS silkworms. Immunofluorescence staining revealed a significant reduction in the size of silk gland cells. The expression of the fibroin genes (FibH, FibL, and P25) was nearly silent, whereas that of the sericin genes (Ser1, Ser2, and Ser3) was significantly downregulated. Moreover, EdU staining indicated a marked impairment of endoreplication in PSG cells, accompanied by a significant downregulation of endoreplication-associated minichromosome maintenance genes (MCM3, MCM5, MCM6, and MCM7). Cell cycle- and growth-related genes (CDK2, CyclinE, and Yki) were also significantly downregulated, whereas apoptosis-related genes (Fadd, Daxx, and Dredd) were significantly upregulated. Collectively, these findings indicate that BmPriS regulates silk gland growth and reduces silk production by modulating PSG endoreplication, and by interfering with the cell cycle and apoptosis processes. This study offers novel perspectives on the involvement of PriS in silk gland development.},
}
@article {pmid40479615,
year = {2025},
author = {Koshizuka, K and Wu, X and Sato, K and Vo, PTT and Murawska, GM and Ishikawa, T and Wang, Z and Molinolo, AA and Dennis, EA and Nathan, CO and Mali, P and Gutkind, JS},
title = {Genome-Wide CRISPR Screening Reveals That mTOR Inhibition Initiates Ferritinophagy and Ferroptosis in Head and Neck Cancer.},
journal = {Cancer research},
volume = {85},
number = {16},
pages = {3032-3051},
pmid = {40479615},
issn = {1538-7445},
support = {R35 GM139641/GM/NIGMS NIH HHS/United States ; R01 GM020501/GM/NIGMS NIH HHS/United States ; R01 DE030497/DE/NIDCR NIH HHS/United States ; U54 CA274502/CA/NCI NIH HHS/United States ; S10 OD026929/OD/NIH HHS/United States ; R01 CA247551/CA/NCI NIH HHS/United States ; R01 DE026870/DE/NIDCR NIH HHS/United States ; R01CA247551//National Cancer Institute (NCI)/ ; NCI U54CA274502//National Cancer Institute (NCI)/ ; R01DE030497//National Institute of Dental and Craniofacial Research (NIDR)/ ; R01DE026870//National Institute of Dental and Craniofacial Research (NIDR)/ ; the JSPS Overseas Research Fellowships//Japan Society for the Promotion of Science London (JSPS)/ ; the Uehara Memorial Foundation Research Fellowship//Uehara Memorial Foundation (UMF)/ ; Study abroad support of Mochida Memorial Foundation for Medical and Pharmaceutical Research//Mochida Memorial Foundation for Medical and Pharmaceutical Research ()/ ; GM RO1 GM20501-44//National Institute of General Medical Sciences (NIGMS)/ ; R35 GM139641-03//National Institute of General Medical Sciences (NIGMS)/ ; },
mesh = {*Ferroptosis/drug effects/genetics ; Humans ; *Head and Neck Neoplasms/genetics/drug therapy/pathology/metabolism ; *TOR Serine-Threonine Kinases/antagonists &amp; inhibitors/metabolism/genetics ; *MTOR Inhibitors/pharmacology ; *Ferritins/metabolism ; Autophagy/drug effects/genetics ; Animals ; Mice ; *Squamous Cell Carcinoma of Head and Neck/genetics/drug therapy/pathology/metabolism ; Signal Transduction/drug effects ; Cell Line, Tumor ; Xenograft Model Antitumor Assays ; CRISPR-Cas Systems ; Iron/metabolism ; },
abstract = {UNLABELLED: Genomic alterations converging on persistent activation of the PI3K/mTOR pathway represent one of the most frequently altered signaling circuitries in cancer. However, the clinical efficacy of mTOR inhibitors (mTORi) has been limited. In this study, we took advantage of the widespread activation of PI3K/mTOR signaling in head and neck squamous cell carcinoma (HNSCC) and the promising effects of mTORi in HNSCC experimental models and recent clinical trials to gain a mechanistic understanding of the antitumoral activity of mTORi. A genome-wide CRISPR screen revealed that treatment with mTORi promotes the autophagic degradation of ferritin (ferritinophagy), consequently increasing free intracellular iron, inducing lipid peroxidation, and ultimately driving cancer cell demise by ferroptosis. These findings provide a rationale for synergistic combinations repurposing approved drugs that disable cellular ferroptotic defense mechanisms. Together, this study provides a molecular framework underlying the antitumor activity of mTORi in HNSCC, thereby revealing multimodal precision therapies for HNSCC and many human malignancies displaying overactive PI3K/mTOR signaling.<br><br>SIGNIFICANCE: Inhibition of mTOR induces ferritinophagy that increases free iron and stimulates ferroptosis, suggesting that this axis could be harnessed to help predict responses and to develop rational combination therapies to overcome resistance.},
}
@article {pmid40466595,
year = {2025},
author = {Xiang, Z and Duan, M and Wang, S and Zhao, H and Zhao, W and Li, X and Li, X and Zeng, W and Wu, Y and Yang, F and Liu, X and Tang, C and Cui, L and Yang, Z},
title = {Three Asparagine insertions in the K13-propeller led to Plasmodiumfalciparum becoming resistant to multiple antimalarial drugs.},
journal = {International journal for parasitology. Drugs and drug resistance},
volume = {28},
number = {},
pages = {100590},
pmid = {40466595},
issn = {2211-3207},
mesh = {*Antimalarials/pharmacology ; *Plasmodium falciparum/drug effects/genetics ; *Asparagine/genetics ; *Protozoan Proteins/genetics ; CRISPR-Cas Systems ; Mutagenesis, Insertional ; Humans ; *Drug Resistance, Multiple/genetics ; Drug Resistance/genetics ; Malaria, Falciparum/parasitology/drug therapy ; Parasitic Sensitivity Tests ; Artemisinins/pharmacology ; },
abstract = {Drug resistance in Plasmodium falciparum represents a significant challenge in malaria treatment. Identifying the molecular markers associated with P. falciparum resistance will effectively detect resistance and enhance treatment efficiency. In this study, we utilized the advanced CRISPR/Cas9 technology to precisely insert one, two, or three asparagine residues into the Kelch 13(K13) gene of the 3D7 strain, positioned after the 142nd amino acid residue, resulting in 1N-3D7, 2N-3D7, and 3N-3D7. Using ring-stage survival assays (RSA), drug sensitivity evaluations, and in vitro developmental assessments, our findings revealed a trend: 1) the insertion of asparagine residues into the parasite genome increased RSA, with more asparagine insertions leading to higher RSA. 2) According to the IC50 values, 1N-3D7 and 2N-3D7 exhibited similar sensitivity profiles across all ten tested drugs, with both demonstrating resistance to Naphthoquine, indicating that the insertions of one or two asparagines played an equivalent role in conferring resistance. However, the insertion of three asparagine residues resulted in significantly higher IC50 values compared to the first two forms when tested with Artesunate, Artemether, Dihydroartemisinin, Pyronaridine Phosphate, and Naphthoquine, showing resistance to all five drugs. Furthermore, 3N-3D7 exhibited a prolonged ring phase and a shortened trophozoite phase within red blood cells; the schizont phase appeared synchronous with the others, yet its mature schizonts contained fewer merozoites. Additionally, 3N-3D7 exhibited a fitness defect, with the proportion decreasing gradually during co-culture with 3D7, its fitness cost calculated as 14.88 ± 2.87. All these results support the opinion that the insertion of three asparagines was a molecular marker of resistance to artemisinin derivatives, Pyronaridine Phosphate, and Naphthoquine in P. falciparum.},
}
@article {pmid40403231,
year = {2025},
author = {Morelli, E and Aktas-Samur, A and Maisano, D and Gao, C and Favasuli, V and Papaioannou, D and De Nola, G and Henninger, JE and Liu, N and Turi, M and Folino, P and Vreux, L and Cumerlato, M and Chen, L and Aifantis, I and Fulciniti, M and Anderson, KC and Lytton-Jean, AKR and Gullà, A and Young, RA and Samur, MK and Munshi, NC},
title = {CRISPR-Cas13d functional transcriptomics reveals widespread isoform-selective cancer dependencies on lncRNAs.},
journal = {Blood},
volume = {146},
number = {7},
pages = {847-860},
doi = {10.1182/blood.2025028746},
pmid = {40403231},
issn = {1528-0020},
support = {P01 CA155258/CA/NCI NIH HHS/United States ; },
mesh = {Humans ; *RNA, Long Noncoding/genetics/metabolism ; *CRISPR-Cas Systems ; *Transcriptome ; Animals ; Mice ; *Multiple Myeloma/genetics/pathology/metabolism ; Cell Line, Tumor ; Gene Expression Regulation, Neoplastic ; *Neoplasms/genetics ; RNA Isoforms/genetics ; },
abstract = {Long noncoding RNAs (lncRNAs) are a significant yet largely uncharted component of the cancer transcriptome, with their isoform-specific functions remaining poorly understood. In this study, we used RNA-targeting CRISPR-Cas13d to uncover and characterize hundreds of tumor-essential lncRNA (te-lncRNA) isoforms with clinical relevance. Focusing on multiple myeloma (MM), we targeted the lncRNA transcriptome expressed in tumor cells from patients with MM and revealed both MM-specific and pan-cancer dependencies across diverse cancer cell lines, which we further validated in animal models. Additionally, we mapped the subcellular localization of these te-lncRNAs, identifying >30 cytosolic isoforms that proved essential when targeted by cytosol-localized Cas13d. Notably, a specific isoform of small nucleolar RNA host gene 6, enriched in the endoplasmic reticulum, interacts with heat shock proteins to maintain cellular proteostasis. We also integrated functional and clinical data into the publicly accessible LongDEP Portal, providing a valuable resource for the research community. Our study offers a comprehensive characterization of te-lncRNAs, underscoring their oncogenic roles and therapeutic potential.},
}
@article {pmid40378078,
year = {2025},
author = {Ding, Q and Gan, P and Xu, Z and Li, H and Guo, L and MacDonald, C and Tan, W and Sanchez-Ortiz, E and McAnally, JR and Zhang, Y and Karri, D and Xu, L and Liu, N and Olson, EN},
title = {Genomic Editing of a Pathogenic Sequence Variant in ACTA2 Rescues Multisystemic Smooth Muscle Dysfunction Syndrome in Mice.},
journal = {Circulation},
volume = {152},
number = {7},
pages = {465-483},
pmid = {40378078},
issn = {1524-4539},
support = {R01 HL157281/HL/NHLBI NIH HHS/United States ; R01 HL130253/HL/NHLBI NIH HHS/United States ; 25POST1372779/AHA/American Heart Association-American Stroke Association/United States ; U54 HD087351/HD/NICHD NIH HHS/United States ; P50 HD087351/HD/NICHD NIH HHS/United States ; },
mesh = {Animals ; *Gene Editing/methods ; Humans ; Mice ; *Actins/genetics/metabolism ; *Muscle, Smooth, Vascular/metabolism/pathology/physiopathology ; *Myocytes, Smooth Muscle/metabolism/pathology ; Induced Pluripotent Stem Cells/metabolism ; CRISPR-Cas Systems ; Disease Models, Animal ; *Genetic Therapy/methods ; },
abstract = {BACKGROUND: Vascular smooth muscle cells (SMCs), the predominant cell type in the aortic wall, play a crucial role in maintaining aortic integrity, blood pressure, and cardiovascular function. Vascular SMC contractility and function depend on ACTA2 (smooth muscle α-actin 2). The pathogenic variant ACTA2 c.536G>A (p.R179H) causes multisystemic smooth muscle dysfunction syndrome, a severe disorder marked by widespread smooth muscle abnormalities, resulting in life-threatening aortic disease and high risk of early death from aneurysms or stroke. No effective treatments exist for multisystemic smooth muscle dysfunction syndrome.<br><br>METHODS: To develop a comprehensive therapy for multisystemic smooth muscle dysfunction syndrome, we used CRISPR (clustered regularly interspaced short palindromic repeats)-Cas9 (CRISPR-associated protein 9) adenine base editing to correct the ACTA2 R179H sequence variant. We generated isogenic human induced pluripotent stem cell lines and humanized mice carrying this pathogenic missense sequence variant. Induced pluripotent stem cell-derived SMCs were evaluated for key functional characteristics, including proliferation, migration, and contractility. The adenine base editor ABE8e-SpCas9-VRQR under control of either an SMC-specific promoter or a cytomegalovirus promoter, and an optimized single guide RNA under control of a U6 promoter were delivered intravenously to humanized R179H mice using adeno-associated virus serotype 9 and phenotypic outcomes were evaluated.<br><br>RESULTS: The R179H sequence variant causes a dramatic phenotypic switch in human induced pluripotent stem cell-derived SMCs from a contractile to a synthetic state, a transition associated with aneurysm formation. Base editing prevented this pathogenic phenotypic switch and restored normal SMC function. In humanized mice, the ACTA2[R179H/+] sequence variant caused widespread smooth muscle dysfunction, manifesting as decreased blood pressure, aortic dilation and dissection, bladder enlargement, gut dilation, and hydronephrosis. In vivo base editing rescued these pathological abnormalities, normalizing smooth muscle function.<br><br>CONCLUSIONS: This study demonstrates the effectiveness of adenine base editing to treat multisystemic smooth muscle dysfunction syndrome and restore aortic smooth muscle function. By correcting the ACTA2 R179H sequence variant, the pathogenic phenotypic shift in SMCs was prevented, key aortic smooth muscle functions were restored, and life-threatening aortic dilation and dissection were mitigated in humanized mice. These findings underscore the promise of gene-editing therapies in addressing the underlying genetic causes of smooth muscle disorders and offer a potential transformative treatment for patients facing severe vascular complications.},
}
@article {pmid40289704,
year = {2025},
author = {Sun, M and Gao, J and Tang, H and Wang, H and Zhou, L and Song, C and Tian, Y and Li, Q},
title = {D-CAPS: an efficient CRISPR-Cas9-based phage defense system for E. coli.},
journal = {Acta biochimica et biophysica Sinica},
volume = {57},
number = {8},
pages = {1244-1251},
pmid = {40289704},
issn = {1745-7270},
mesh = {*Escherichia coli/virology/genetics ; *CRISPR-Cas Systems ; *Bacteriophage T7/genetics ; },
abstract = {Escherichia coli is widely used in industrial chemical synthesis but faces significant challenges due to bacteriophage contamination, which reduces product quality and yield. Therefore, developing an efficient antiphage system is essential. In this study, we develop a CRISPR-Cas9-based antiphage system (CAPS) targeting essential genes of the T7 phage (gene 5 and gene 19) with single gRNAs transformed into MG1655 strains expressing Cas9. While CAPS provides limited resistance, with plating efficiencies ranging from 10 [-5] to 10 [-1], further optimization is needed. To enhance efficacy, we design a double-site-targeting CRISPR-Cas9-based antiphage system (D-CAPS). D-CAPS demonstrates complete resistance, with no plaques observed even at a high multiplicity of infection (MOI of 2), and growth curve analysis reveals that antiphage E. coli strains grow normally, similar to the wild-type strain, even at a high multiplicity of infection. Furthermore, D-CAPS is effective against BL21(DE3) strains, showing strong resistance and demonstrating its versatility across different E. coli strains. Protein expression analysis via green fluorescent protein confirms that E. coli carrying D-CAPS could maintain normal protein expression levels even in the presence of phages, comparable to wild-type strains. Overall, D-CAPS offers a robust and versatile approach to enhancing E. coli resistance to phages, providing a practical solution for protecting industrial E. coli strains and improving fermentation processes.},
}
@article {pmid39871553,
year = {2025},
author = {Roy, AA and Pokale, R and Mukharya, A and Nikam, AN and Dua, K and Rao, BSS and Seetharam, RN and Mutalik, S},
title = {Synergizing CRISPR-Cas9 with Advanced Artificial Intelligence and Machine Learning for Precision Drug Delivery: Technological Nexus and Regulatory Insights.},
journal = {Current gene therapy},
volume = {25},
number = {4},
pages = {467-496},
pmid = {39871553},
issn = {1875-5631},
support = {5/13/90/2020/NCD-III//Indian Council for Medical Research (ICMR), Government of India, New Delhi/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; Humans ; *Gene Editing/methods ; *Artificial Intelligence ; *Machine Learning ; *Drug Delivery Systems/methods ; Genetic Therapy/methods ; RNA, Guide, CRISPR-Cas Systems/genetics ; Precision Medicine ; },
abstract = {The evolution of genetic exploration tools, from laborious methods like radiationinduced mutations to the transformative CRISPR-Cas9 system, has fundamentally reshaped genetic research and gene editing capabilities. This journey, initiated by foundational techniques such as ZFNs and TALENs and culminating in the groundbreaking work of Doudna and Charpentier in 2012, has ushered in an era of precise DNA alteration and profound insights into gene functions. The CRISPR/Cas9 system uses the Cas9 enzyme and guides RNA (gRNA) to precisely target and cleave DNA, with subsequent repair via error-prone NHEJ or precise HDR, enabling versatile gene editing. Complementary computational tools like E-CRISP and Azimuth 2.0, alongside advanced deep learning models like DeepCRISPR, have significantly contributed to refining CRISPR experiments, optimizing gRNA efficiency, and predicting outcomes with greater precision. In clinical applications, CRISPR-Cas9 shows great promise for treating complex genetic disorders like sickle cell disease and β-thalassemia, but faces challenges such as off-target effects, immune responses to viral vectors, and ethical issues in germline editing. Overcoming these challenges requires meticulous experimentation and robust regulatory frameworks to ensure responsible and beneficial utilization of the CRISPR-Cas9 technology across diverse fields, including cancer treatment, genetic disease therapies, agriculture, and synthetic biology, while continually addressing ethical, safety, and legal considerations for its advancement and widespread adoption.},
}
@article {pmid40849301,
year = {2025},
author = {Buonaiuto, G and Desideri, F and Setti, A and Palma, A and D'Angelo, A and Storari, G and Santini, T and Laneve, P and Trisciuoglio, D and Ballarino, M},
title = {LncRNA HSCHARME is altered in human cardiomyopathies and promotes stem cell-derived cardiomyogenesis via splicing regulation.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {7880},
pmid = {40849301},
issn = {2041-1723},
support = {RM123188F6B80CE4//Sapienza Universit&#xe0; di Roma (Sapienza University of Rome)/ ; AR223188B40CB2D0//Sapienza Universit&#xe0; di Roma (Sapienza University of Rome)/ ; B83C22002870006//Ministero dell'Istruzione, dell'Universit&#xe0; e della Ricerca (Ministry of Education, University and Research)/ ; B53D23016090006//Ministero dell'Istruzione, dell'Universit&#xe0; e della Ricerca (Ministry of Education, University and Research)/ ; B53D23026140001//Ministero dell'Istruzione, dell'Universit&#xe0; e della Ricerca (Ministry of Education, University and Research)/ ; B83C22002860006//Ministero dell'Istruzione, dell'Universit&#xe0; e della Ricerca (Ministry of Education, University and Research)/ ; B53D23016090006//Ministero dell'Istruzione, dell'Universit&#xe0; e della Ricerca (Ministry of Education, University and Research)/ ; DBA.AD005.225-NUTRAGE-FOE2021//Consiglio Nazionale delle Ricerche (National Research Council)/ ; DSB.AD006.371-InvAt-FOE2022//Consiglio Nazionale delle Ricerche (National Research Council)/ ; },
mesh = {Humans ; *RNA, Long Noncoding/genetics/metabolism ; *Myocytes, Cardiac/metabolism/cytology ; Polypyrimidine Tract-Binding Protein/metabolism/genetics ; Animals ; Cell Differentiation/genetics ; Induced Pluripotent Stem Cells/metabolism/cytology ; Mice ; *Cardiomyopathies/genetics/metabolism/pathology ; *RNA Splicing/genetics ; Heterogeneous-Nuclear Ribonucleoproteins/metabolism/genetics ; CRISPR-Cas Systems ; },
abstract = {A growing body of evidence suggests that tissue-specific lncRNAs play pivotal roles in the heart. Here, we exploit the synteny between the mouse and human genomes to identify the human lncRNA HSCHARME and combine single-cell transcriptomics, CAGE-seq data, RNA-FISH imaging and CRISPR/Cas9 genome editing to document its role in cardiomyogenesis. By investigating the mechanism of action of HSCHARME in hiPSC-derived cardiomyocytes, we report that the locus produces the major pCHARME isoform that associates with SC35-containing speckles and interacts with the splicing regulator PTBP1. Consistently, the functional inactivation of pCHARME influences the splicing of cardiac-specific pre-mRNAs and impacts their expression, which reflects a decline in cardiomyocyte differentiation and physiology. In line with a possible association with disease, large-scale analysis of the lncRNA expression across cardiomyopathy patients reveals increased levels of pCHARME in hypertrophic and dilated hearts. We also find that HSCHARME dosage can modulate the expression of a subset of disease-associated targets. Our findings provide mechanistic insights into the role of pCHARME in cardiac cells with potential implications for disease.},
}
@article {pmid40848256,
year = {2025},
author = {Guo, JA and Gong, D and Evans, K and Takahashi, K and Jambhale, AD and Shiau, C and Yu, P and Wang, S and Wu, WW and Chugh, S and Kapner, KS and Dilly, J and Zhao, D and Chen, P and Smith, EL and Mancias, JD and Vazquez, F and Singh, H and Hwang, WL and Aguirre, AJ},
title = {Integrative genomic identification of therapeutic targets for pancreatic cancer.},
journal = {Cell reports},
volume = {44},
number = {9},
pages = {116191},
doi = {10.1016/j.celrep.2025.116191},
pmid = {40848256},
issn = {2211-1247},
abstract = {Pancreatic ductal adenocarcinoma (PDAC) is a deadly disease, and new therapeutic strategies are urgently needed. Here, we conduct an integrative, genome-scale examination of genetic dependencies and cell surface targets using CRISPR-Cas screening and multi-omic data, including single-nucleus and spatial transcriptomic data from patient tumors. We systematically identify clinically tractable and biomarker-linked PDAC dependencies, including CDS2 as a synthetic lethal target in cancer cells expressing signatures of epithelial-to-mesenchymal transition. We examine biomarkers and co-dependencies of the KRAS oncogene, defining gene expression signatures of sensitivity and resistance associated with response to pharmacological inhibition of KRAS. mRNA and protein profiling reveal cell surface protein-encoding genes with robust expression in patient tumors and minimal expression in non-malignant tissues. Furthermore, we define intratumoral and interpatient heterogeneity of target gene expression and identify orthogonal targets that suggest combinatorial strategies. Collectively, this work identifies multiple targets that may inform therapeutic strategies for patients with PDAC.},
}
@article {pmid40845808,
year = {2025},
author = {Yang, KK and Amini, AP},
title = {Simplifying protein engineering with deep learning.},
journal = {Cell},
volume = {188},
number = {17},
pages = {4477-4479},
doi = {10.1016/j.cell.2025.07.037},
pmid = {40845808},
issn = {1097-4172},
mesh = {*Deep Learning ; *Protein Engineering/methods ; Gene Editing/methods ; Humans ; CRISPR-Cas Systems ; Proteins/genetics ; },
abstract = {When it comes to deep learning for protein engineering, there is strength in simplicity. In this issue of Cell, through thoughtful deployment of existing fixed-backbone sequence design models, Caixia Gao and colleagues engineer diverse genome editing systems with improved functionality, enabling powerful capabilities in fine-grained and large-scale genome editing as demonstrated through strong experimental validation.},
}
@article {pmid40842235,
year = {2025},
author = {Moon, SY and Kim, MR and An, NY and Noh, MH and Lee, JY},
title = {Dual-mode CRISPRa/i for genome-scale metabolic rewiring in Escherichia coli.},
journal = {Nucleic acids research},
volume = {53},
number = {15},
pages = {},
doi = {10.1093/nar/gkaf818},
pmid = {40842235},
issn = {1362-4962},
support = {//Ministry of Science and ICT/ ; //National Research Foundation/ ; 2022M3A9B6082671//Republic of Korea: Bio &amp; Medical Technology Development Program/ ; 2024M3J5A1045980//Artificial Cellular Organelles Project/ ; //Korea-US Collaborative Research Fund (KUCRF)/ ; RS-2024-00468410//MSIT and the Ministry of Health &amp; Welfare, Republic of Korea/ ; },
mesh = {*Escherichia coli/genetics/metabolism ; Cyclic AMP Receptor Protein/genetics/metabolism ; *CRISPR-Cas Systems ; Gene Expression Regulation, Bacterial ; *Escherichia coli Proteins/genetics/metabolism ; *Metabolic Engineering/methods ; Genome, Bacterial ; RNA, Guide, CRISPR-Cas Systems/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {CRISPR (clustered regularly interspaced palindromic repeats)-mediated transcriptional regulation is a powerful and programmable approach for controlling gene expression. While CRISPR-based gene repression is well established in bacteria, simultaneous activation and repression remain challenging due to the limited availability of effective bacterial activation domains. Here, we provide an efficient dual-mode CRISPR activation and interference (CRISPRa/i) system that integrates an evolved protospacer adjacent motif (PAM)-flexible dxCas9 with an engineered Escherichia coli cAMP receptor protein (CRP). Through systematic optimization of the CRP domains and linkers, we developed a versatile effector capable of precise gene expression control when combined with dxCas9. Our dxCas9-CRP system demonstrated robust activation of upstream regulatory regions and effective repression of coding sequences, enabling targeted and programmable gene regulation. Using dual-fluorescent reporters, we validated the ability of this system to concurrently regulate multiple genes. Furthermore, with pooled guide RNA libraries, we applied the dxCas9-CRP system to increase violacein production in E. coli via genome-scale activation and repression in a coordinated manner, successfully identifying key regulatory targets that significantly increase production. Overall, this dual-mode CRISPRa/i system advances the potential for bacterial metabolic pathway rewiring, providing precise and flexible control for a wide range of biotechnological applications.},
}
@article {pmid40841511,
year = {2025},
author = {Potts, MA and Mizutani, S and Deng, Y and Vaidyanathan, S and Ting, KE and Giner, G and Sridhar, S and Shenoy, G and Liao, Y and Diepstraten, ST and Kueh, AJ and Pal, M and Healey, G and Tai, L and Wang, Z and König, C and Kaloni, D and Whelan, L and Milevskiy, MJG and Coughlan, HD and Pomilio, G and Wei, AH and Visvader, JE and Papenfuss, AT and Wilcox, S and Jeyasekharan, AD and Shi, W and Lelliott, EJ and Kelly, GL and Brown, KK and Strasser, A and Herold, MJ},
title = {Genome-wide in vivo CRISPR screens identify GATOR1 complex as a tumor suppressor in Myc-driven lymphoma.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {7582},
pmid = {40841511},
issn = {2041-1723},
mesh = {Animals ; *Lymphoma/genetics/metabolism/pathology ; Mice ; Mechanistic Target of Rapamycin Complex 1/metabolism/genetics ; *Proto-Oncogene Proteins c-myc/genetics/metabolism ; Humans ; CRISPR-Cas Systems ; TOR Serine-Threonine Kinases/metabolism ; *Genes, Tumor Suppressor ; Mice, Knockout ; Clustered Regularly Interspaced Short Palindromic Repeats ; Cell Line, Tumor ; Signal Transduction ; Tumor Suppressor Proteins/genetics/metabolism ; GTPase-Activating Proteins/genetics/metabolism ; Mice, Transgenic ; },
abstract = {Identifying tumor suppressor genes is predicted to inform on the development of novel strategies for cancer therapy. To identify new lymphoma driving processes that cooperate with oncogenic MYC, which is abnormally highly expressed in ~70% of human cancers, we use a genome-wide CRISPR gene knockout screen in Eµ-Myc;Cas9 transgenic hematopoietic stem and progenitor cells in vivo. We discover that loss of any of the GATOR1 complex components - NPRL3, DEPDC5, NPRL2 - significantly accelerates c-MYC-driven lymphoma development in mice. MYC-driven lymphomas lacking GATOR1 display constitutive mTOR pathway activation and are highly sensitive to mTOR inhibitors, both in vitro and in vivo. These findings identify GATOR1 suppression of mTORC1 as a tumor suppressive mechanism in MYC-driven lymphomagenesis and suggest an avenue for therapeutic intervention in GATOR1-deficient lymphomas through mTOR inhibition.},
}
@article {pmid40838896,
year = {2025},
author = {Losier, TT and King, KE and Rousseaux, MWC and Russell, RC},
title = {Identification of organelle-specific autophagy regulators from tandem CRISPR screens.},
journal = {The Journal of cell biology},
volume = {224},
number = {10},
pages = {},
doi = {10.1083/jcb.202405138},
pmid = {40838896},
issn = {1540-8140},
support = {#153034/CAPMC/CIHR/Canada ; PJT-169097/CAPMC/CIHR/Canada ; #2023-05587//Natural Sciences and Engineering Research Council of Canada/ ; RGPIN-2019-04133//Natural Sciences and Engineering Research Council of Canada/ ; DGECR-2019-00369//Natural Sciences and Engineering Research Council of Canada/ ; 201911CGV-434032-74238//Natural Sciences and Engineering Research Council of Canada/ ; //Natural Sciences and Engineering Research Council Postgraduate Scholarship/ ; },
mesh = {*Autophagy/genetics ; Humans ; Signal Transduction ; Endoplasmic Reticulum/metabolism/genetics ; *CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; *Organelles/metabolism ; *Autophagy-Related Proteins/metabolism/genetics ; HeLa Cells ; },
abstract = {Autophagy is a conserved degradative process that promotes cellular homeostasis under stress conditions. Under nutrient starvation, autophagy is nonselective, promoting indiscriminate breakdown of cytosolic components. Conversely, selective autophagy is responsible for the specific turnover of damaged organelles. We hypothesized that selective autophagy may be regulated by signaling pathways distinct from those controlling starvation-induced autophagy, thereby promoting organelle turnover. To address this question, we conducted kinome-wide CRISPR screens to identify distinct signaling pathways responsible for the regulation of basal autophagy, starvation-induced autophagy, and two types of selective autophagy, ER-phagy and pexophagy. These parallel screens identified both known and novel autophagy regulators, some common to all conditions and others specific to selective autophagy. More specifically, CDK11A and NME3 were further characterized to be selective ER-phagy regulators. Meanwhile, PAN3 and CDC42BPG were identified as an activator and inhibitor of pexophagy, respectively. Collectively, these datasets provide the first comparative description of the kinase signaling that defines the regulation of selective autophagy and bulk autophagy.},
}
@article {pmid40828859,
year = {2025},
author = {Saunier, M and Humbert, A and Kreis, V and Peltier, J and Tisba, A and Auxilien, S and Blum, M and Caldelari, I and Lucier, JF and Ueda, J and Gautheret, D and Toffano-Nioche, C and Andreani, J and Fortier, LC and Soutourina, O},
title = {Deciphering the RNA-based regulation mechanism of the phage-encoded AbiF system in Clostridioides difficile.},
journal = {PLoS genetics},
volume = {21},
number = {8},
pages = {e1011831},
doi = {10.1371/journal.pgen.1011831},
pmid = {40828859},
issn = {1553-7404},
mesh = {*Clostridioides difficile/genetics/virology/pathogenicity ; Prophages/genetics ; CRISPR-Cas Systems/genetics ; *Bacteriophages/genetics ; Toxin-Antitoxin Systems/genetics ; Clostridium Infections/microbiology/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Bacterial Proteins/genetics ; Humans ; RNA, Bacterial/genetics ; Gene Expression Regulation, Bacterial ; },
abstract = {Clostridioides difficile is the major cause of nosocomial infections associated with antibiotic therapy. The severity of C. difficile infections increased worldwide with the emergence of hypervirulent strains, including 027 ribotype epidemic strains. Many aspects of C. difficile adaptation strategies during pathogenesis remain poorly understood. This pathogen thrives in gut communities that are rich in microbes and phages. To regulate horizontal transfer of genetic material during its infection cycle, C. difficile relies on diverse mechanisms. More specifically, CRISPR (clustered regularly interspaced short palindromic repeats)-Cas and Toxin-Antitoxin (TA) systems contribute to prophage maintenance, prevention of phage infection, and stress response. Abortive infection (Abi) systems can provide additional lines of anti-phage defense. RNAs have emerged as key components of these systems including CRISPR RNAs and antitoxin RNAs within type I and type III TA. We report here the identification of a new AbiF-like system within a prophage of the hypervirulent C. difficile strain R20291. It is associated with an Abi_2/AbiD/F protein family largely distributed in Bacillota and Pseudomonadota with structural links to ancestral Cas13 proteins at the origin of the RNA-targeting CRISPR-Cas13 systems. We demonstrated toxic activity of the AbiFCd protein in C. difficile and in Escherichia coli and negative regulation of the abiFCd expression by an associated non-coding RNA RCd22. RCd22 contains two conserved abiF motifs and is active both in cis and in trans to neutralize the toxin by direct RNA-protein interaction, similar to RNA antitoxin in type III TA. A mass spectrometry interactomics analysis of protein fractions from MS2-Affinity Purification coupled with RNA sequencing (MAPS) revealed the AbiFCd protein among the most enriched RCd22 partners in C. difficile. Structural modeling of the RNA-protein complex and mutagenesis analysis revealed key positions on both protein and RNA partners for this interaction and toxic activity. In summary, these findings provide valuable insights into the mechanisms of interaction between bacteria and phages, which are pertinent to the advancement of phage therapy, genome editing, epidemiological surveillance, and the formulation of novel therapeutic approaches.},
}
@article {pmid40728278,
year = {2025},
author = {Joung, Y and Han, DK and Jang, H and Kang, T and Chen, L and Choo, J},
title = {Dual-Pathway Lateral Flow Assay for Rapid and Sensitive SARS-CoV-2 RNA Detection via CRISPR/Cas13a-Mediated SERS.},
journal = {ACS sensors},
volume = {10},
number = {8},
pages = {6253-6262},
doi = {10.1021/acssensors.5c02084},
pmid = {40728278},
issn = {2379-3694},
mesh = {*Spectrum Analysis, Raman/methods ; *SARS-CoV-2/genetics/isolation &amp; purification ; *RNA, Viral/analysis/genetics ; *CRISPR-Cas Systems/genetics ; Humans ; *COVID-19/diagnosis/virology ; *COVID-19 Nucleic Acid Testing/methods ; },
abstract = {Reverse transcription-polymerase chain reaction (RT-PCR) has been the gold standard for SARS-CoV-2 detection during the COVID-19 pandemic. However, its requirement for RNA-to-DNA conversion, reliance on centralized laboratory infrastructure, and lengthy turnaround times have limited its application in point-of-care (POC) settings. CRISPR/Cas13a-mediated lateral flow assays (LFAs) have emerged as promising alternatives for direct RNA analysis, yet their two-step workflows introduce procedural complexity and reduce sensitivity. To overcome these limitations, we developed a dual-pathway LFA strip based on surface-enhanced Raman scattering (SERS), which integrates CRISPR/Cas13a-mediated RNA cleavage and SERS detection into a single, portable platform. The device utilizes five vertically stacked paper layers with distinct geometries, enabling sequential CRISPR reaction and SERS quantification through two independent pathways. When tested with SARS-CoV-2 ORF1ab RNA targets, the system exhibited an 80-fold increase in sensitivity and a 10 min reduction in assay time compared to conventional fluorescence assays. Clinical validation using 18 samples (13 positives and 5 negatives) demonstrated high diagnostic accuracy, fully consistent with RT-PCR results. By unifying CRISPR-based RNA recognition and SERS signal amplification in a user-friendly format, this dual-pathway LFA strip offers a rapid, ultrasensitive, and practical diagnostic tool for infectious diseases in POC settings.},
}
@article {pmid40675426,
year = {2025},
author = {Li, W and Tang, Z and Zhu, X and Wang, B and Zhang, X and Ji, Z and Cheng, S},
title = {Ultrasensitive detection of MMP-2 via T7 RNA polymerase and CRISPR/Cas13a-Enhanced electrochemiluminescence biosensor for COPD diagnosis.},
journal = {Methods (San Diego, Calif.)},
volume = {242},
number = {},
pages = {80-88},
doi = {10.1016/j.ymeth.2025.07.005},
pmid = {40675426},
issn = {1095-9130},
mesh = {*Matrix Metalloproteinase 2/analysis/blood/genetics ; *Biosensing Techniques/methods ; Humans ; CRISPR-Cas Systems/genetics ; Limit of Detection ; Luminescent Measurements/methods ; *Electrochemical Techniques/methods ; *Pulmonary Disease, Chronic Obstructive/diagnosis/blood ; *DNA-Directed RNA Polymerases/genetics/metabolism ; *Viral Proteins/genetics/metabolism ; Metal Nanoparticles/chemistry ; Gold/chemistry ; Biomarkers ; Nucleic Acid Amplification Techniques/methods ; },
abstract = {In this work, an electrochemiluminescence (ECL) biosensor integrating T7 RNA polymerase amplification and CRISPR/Cas13a-mediated signal enhancement was developed for the ultrasensitive detection of matrix metalloproteinase-2 (MMP-2), a key biomarker associated with chronic inflammatory diseases such as COPD. A peptide nucleic acid (PNA) probe was designed to respond specifically to MMP-2 cleavage, enabling the release of DNA templates for subsequent T7 RNA polymerase-driven transcription amplification. The generated RNA triggers the collateral cleavage activity of CRISPR/Cas13a, resulting in a significant amplification of the ECL signal. The biosensor's surface was constructed using a AuNPs/Ti3C2Tx/Ru(II)-PEI nanocomposite, which enhanced signal transduction and stability. Under optimized conditions, the proposed biosensor achieved a detection limit as low as 62.05 fM, demonstrating superior sensitivity compared to conventional methods, as summarized in Table 1. The platform also exhibited excellent specificity and anti-interference capability, ensuring reliable detection of MMP-2 in complex biological samples. This study provides a simple yet highly efficient strategy for enzymatic biomarker detection, offering great potential for clinical applications in early disease diagnosis and monitoring.},
}
@article {pmid40626419,
year = {2025},
author = {Jacobebbinghaus, N and Bigge, F and Saudhof, M and Hübner, W and Kruse, O and Baier, T},
title = {Transcriptional gene fusions via targeted integration at safe harbors for high transgene expression in Chlamydomonas reinhardtii.},
journal = {The New phytologist},
volume = {247},
number = {6},
pages = {2665-2677},
doi = {10.1111/nph.70368},
pmid = {40626419},
issn = {1469-8137},
support = {2221NR063X//Federal Ministry of Agriculture, Food and Regional Identity/ ; },
mesh = {*Chlamydomonas reinhardtii/genetics/metabolism ; *Transgenes/genetics ; *Gene Fusion ; *Transcription, Genetic ; CRISPR-Cas Systems/genetics ; Gene Editing ; Terpenes/metabolism ; },
abstract = {Conventional genetic engineering in green microalgae employs error-prone nonhomologous end joining to integrate recombinant DNA at double-strand breaks generated at random positions across the nuclear genome. This typically results in variable transcription strength and requires a labor-intensive screening procedure to identify transformants with sufficient expression. Current advances in genome editing enable scar-less integration of DNA at any desired locus for engineered bioproduction. We optimized construct design for predictable transgene expression at a high level, significantly improved scar-less integration rates into the nuclear genome via homology arm length optimization and quantified endogenous gene expression in vivo. Subsequently, endogenous genes were successfully targeted via Clustered Regularly Interspaced Short Palindromic Repeats and CRISPR-associated protein 9 (CRISPR/Cas9) to evaluate their capacity for high transgene expression and terpenoid production. Highest scar-less homology-directed repair efficiency was achieved with 50 bp homology arms. The Light harvesting Chl a/b binding protein of LHCII (LHCBM1) locus was found to be differentially expressed under several light intensities and allows an 8.6-fold increase in transgenic protein accumulation compared with random insertion approaches. Co-expression of a functional sesquiterpene synthase achieved a 60-fold increase in valencene production compared with previous attempts. We showed LHCBM1 locus constitutes a genetic safe harbor for transgene expression and demonstrates the potential of C. reinhardtii as a green cell factory.},
}
@article {pmid40338215,
year = {2025},
author = {Wu, S and Yuan, J and Xi, X and Wang, L and Li, Y and Wang, Y and Lin, J},
title = {A Colorimetric Biosensor Integrating Rotifer-Mimicking Magnetic Separation with RAA/CRISPR-Cas12a for Rapid and Sensitive Detection of Salmonella.},
journal = {ACS sensors},
volume = {10},
number = {8},
pages = {5473-5483},
doi = {10.1021/acssensors.4c03356},
pmid = {40338215},
issn = {2379-3694},
mesh = {*Salmonella/isolation &amp; purification/genetics ; *Biosensing Techniques/methods ; *Colorimetry/methods ; *CRISPR-Cas Systems ; Limit of Detection ; Milk/microbiology ; *Rotifera/chemistry ; Gold/chemistry ; Animals ; Food Microbiology ; Nucleic Acid Amplification Techniques ; Metal Nanoparticles/chemistry ; Food Contamination/analysis ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {Efficient detection of foodborne bacteria is crucial for ensuring food safety, yet current methods often fall short in balancing speed, accuracy, sensitivity, and cost. This study presents an integrated biosensing platform for the rapid and sensitive detection of Salmonella in large-volume food samples. The platform incorporates a Rotifer-Mimicking Magnetic Separator (RMMS) that enhances the sample pretreatment by effectively mixing and isolating the bacteria from the sample. Coupled with this, the colorimetric biosensor utilizes a streamlined one-pot system that combines Recombinase Aided Amplification (RAA), betaine, and CRISPR-Cas12a to enable efficient pathogen detection. Initially, phenylboronic acid-modified magnetic beads (PBA-MBs) capture Salmonella, forming bacteria-PBA-MB complexes, which are then isolated using the RMMS. Target DNA amplicons activate ribonucleoprotein complexes, and Au@PtNPs-MBs with linker single DNAs are cleaved to release Au@PtNPs. The Au@PtNPs catalyze the H2O2-3,3',5,5'-tetramethylbenzidine, producing a visible blue color that indicates Salmonella concentration. This biosensor successfully detects Salmonella in 40 mL spiked milk samples within 75 min, achieving a detection limit of 89 CFU/mL. This work offers a simple, sensitive, low-cost detection method with potential applications in on-site testing, significantly enhancing food safety monitoring.},
}
@article {pmid40838720,
year = {2025},
author = {Ferronato, GA and Silveira, JC and Ferraz, MAMM},
title = {Potential of small extracellular vesicles as Cas9 delivery tool: A promising approach for gene editing livestock gametes and embryos.},
journal = {Biology of reproduction},
volume = {},
number = {},
pages = {},
doi = {10.1093/biolre/ioaf195},
pmid = {40838720},
issn = {1529-7268},
abstract = {Genome editing is a rapidly advancing technology with transformative potential in livestock, offering opportunities that range from enhanced production traits to the generation of biomedical models for human disease and xenotransplantation. The CRISPR/Cas9 system, originally identified as a bacterial defense mechanism, has become the most widely used tool for precise genome editing. In this review, we first summarize the potential applications of CRISPR/Cas9 in livestock and highlight notable successes to date. We then address the ongoing challenges associated with delivering CRISPR/Cas9 into gametes and embryos, as current methods such as microinjection and electroporation often result in high mosaicism and cellular damage. We subsequently introduce extracellular vesicles (EVs) as a promising alternative delivery system. Secreted by virtually all cell types, EVs can efficiently transport bioactive molecules and are readily internalized by gametes and embryos. Although EV-mediated delivery of CRISPR/Cas9 has shown success in somatic cells, its use in reproductive cells remains largely unexplored. We review emerging strategies for loading EVs with CRISPR/Cas components and discuss the potential advantages of combining this approach with recently developed smaller Cas variants to overcome delivery barriers. Collectively, these innovations support the promise of EVs as a biologically compatible, efficient, and minimally invasive system for targeted genome editing in livestock reproduction.},
}
@article {pmid40838475,
year = {2025},
author = {},
title = {RETRACTION: Spider Eye Development Editing and Silk Fiber Engineering Using CRISPR-Cas.},
journal = {Angewandte Chemie (International ed. in English)},
volume = {},
number = {},
pages = {e202517416},
doi = {10.1002/anie.202517416},
pmid = {40838475},
issn = {1521-3773},
}
@article {pmid40835841,
year = {2025},
author = {Watson, LC and Sala, KA and Bernitz, N and Baumgärtel, L and Pallett, MA and Marzook, NB and Straker, LC and Peng, D and Collinson, L and Sateriale, A},
title = {Targeted CRISPR screens reveal genes essential for Cryptosporidium survival in the host intestine.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {7749},
pmid = {40835841},
issn = {2041-1723},
support = {CC2063/WT_/Wellcome Trust/United Kingdom ; CC2063/WT_/Wellcome Trust/United Kingdom ; CC2063/WT_/Wellcome Trust/United Kingdom ; },
mesh = {Animals ; *Cryptosporidiosis/parasitology ; *Cryptosporidium/genetics/pathogenicity ; *CRISPR-Cas Systems ; *Intestines/parasitology ; Mice ; *Protozoan Proteins/genetics/metabolism ; *Genes, Essential ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Humans ; Gene Knockout Techniques ; Cryptosporidium parvum/genetics ; Protozoan Vaccines/immunology ; },
abstract = {The Cryptosporidium parasite is one of the leading causes of diarrheal morbidity and mortality in children, and adolescent infections are associated with chronic malnutrition. There are no vaccines available for protection and only one drug approved for treatment that has limited efficacy. A major barrier to developing new therapeutics is a lack of foundational knowledge of Cryptosporidium biology, including which parasite genes are essential for survival and virulence. Here, we iteratively improve the tools for genetically manipulating Cryptosporidium and develop a targeted CRISPR-based screening method to rapidly assess how the loss of individual parasite genes influence survival in vivo. Using this method, we examine the parasite's pyrimidine salvage pathway and a set of leading Cryptosporidium vaccine candidates. From this latter group, using inducible knockout, we determined the parasite gene known as Cp23 to be essential for survival in vivo. Parasites deficient in Cp23 were able to replicate within and emerge from infected epithelial cells, yet unable to initiate gliding motility which is required for the reinfection of neighbouring cells. The targeted screening method presented here is highly versatile and will enable researchers to more rapidly expand the knowledge base for Cryptosporidium infection biology, paving the way for new therapeutics.},
}
@article {pmid40835811,
year = {2025},
author = {Luzics, S and Baka, E and Otto, M and Kosztik, J and Szalontai, H and Bata-Vidács, I and Nagy, I and Tóth, &#xc1; and Táncsics, A and Pápai, M and Nagy, I and Orsini, M and Kukolya, J},
title = {High-quality de novo genome assembly and functional genomic insights into Thermobifida alba DSM43795[T], a mesophilic actinobacterium isolated from garden soil.},
journal = {Biologia futura},
volume = {},
number = {},
pages = {},
pmid = {40835811},
issn = {2676-8607},
support = {K142686//National Research, Development and Innovation Office/ ; EK&#xd6;P- 24- VI/MATE-3//Ministry for Culture and Innovation from the source of the National Research, Development and Innovation Fund/ ; EK&#xd6;P-MATE/2024/25/D//Ministry for Culture and Innovation from the source of the National Research, Development and Innovation Fund/ ; },
abstract = {Thermobifida alba DSM43795[T], a mesophilic actinobacterium isolated from garden soil, plays a vital role in lignocellulose degradation and holds biotechnological and pharmaceutical potential. We present a high-quality, complete de novo genome assembly of T. alba DSM43795[T] using combined PacBio long-read and Illumina short-read sequencing, resulting in a single circular chromosome of 4.9 Mbp with 72.1% GC content. Comparative genomics with the thermophilic relative T. fusca YX revealed 83.39% average nucleotide identity and extensive genome synteny alongside niche-specific differences. Functional annotation identified 4345 genes, including a rich complement of carbohydrate-active enzymes (CAZymes) such as glycoside hydrolases (GHs), esterases, and polysaccharide lyases, supporting versatile plant biomass degradation. GH gene sets were largely conserved between the species in both gene number and distribution, but T. alba uniquely encodes a novel GH10 endo-xylanase near a characterised palindrome regulatory sequence, indicating species-specific regulation. We hypothesise that thermophilic adaptation in T. fusca requires more proteins for ribosome integrity and amino acid metabolism, with reduced emphasis on carbohydrate metabolism and defence compared to T. alba. Moreover, T. alba harbours a broader array of defence-related genes and mobile genetic elements, including integrases and transposases. Although lacking a complete CRISPR-Cas system, two CRISPR arrays were detected, suggesting alternative immune strategies. Virulence factor homologs shared by both species likely reflect environmental survival rather than pathogenicity. This genomic characterisation elucidates T. alba's metabolic versatility and ecological adaptations, laying the groundwork for its potential applications in biomass conversion, environmental biotechnology, and drug discovery.},
}
@article {pmid40835333,
year = {2025},
author = {Sanders, J and Lin, S},
title = {Molecular techniques for understanding harmful algal blooms: A review.},
journal = {Harmful algae},
volume = {148},
number = {},
pages = {102909},
doi = {10.1016/j.hal.2025.102909},
pmid = {40835333},
issn = {1878-1470},
mesh = {*Harmful Algal Bloom ; Dinoflagellida/classification/genetics ; Cyanobacteria/classification/genetics ; Seawater ; Fresh Water ; *Water Pollution/analysis/prevention &amp; control ; Molecular Probe Techniques ; Enzyme-Linked Immunosorbent Assay ; Molecular Diagnostic Techniques ; CRISPR-Cas Systems ; In Situ Hybridization, Fluorescence ; DNA Barcoding, Taxonomic ; *Molecular Typing/methods ; },
abstract = {Harmful algal blooms (HABs) are intricate ecological events caused by diverse algal species and are influenced by a myriad of biotic and abiotic factors. The urgently needed development of effective prevention and control techniques face two primary challenges. The first challenge is the technical shortfalls in rapidly identifying and monitoring the causative species. The second challenge is the absence of research frameworks and technologies for accurately diagnosing the primary drivers of these blooms. Molecular techniques offer promising solutions to these issues, and research in this field has seen significant growth over the past two decades. Previous reviews have predominantly focused on species identification and monitoring, leaving the status of bloom driver studies less clear. This review provides a comprehensive overview of molecular techniques for HAB identification and driver analysis. HAB-specific use cases of techniques and comparison between them based on technical specifications are provided. Nucleic acid-based techniques presently dominate over antibody-based techniques due to their tunable taxon-specificity and ease to prepare probes. In situ applications and monitoring platforms still have a large room for improvement. The omics approach is the most promising choice for unraveling HAB drivers but requires a framework and a quantitative model for estimating the contribution of potential responsible factors. Future prospects relating to particular needs in HAB research and emerging technologies are also discussed.},
}
@article {pmid40782800,
year = {2025},
author = {Traxler, P and Reichl, S and Folkman, L and Shaw, L and Fife, V and Nemc, A and Pasajlic, D and Kusienicka, A and Barreca, D and Fortelny, N and Rendeiro, AF and Halbritter, F and Weninger, W and Decker, T and Farlik, M and Bock, C},
title = {Integrated time-series analysis and high-content CRISPR screening delineate the dynamics of macrophage immune regulation.},
journal = {Cell systems},
volume = {16},
number = {8},
pages = {101346},
doi = {10.1016/j.cels.2025.101346},
pmid = {40782800},
issn = {2405-4720},
mesh = {*Macrophages/immunology/metabolism ; Animals ; Mice ; Signal Transduction ; CRISPR-Cas Systems/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Gene Expression Regulation ; },
abstract = {Macrophages are innate immune cells involved in host defense. Dissecting the regulatory landscape that enables their swift and specific response to pathogens, we performed time-series analysis of gene expression and chromatin accessibility in murine macrophages exposed to various immune stimuli, and we functionally evaluated gene knockouts at scale using a combined CROP-seq and CITE-seq assay. We identified new roles of transcription regulators such as Spi1/PU.1 and JAK-STAT pathway members in immune cell homeostasis and response to pathogens. Macrophage activity was modulated by splicing proteins SFPQ and SF3B1, histone acetyltransferase EP300, cohesin subunit SMC1A, and mediator complex proteins MED8 and MED14. We further observed crosstalk among immune signaling pathways and identified molecular drivers of pathogen-induced dynamics. In summary, this study establishes a time-resolved regulatory map of pathogen response in macrophages, and it describes a broadly applicable method for dissecting immune-regulatory programs through integrative time-series analysis and high-content CRISPR screening. A record of this paper's transparent peer review process is included in the supplemental information.},
}
@article {pmid40781772,
year = {2025},
author = {Kweon, J and Park, S and Jeon, MY and Lim, K and Jang, G and Jang, AH and Lee, M and Seok, C and Lee, C and Park, S and Ahn, J and Jang, J and Kim, N and Sung, YH and Kim, D and Kim, Y},
title = {High-efficiency base editing for nuclear and mitochondrial DNA with an optimized DYW-like deaminase.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.ymthe.2025.08.007},
pmid = {40781772},
issn = {1525-0024},
abstract = {CRISPR-based cytosine base editors enable precise genome editing without inducing double-stranded DNA breaks yet traditionally depend on a limited selection of deaminases from the APOBEC/AID or TadA families. Here, we present SsCBE, a CRISPR-based cytosine base editor utilizing SsdAtox, a DYW-like deaminase derived from the toxin of Pseudomonas syringae. Strategic engineering of SsdAtox has led to remarkable improvements in the base editing efficiency (by up to 8.4-fold) and specificity for SsCBE, while concurrently reducing cytotoxicity. Exhibiting exceptional versatility, SsCBE was delivered and efficiently applied using diverse delivery methods, including engineered virus-like particles. Its application has enabled targeted cytosine base editing in mouse zygotes and pioneering edits in mitochondrial DNA. SsCBE expands the genome editing toolbox by introducing a distinct deaminase scaffold with broad utility for both basic research and potential therapeutic applications.},
}
@article {pmid40763746,
year = {2025},
author = {Sun, Q and Ma, X and Ning, Q and Li, S and Wang, P and Tan, X and Jin, Q and Zheng, J and Li, Y and Dong, D},
title = {Systematic screening for functional exon-skipping isoforms using the CRISPR-RfxCas13d system.},
journal = {Cell systems},
volume = {16},
number = {8},
pages = {101351},
doi = {10.1016/j.cels.2025.101351},
pmid = {40763746},
issn = {2405-4720},
mesh = {Humans ; *Exons/genetics ; Protein Isoforms/genetics ; *Alternative Splicing/genetics ; Colorectal Neoplasms/genetics/pathology ; *CRISPR-Cas Systems/genetics ; Cell Line, Tumor ; Animals ; Mice ; RNA, Guide, CRISPR-Cas Systems/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Cell Proliferation/genetics ; },
abstract = {Exon skipping (ES) is the most prevalent form of alternative splicing and a hallmark of tumorigenesis, yet its functional roles remain underexplored. Here, we present a CRISPR-RfxCas13d-based platform for transcript-specific silencing of ES-derived isoforms using guide RNAs (gRNAs) targeting exon-exon junctions. We designed a transcriptome-wide gRNA library against 3,744 human ES events and conducted loss-of-function screens in colorectal cancer (CRC) cells in vitro and in vivo. This screen uncovered multiple ES events essential for CRC growth, notably HMGN3 Δ6, an isoform arising from exon 6 skipping, which enhanced tumor proliferation. Functional validation confirmed the oncogenic role of HMGN3 Δ6 and its necessity for CRC progression. Our study establishes CRISPR-RfxCas13d as a powerful tool for isoform-specific functional genomics and reveals a widespread, previously uncharacterized layer of tumor biology driven by ES. These findings position ES-derived transcripts as promising targets for therapeutic intervention in cancer.},
}
@article {pmid40623538,
year = {2025},
author = {Peng, Z and Yang, T and Xu, S and Yang, B and Zhang, Z and Ding, M and Gu, W and Zheng, L},
title = {Aerobic exercise ameliorates skeletal muscle atrophy in atic knockout zebrafish through the oxidative phosphorylation pathway.},
journal = {Free radical biology &amp; medicine},
volume = {238},
number = {},
pages = {653-668},
doi = {10.1016/j.freeradbiomed.2025.07.007},
pmid = {40623538},
issn = {1873-4596},
mesh = {Animals ; Zebrafish/genetics ; *Muscular Atrophy/genetics/metabolism/pathology/therapy ; Oxidative Phosphorylation ; *Muscle, Skeletal/metabolism/pathology ; Aminoimidazole Carboxamide/analogs &amp; derivatives/metabolism ; Reactive Oxygen Species/metabolism ; Ribonucleotides/metabolism ; *Physical Conditioning, Animal ; *Zebrafish Proteins/genetics/metabolism ; *Hydroxymethyl and Formyl Transferases/genetics/metabolism ; Gene Knockout Techniques ; *Nucleotide Deaminases/genetics/metabolism ; Mice ; Mitochondria/metabolism ; CRISPR-Cas Systems ; Purines/metabolism ; Multienzyme Complexes ; },
abstract = {The mechanisms linking purine metabolism disorders to skeletal muscle pathology are unclear. This study constructed a CRISPR/Cas9-mediated zebrafish atic knockout model and a siRNA-interfered C2C12 myoblast cell model. We revealed a novel mechanism by which ATIC (5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase/IMP cyclohydrolase) deletion drove the atrophy of skeletal muscle through the downregulation of the oxidative phosphorylation of mitochondria (OXPHOS) pathway. It was found that atic/Atic knockout/knockdown led to the interruption of purine de novo synthesis, abnormal 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) accumulation, and blockage of inosine monophosphate (IMP) synthesis, which in turn triggered mitochondrial structural damage, dysfunction of complex I-V function, and a burst of reactive oxygen species (ROS), and ultimately triggered muscle atrophy through activation of the ubiquitin-proteasome system. The progressive aerobic intervention revealed that 8 weeks of training significantly restored skeletal muscle function in zebrafish atic[-/-] mutants, and the mechanism was related to the enhancement of mitochondrial biogenesis, up-regulation of the core complex expression of the OXPHOS pathway, and the improvement of ROS scavenging ability. These findings reveal that ATIC deficiency disrupts mitochondrial function through purine metabolism dysregulation, linking aberrant AICAR accumulation to OXPHOS impairment, which provides a theoretical basis for the early warning of muscular toxicity of targeted purine metabolizing drugs and lays a molecular foundation for the exercise rehabilitation strategy of metabolic myopathies.},
}
@article {pmid40456437,
year = {2025},
author = {Yoshida, S and Onozawa, M and Yokoyama, S and Matsukawa, T and Goto, H and Hirabayashi, S and Kondo, T and Hashimoto, D and Onodera, Y and Teshima, T},
title = {Peposertib suppresses generation of FLT3-internal tandem duplication formed by contralateral double nicks.},
journal = {Experimental hematology},
volume = {149},
number = {},
pages = {104819},
doi = {10.1016/j.exphem.2025.104819},
pmid = {40456437},
issn = {1873-2399},
mesh = {*fms-Like Tyrosine Kinase 3/genetics/antagonists &amp; inhibitors ; Humans ; Mice ; Animals ; Phthalazines/pharmacology ; *Gene Duplication/drug effects ; *Leukemia, Myeloid, Acute/genetics/drug therapy ; *Tandem Repeat Sequences/drug effects ; Piperazines/pharmacology ; *DNA Breaks, Double-Stranded/drug effects ; CRISPR-Cas Systems ; Cell Line, Tumor ; },
abstract = {Fms-like tyrosine kinase 3-internal tandem duplication (FLT3-ITD) is the most frequent gene mutation in acute myeloid leukemia. The consequences of FLT3-ITD have been analyzed in detail; however, the molecular mechanisms underlying the generation of FLT3-ITD remain to be elucidated. We analyzed FLT3-ITDs in clinical samples using deep sequencing and identified not only oligoclonal ITDs but also rare deletion clones clustered at the palindrome-like sequence at FLT3 exon 14. We hypothesized that FLT3 exon 14 is genetically unstable due to the palindrome-like sequence at the region and that genomic damage at the site initiates FLT3-ITD formation. We used clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 to induce DNA damage for creating artificial FLT3-ITDs in human and mouse cell lines. We found that double nicks on the adjacent contralateral strand most efficiently generate ITDs. The artificial ITDs resembled clinical ITDs in the length distribution and characteristics at the joint. We further compared the inhibitory effects of olaparib and peposertib, specific inhibitors of single-strand break (SSB) and double-strand break (DSB) repair, respectively. Peposertib remarkably reduced ITD formation, but olaparib did not affect the mutation pattern. The findings indicated that nonhomologous end joining has a crucial role in the generation of ITDs. Our data shed light to the new role of peposertib, which potentially suppresses the generation of de novo FLT3-ITDs caused by mis-repair events of the DNA damages in a clinical course.},
}
@article {pmid40397098,
year = {2025},
author = {Williams, PJ},
title = {The New Old Colonialism.},
journal = {The CRISPR journal},
volume = {8},
number = {4},
pages = {296-299},
doi = {10.1089/crispr.2025.0059},
pmid = {40397098},
issn = {2573-1602},
mesh = {Humans ; *Colonialism ; *Gene Editing/legislation &amp; jurisprudence/ethics ; Genome, Human ; CRISPR-Cas Systems ; },
abstract = {The question of how law should regulate the manipulation of the human genome or germline is inflected by the interconnected, intersectional parrying among different systems of moral value. Contract law and constitutional law reflect two poles of interest: the transactional aspects of market valuation and the relational aspects of the web of life that acknowledge "pricelessness." In the decades from the initial decoding of the human genome in 2000 to the emergence of CRISPR technologies, powerful companies and powerful individuals now all but own the fate of our species and the health of our planet. The destructive effects of the realignments we are undergoing are still largely invisible (if not for long) and largely unresponsive to conventional checks.},
}
@article {pmid40397097,
year = {2025},
author = {Musunuru, K and Urnov, F},
title = {Moving Therapeutic Genome Editing into Global Clinical Trials and Medicine.},
journal = {The CRISPR journal},
volume = {8},
number = {4},
pages = {228-231},
doi = {10.1089/crispr.2025.0049},
pmid = {40397097},
issn = {2573-1602},
mesh = {Humans ; Clinical Trials as Topic ; CRISPR-Cas Systems ; *Gene Editing/methods/trends ; *Genetic Therapy/methods/trends ; },
abstract = {Moving CRISPR-based therapies from discovery to dosing patients in clinical trials and ultimately to approval involves navigating a challenging terrain of highs and lows. In this interview, physician-scientist Kiran Musunuru and genome editor Fyodor Urnov reflect on the past 20 years of their nonclinical and clinical programs in the field, the current landscape of innovation, and what they see on the horizon.},
}
@article {pmid40397096,
year = {2025},
author = {Chandru, V and Gupta, V and Hegde, V and Venkatesan, A and Arora, R},
title = {Intent to Cure: The Need for a Rare Disease Platform in India and Across the Global South.},
journal = {The CRISPR journal},
volume = {8},
number = {4},
pages = {277-281},
doi = {10.1089/crispr.2025.0046},
pmid = {40397096},
issn = {2573-1602},
mesh = {Humans ; India ; *Rare Diseases/therapy/genetics ; *Genetic Therapy/methods ; Gene Editing/methods ; Genomics/methods ; Artificial Intelligence ; CRISPR-Cas Systems ; },
abstract = {The democratization of genomic technologies presents substantial opportunities for addressing rare genetic diseases, particularly in collaborations between the Global South and North. In this Perspective, we describe the current progress in gene therapy, including CRISPR, in India and see an upward trajectory of innovation. We propose the establishment of a rare disease platform in India and across the Global South designed to bridge scientific, clinical, and economic gaps, transforming untapped genetic diversity into shared opportunities for therapeutic innovation and health care equity. This platform would encompass a comprehensive data infrastructure capturing clinical, genomic, and biosample data, complemented by an artificial intelligence-powered analytics layer to enhance patient engagement and clinical trial matching, ultimately enabling cost-effective research and development (R&amp;D) of novel therapies.},
}
@article {pmid40397095,
year = {2025},
author = {Qiu, J},
title = {From 'Frankenstein Science' to Cosmopolitan Ethics: Overlooked Perspectives on the 'CRISPR Babies' Scandal.},
journal = {The CRISPR journal},
volume = {8},
number = {4},
pages = {257-261},
doi = {10.1089/crispr.2025.0057},
pmid = {40397095},
issn = {2573-1602},
mesh = {Humans ; *Gene Editing/ethics ; *Bioethics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; *CRISPR-Cas Systems ; China ; },
abstract = {In November 2018, Chinese biophysicist He Jiankui stunned the world by announcing that he had created the first genetically-modified babies. Is he a rogue scientist? What are the socio-cultural contexts that motivated him to commit an act widely regarded as morally indefensible? What does it say about Chinese bioethics? How should we determine whether it can ever be justified to permanently alter the human gene pool? This article highlights the global institutional failures that enabled this unfortunate episode, including the prevailing international scientific culture and the persistent Western bias against scientific work originated in the Global South. It calls for systemic efforts-including regulatory reforms, increased transparency, public engagement, and international cooperation-to strengthen ethics governance both within nations and across borders. Finally, it advocates for decolonizing bioethics, advancing the sociology of bioethics, and fostering a cosmopolitan approach to ethics grounded in diversity, equity, inclusion, and our shared humanity.},
}
@article {pmid40397093,
year = {2025},
author = {Jasanoff, S and Hurlbut, JB and Saha, K and Moses, JD and Affsprung, D and Austin, H and Baylis, F and Evans, JH and Hunt, T and Kysar, DA and Lwoff, L and Mills, P and Phalkey, J and Porteus, MH and Snead, OC and Sunder Rajan, K and Wolinetz, CD},
title = {A Reset for Bioethics: A Statement from the Global Observatory for Genome Editing.},
journal = {The CRISPR journal},
volume = {8},
number = {4},
pages = {232-238},
doi = {10.1089/crispr.2025.0058},
pmid = {40397093},
issn = {2573-1602},
mesh = {*Gene Editing/ethics ; Humans ; *Bioethics/trends ; CRISPR-Cas Systems ; },
abstract = {How should we govern our increasing power to intervene in the processes of life? Genome editing, especially of the human germline, has brought this question to the forefront of global debate. We must seek to rectify shortcomings of earlier deliberative approaches by setting aside a science-and-technology first approach; expanding the range of questions for deliberation; revisiting the distribution of innovation's benefits and risks; and reimagining the limits of research. This Perspective from the Organizing Committee of the 2025 Global Observatory for Genome Editing International Summit calls for a new social compact, recognizing and rendering accountable the constitutive role of science and technology in shaping the meaning of human life in the 21st century.},
}
@article {pmid40397087,
year = {2025},
author = {Newman, SA},
title = {Opposing Human Genetic Engineering.},
journal = {The CRISPR journal},
volume = {8},
number = {4},
pages = {252-256},
doi = {10.1089/crispr.2025.0043},
pmid = {40397087},
issn = {2573-1602},
mesh = {Humans ; *Genetic Engineering/ethics/methods ; *Gene Editing/ethics ; CRISPR-Cas Systems ; Germ Cells ; },
abstract = {The past five decades have been a time of substantial change in the technological capacity to modify genetic material. During this period, I have maintained an unwavering stance against human germline modification. As a biologist who has researched the complexities of genotype-phenotype relationships, I remain convinced embryo-stage human genetic modification will always remain in the realm of uncontrolled experimentation. Based on my observations and participation in the twists and turns of genetics and society, I point to the limits of calls for "broad societal consensus."},
}
@article {pmid40397084,
year = {2025},
author = {Baylis, F},
title = {Summitting CRISPR for Human Heritable Genome Editing.},
journal = {The CRISPR journal},
volume = {8},
number = {4},
pages = {239-244},
doi = {10.1089/crispr.2025.0051},
pmid = {40397084},
issn = {2573-1602},
mesh = {Humans ; *Gene Editing/ethics/methods ; *CRISPR-Cas Systems/genetics ; *Genome, Human ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; },
abstract = {The ethical issues of human heritable genome editing have been discussed at international summits held since 2015. In this Perspective, I consider how the discussions evolved over three summits held in Washington, DC (2015), Hong Kong (2018), and London (2023). The significance of safety and efficacy, meanings of a moratorium, and place of broad societal consensus are traced through publications produced surrounding these summits. Looking ahead, I highlight the difference between two fundamentally distinct ethical questions: Is human heritable genome editing ethical? Can human heritable genome editing be done ethically?},
}
@article {pmid39428567,
year = {2025},
author = {Liu, Y and Yang, X and Wu, P and Guo, X and Liu, Z and Huang, Y and Xu, X},
title = {Multi-targets cleavage of BmNPV genome through genome-wide repeat sequence using CRISPR/Cas9 antiviral system.},
journal = {Insect science},
volume = {32},
number = {4},
pages = {1174-1184},
doi = {10.1111/1744-7917.13462},
pmid = {39428567},
issn = {1744-7917},
support = {32100381//National Natural Science Foundation of China/ ; 31830093//National Natural Science Foundation of China/ ; 32021001//National Natural Science Foundation Innovation Group Project/ ; },
mesh = {Animals ; *CRISPR-Cas Systems ; *Bombyx/virology/genetics ; *Nucleopolyhedroviruses/genetics/physiology ; *Genome, Viral ; Gene Editing ; Animals, Genetically Modified ; },
abstract = {The escalating severity of Bombyx mori nuclear polyhedrosis virus (BmNPV) infections poses significant challenges to the silkworm industry, especially when massive production shifts occur from the eastern regions to western regions with lower labor costs. Education and experience levels are different and disease control is badly needed. To solve the problems, we have developed an innovative CRISPR/Cas9 system specifically targeting BmNPV to enhance viral resistance. For the system, we selected BmNPV genes linked to virus replication and proliferation as targets, designing 2 sites for each gene. Mutating the target sequence renders the system incapable of efficiently cleaving the virus genome, hence decreasing cleavage efficiency. We conducted a search for "NGG" or "CCN" target sequences in the BmNPV genome, excluding non-recurring and potential targets in the B. mori genome. We successfully identified 2 distinct target sequences in the BmNPV genome-one being repeated 12 times and the other three times. These sequences lead to fragmentation of virus genome into multiple large segments that are difficult to repair. Transgenic silkworms demonstrate robust resistance to viruses, significantly boosting their survival rates compared with wild-type silkworms under various virus infection concentrations. Our system efficiently targets dozens of viral genomes with just 2 sequences, minimizing transposable elements while ensuring cutting effectiveness. This marks a pioneering advancement by using repetitive elements within the virus genome for targeted CRISPR cleavage, aiming for antiviral effects through genome fragmentation rather than disrupting essential viral genes. Our research introduces innovative concepts to CRISPR antiviral investigations and shows promise for the practical application of gene editing in industrial silkworm strains.},
}
@article {pmid39300921,
year = {2025},
author = {Liu, ZL and Zhou, YY and Xu, QX and Wang, XC and Liu, TX and Tian, HG},
title = {Efficient CRISPR-mediated genome editing can be initiated by embryonic injection but not by ovarian delivery in the beetle Tribolium castaneum.},
journal = {Insect science},
volume = {32},
number = {4},
pages = {1185-1200},
doi = {10.1111/1744-7917.13447},
pmid = {39300921},
issn = {1744-7917},
support = {2017YFD0200900//National Key Research and Development Program of China/ ; 31101432//National Natural Science Foundation of China/ ; },
mesh = {Animals ; *Tribolium/genetics/embryology ; *Gene Editing/methods ; Female ; *CRISPR-Cas Systems ; Ovary ; Embryo, Nonmammalian ; Microinjections ; RNA, Guide, CRISPR-Cas Systems ; },
abstract = {The clustered regularly interspaced small palindromic repeats (CRISPR) / CRISPR-associated nuclease 9 (Cas9)-mediated gene editing technology has revolutionized the study of fundamental biological questions in various insects. Diverse approaches have been developed to deliver the single-guide RNA (sgRNA) and Cas9 to the nucleus of insect embryos or oocytes to achieve gene editing, including the predominant embryonic injection methods and alternative protocols through parental ovary delivery. However, a systematic comparative study of these approaches is limited, especially within a given insect. Here, we focused on revealing the detailed differences in CRISPR/Cas9-mediated gene editing between the embryo and ovary delivery methods in the beetle Tribolium castaneum, using the cardinal and tyrosine hydroxylase (TH) as reporter genes. We demonstrated that both genes could be efficiently edited by delivering Cas9/sgRNA ribonucleoproteins to the embryos by microinjection, leading to the mutant phenotypes and indels in the target gene sites. Next, the Cas9/sgRNA complex, coupled with a nanocarrier called Branched Amphiphilic Peptide Capsules (BAPC), were delivered to the ovaries of parental females to examine the efficacy of BAPC-mediated gene editing. Although we observed that a small number of beetles' progeny targeting the cardinal exhibited the expected white-eye phenotype, unexpectedly, no target DNA indels were found following subsequent sequencing analysis. In addition, we adopted a novel approach termed "direct parental" CRISPR (DIPA-CRISPR). However, we still failed to find gene-editing events in the cardinal or TH gene-targeted insects. Our results indicate that the conventional embryonic injection of CRISPR is an effective method to initiate genome editing in T. castaneum. However, it is inefficient by the parental ovary delivery approach.},
}
@article {pmid40838063,
year = {2023},
author = {Maddalena, A and Kleinlogel, S},
title = {CRISPR-mediated optogene expression from a cell-specific endogenous promoter in retinal ON-bipolar cells to restore vision.},
journal = {Frontiers in drug delivery},
volume = {3},
number = {},
pages = {934394},
pmid = {40838063},
issn = {2674-0850},
abstract = {Retinitis pigmentosa, an inherited form of retinal degeneration, is characterized by a progressive loss of rods and subsequent degeneration of cones, leading to blindness. However, the remaining neural portion of the retina (bipolar and ganglion cells) remains anatomically and functionally intact for an extended time. A possible treatment to restore the light sensitivity of the retina consists of rendering the remaining retinal cells photosensitive using optogenetic tools like, for example, Opto-mGluR6, a light-sensitive mGluR6 receptor. We have previously demonstrated that AAV vector-mediated expression of Opto-mGluR6 in ON-bipolar cells restores visual function in otherwise blind mice. However, classical gene supplementation therapy still suffers from high off-target expression rates and uncontrollable target gene expression levels that may lead to either cytotoxicity or lack of functional restoration. To address these issues and achieve cell-specific and endogenously controlled Opto-mGluR6 expression, we employed the CRISPR/Cas technology-in particular, homology-independent targeted integration (HITI) and microhomology-dependent targeted integration (MITI)-to knock-in the Opto-mGluR6 gene behind the ON-bipolar cell-specific GRM6 promoter. We compared four Cas systems in vitro and show that SpCas9 for HITI and LbCpf1 for MITI are well suited to promoting knock-in. As AAV2-mediated ON-bipolar cell transduction resulted in inefficiency, we evaluated Exo-AAVs as delivery vehicles and found Exo-AAV1 efficient for targeting ON-bipolar cells. We demonstrate that intravitreal injection of Exo-AAV1 carrying vectors that promote MITI significantly improved visual acuity in otherwise blind rd1 mice. We conclude by confirming and providing a qualitative evaluation of the MITI-mediated knock-in in the correct genomic locus.},
}
@article {pmid40835744,
year = {2025},
author = {Sinaga, DS and Huang, PY and Huang, CK and Lu, CA and Huang, LF},
title = {A single donor cassette enables site-specific knock-in at either the αAmy3 or αAmy8 locus in rice cells via CRISPR/Cas9.},
journal = {Applied microbiology and biotechnology},
volume = {109},
number = {1},
pages = {190},
pmid = {40835744},
issn = {1432-0614},
support = {112-2313-B-008-002-MY3//the National Science and Technology Council of the Republic of China/ ; 112-2313-B-008-002-MY3//the National Science and Technology Council of the Republic of China/ ; 111-2313-B-155-001-MY3//the National Science and Technology Council of the Republic of China/ ; },
mesh = {*Oryza/genetics ; *CRISPR-Cas Systems ; Gene Editing/methods ; *Gene Knock-In Techniques/methods ; Promoter Regions, Genetic ; Introns ; },
abstract = {CRISPR/Cas9 gene editing is widely used to manipulate gene expression and integrate transgenes into specific target sites, making it a powerful tool for recombinant protein expression. In this study, we generated a single donor cassette for CRISPR/Cas9-mediated knock-in at either the αAmy3 or αAmy8 locus in rice cells. The transgene was inserted downstream of the promoter and first exon of the rice αAmy3 or αAmy8 genes, which are highly expressed under sugar-starved conditions in rice suspension cultures. We constructed a simple vector with the homologous intron sequences of the αAmy3 and αAmy8, along with rice codon-optimized recombinant receptor binding domain (rcRBD) of the SARS-CoV-2 spike protein, a functional domain responsible for binding to the angiotensin-converting enzyme 2 (ACE2) receptor on host cells. Using this construct, rcRBD was successfully integrated into the intron 1 of either the αAmy3 or αAmy8 genes. As a result, rcRBD expression was driven by endogenous αAmy3 or αAmy8 promoter-signal peptide. Under the control of αAmy3-signal peptide, rcRBD proteins was detected in both the soluble cellular protein fraction and culture medium, whereas expression driven by the αAmy8 promoter-signal peptide was exclusively detected in the culture medium of rice suspension cells. The highest secreted protein yield of rcRBD in the rice culture medium under the control of αAmy8 endogenous promoter reached 20.7 mg/L, demonstrating a production efficiency comparable to that driven by the endogenous αAmy3 promoter.},
}
@article {pmid40835660,
year = {2025},
author = {Dong, T and Zhao, Y and Zhang, M and Lang, WY and Liu, DY and Zhang, KS and Wang, YJ and Li, L and Lian, J and Yao, HB and Zhang, HY and Jin, HF and Lu, T and Shen, L and Yue, LL and Lin, Y},
title = {SNTA1-deficient human cardiomyocytes show shorter field potential duration and slower conduction velocity.},
journal = {Scientific reports},
volume = {15},
number = {1},
pages = {30600},
pmid = {40835660},
issn = {2045-2322},
support = {2022-KYYWF-0808//Basic scientific research expenses of colleges and universities in Heilongjiang Province/ ; },
mesh = {Humans ; *Myocytes, Cardiac/metabolism/physiology ; NAV1.5 Voltage-Gated Sodium Channel/metabolism/genetics ; *Calcium-Binding Proteins/genetics/deficiency ; Arrhythmias, Cardiac/genetics ; CRISPR-Cas Systems ; Action Potentials ; Gene Knockout Techniques ; Cell Differentiation ; Cell Line ; *Membrane Proteins/genetics/deficiency ; *Muscle Proteins/genetics/deficiency ; },
abstract = {In clinical settings, patients with α-1-syntrophin point mutations are often associated with rare arrhythmias, including Long QT syndrome, Brugada syndrome, and sudden infant death syndrome. Previous studies on α-1-syntrophin have predominantly utilized nonhuman cardiomyocyte models. This study aims to elucidate the phenotype of α-1-syntrophin deficiency using human cardiomyocytes. Using CRISPR/Cas9 technology, we generated SNTA1 knockout (KO) embryonic stem cell line, which were subsequently differentiated into cardiomyocytes using 2D differentiation method. Genotype analysis identified an adenine (A) insertion in the second exon of SNTA1, resulting in a premature stop codon at the 149th amino acid position and truncation within the PDZ domain. SNTA1-deficient cardiomyocytes exhibited a shortened field potential duration (FPD) and slower conduction velocity, as detected by micro electrode array analysis. Immunofluorescence analysis further revealed disorganized distribution of Nav1.5 in SNTA1-deficient cardiomyocytes. SNTA1 is a susceptibility locus for arrhythmias and plays a critical role as an essential auxiliary protein in the proper localization of Nav1.5 in human cardiomyocytes.},
}
@article {pmid40833724,
year = {2025},
author = {Bhattacharjee, G and Gohil, N and Khambhati, K and Murjani, K and Chu, DT and Le Bui, N and Thi, HV and Mani, I and Bansal, A and Shamili, S and Satish, L and Ramakrishna, S and Alzahrani, KJ and Singh, V},
title = {Advancements in CRISPR-Cas Systems for Genome Editing towards Eradication of Human Microbial Pathogens.},
journal = {Molecular biotechnology},
volume = {},
number = {},
pages = {},
pmid = {40833724},
issn = {1559-0305},
support = {TU-DSPP-2024-05//Taif University/ ; 2021M3A9H3015390//National Science foundation of Korea/ ; BT/PR38411/GET/119/311/2020//Department of Biotechnology/ ; },
abstract = {CRISPR-Cas systems have been explored for targeted genome editing of several organisms. It is rapid, cost-effective, specific, and versatile technology. It requires expression of multidomain single Cas9 protein and single guide RNA (sgRNA) that targets desired nucleic acids in the presence of a protospacer adjacent motif (PAM). This generates a double-stranded break that is repaired by either non-homologous end joining or a homology-directed repair pathway. Currently, several Cas protein variants have been discovered and being used for several biotechnological applications. This review highlights the recent progress of CRISPR-Cas systems for genome editing of mainly human pathogenic microorganisms for their controlling infections.},
}
@article {pmid40833532,
year = {2025},
author = {Tang, K and Wu, L and Hu, Y and Xue, T and Jin, Y and Zhou, X and Luo, C and Zhao, Y and Tong, L and Dai, J and Feng, D and Zeng, Z and Pan, D},
title = {Perturbation of calreticulin potentiates CD8+ T cell-mediated antitumor immunity.},
journal = {The Journal of experimental medicine},
volume = {222},
number = {10},
pages = {},
doi = {10.1084/jem.20242360},
pmid = {40833532},
issn = {1540-9538},
support = {82341026//National Natural Science Foundation of China/ ; 82073163//National Natural Science Foundation of China/ ; 2022YFC2505400//National Key Research and Development Program of China/ ; //Tsinghua University Initiative Scientific Research Program/ ; //Tsinghua-Peking University Center of Life Science/ ; //Tsinghua-Peking Joint Centre for Life Sciences/ ; },
mesh = {*Calreticulin/metabolism/immunology/genetics ; Animals ; *CD8-Positive T-Lymphocytes/immunology ; Humans ; Mice ; Histocompatibility Antigens Class I/immunology/metabolism ; Cell Line, Tumor ; Mice, Inbred C57BL ; *Neoplasms/immunology ; CRISPR-Cas Systems ; Immunotherapy ; *Immunity, Cellular ; },
abstract = {Effective immunotherapy relies on the presentation of tumor-derived neoantigens on the major histocompatibility complex class I (MHC-I) to activate CD8+ T cells. Deficiencies in this process are a key mechanism of immune evasion and resistance to checkpoint blockade. In this study, using an in vivo CRISPR-Cas9 screen, we unexpectedly found that inactivation of calreticulin (CALR), and other selected components of the peptide-loading complex (PLC), induced robust CD8+ T cell-mediated immune responses. We show that this effect is dependent on the expression of classical MHC-I on tumor cells. Mechanistically, loss of CALR reshaped the MHC-I peptide repertoire, favoring the presentation of low-affinity peptides in murine and human cell lines. Genetic or pharmacological inhibition of PDIA3, another PLC component, similarly induced antitumor effects. These findings reveal a previously unrecognized role of CALR and the PLC in regulating antitumor immunity and suggest that targeting this pathway could be a promising strategy to overcome immune resistance and improve the efficacy of cancer immunotherapies.},
}
@article {pmid40831701,
year = {2025},
author = {Liu, W and Jiang, Y and Wang, C and Wang, M and Zhang, W and Ren, H and Xu, S and Qin, J and Liu, P and Jin, L and Zhao, D},
title = {Towards the elimination of infectious HPV: exploiting CRISPR/Cas innovations.},
journal = {Frontiers in cellular and infection microbiology},
volume = {15},
number = {},
pages = {1627668},
pmid = {40831701},
issn = {2235-2988},
mesh = {Humans ; *CRISPR-Cas Systems ; *Papillomavirus Infections/therapy/prevention &amp; control/virology ; *Papillomaviridae/genetics ; *Gene Editing/methods ; *Genetic Therapy/methods ; Genome, Viral ; },
abstract = {HPV has been conclusively associated with various human malignancies, making the development of prevention and treatment strategies for HPV-induced diseases a high priority. Currently, primary prevention methods include HPV immunization and routine screening, which significantly reduce the risk of HPV transmission. However, for patients diagnosed with invasive, advanced, or recurrent malignancies, non-virus-specific therapies frequently lead to drug resistance and adverse effects, resulting in minimal improvement in treatment efficacy for numerous patients. Viral genome-targeting therapy is emerging as a promising avenue for the future management of HPV infections. With the rapid advancement of genetic modification technologies, the CRISPR/Cas system has demonstrated significant potential in treating viral infections. Its ability to selectively target and edit viral genomes for elimination positions it as a highly effective approach for combating HPV. This review will explore the functions and applications of the CRISPR/Cas system as an innovative therapy for HPV. We will illustrate the prospective efficacy of CRISPR/Cas as a groundbreaking and promising cure for HPV infections, while also addressing the opportunities and challenges associated with this novel approach.},
}
@article {pmid40831643,
year = {2025},
author = {Meza, C and Sepulveda, B and Flores-Castañón, N and Valenzuela, F and Ormeño, C and Castillo, A and Echeverría-Vega, A and Jasem Mohammed Breig, S and Alardhi, SM and Gonzalez, A and Mora-Lagos, B and Banerjee, A},
title = {Genomic basis and functional characterization of the exopolysaccharide production by a thermotolerant Bacillus isolated from Tolhuaca hot spring.},
journal = {Frontiers in microbiology},
volume = {16},
number = {},
pages = {1622325},
pmid = {40831643},
issn = {1664-302X},
abstract = {Bacillus licheniformis Tol1, a thermotolerant bacterial strain isolated from the Tolhuaca hot spring in Chile, was investigated for its genomic features and the functional properties of its exopolysaccharide (EPS). The whole-genome sequencing revealed ∼4.25 Mbp genome with a GC content of 45.9% and a rich repertoire of genes associated with environmental stress adaptation, antibiotic resistance, sporulation, biofilm formation, and EPS biosynthesis, including the presence of epsD and epsC. The strain also harbored intact prophage elements and a Type I-A CRISPR-Cas system, indicating potential horizontal gene transfer and genome plasticity. Confocal microscopy revealed robust biofilm formation at 45-55°C under neutral to slightly alkaline pH, with strong EPS matrix development. EPS production was optimized using OFAT and Response Surface Methodology (RSM), achieving a yield of 2.11 g L[-1] under optimized conditions, which was further validated using an Artificial Neural Network (ANN) model (R [2] = 0.9909). The EPS exhibited promising antioxidant activity and significant emulsification potential across various vegetable oils, which were comparable or superior to commercial bacterial EPS xanthan gum. Notably, the EPS also showed cytotoxic effects against AGS gastric adenocarcinoma cells, reducing viability by 38.38 and 37% at 50-100 μg μL[-1] concentrations, respectively, suggesting potential anticancer activity. Altogether, the study highlights B. licheniformis Tol1 as a multifunctional thermophile with valuable biotechnological potential, particularly for applications in food, pharmaceutical, and biomedical industries.},
}
@article {pmid40830872,
year = {2025},
author = {Saffari Natanzi, A and Poudineh, M and Karimi, E and Khaledi, A and Haddad Kashani, H},
title = {Innovative approaches to combat antibiotic resistance: integrating CRISPR/Cas9 and nanoparticles against biofilm-driven infections.},
journal = {BMC medicine},
volume = {23},
number = {1},
pages = {486},
pmid = {40830872},
issn = {1741-7015},
mesh = {*Biofilms/drug effects ; *CRISPR-Cas Systems ; Humans ; *Nanoparticles ; *Anti-Bacterial Agents/administration &amp; dosage/pharmacology ; Gene Editing/methods ; *Drug Resistance, Bacterial/genetics ; *Bacterial Infections/drug therapy ; Pseudomonas aeruginosa/drug effects ; },
abstract = {The increasing prevalence of antibiotic-resistant bacterial infections is a major global health concern, with biofilms playing a key role in bacterial persistence and resistance. Biofilms provide a protective matrix that limits antibiotic penetration, enhances horizontal gene transfer, and enables bacterial survival in hostile environments. Conventional antimicrobial therapies are often ineffective against biofilm-associated infections, necessitating the development of novel therapeutic strategies. The CRISPR/Cas9 gene-editing system has emerged as a revolutionary tool for precision genome modification, offering targeted disruption of antibiotic resistance genes, quorum sensing pathways, and biofilm-regulating factors. However, the clinical application of CRISPR-based antibacterials faces significant challenges, particularly in efficient delivery and stability within bacterial populations. Nanoparticles (NPs) present an innovative solution, serving as effective carriers for CRISPR/Cas9 components while exhibiting intrinsic antibacterial properties. Nanoparticles can enhance CRISPR delivery by improving cellular uptake, increasing target specificity, and ensuring controlled release within biofilm environments. Recent advances have demonstrated that liposomal CRISPR-Cas9 formulations can reduce Pseudomonas aeruginosa biofilm biomass by over 90% in vitro, while gold nanoparticle carriers enhance editing efficiency up to 3.5-fold compared to non-carrier systems. These hybrid platforms also enable co-delivery with antibiotics, producing synergistic antibacterial effects and superior biofilm disruption. Additionally, they can facilitate co-delivery of antibiotics or antimicrobial peptides, further enhancing therapeutic efficacy. This review explores the synergistic integration of CRISPR/Cas9 and nanoparticles in combating biofilm-associated antibiotic resistance. We discuss the mechanisms of action, recent advancements, and current challenges in translating this approach into clinical practice. While CRISPR-nanoparticle hybrid systems hold immense potential for next-generation precision antimicrobial therapies, further research is required to optimize delivery platforms, minimize off-target effects, and assess long-term safety. Understanding and overcoming these challenges will be critical for developing effective biofilm-targeted antibacterial strategies.},
}
@article {pmid40829801,
year = {2025},
author = {Mohseni, A and Nia, RG and Tafrishi, A and López, ML and Liu, XZ and Stajich, JE and Lonardi, S and Wheeldon, I},
title = {Kingdom-wide CRISPR guide design with ALLEGRO.},
journal = {Nucleic acids research},
volume = {53},
number = {15},
pages = {},
doi = {10.1093/nar/gkaf783},
pmid = {40829801},
issn = {1362-4962},
support = {NSF-2225878//Chemical, Bioengineering, Environmental and Transport Systems/ ; DBI-2400327//National Science Foundation/ ; EF-2125066//National Science Foundation/ ; CA-R-PPA-211-5062-H//U.S. Department of Agriculture, National Institute of Food and Agriculture/ ; },
mesh = {*RNA, Guide, CRISPR-Cas Systems/genetics ; *CRISPR-Cas Systems ; Genome, Fungal ; *Gene Editing/methods ; *Algorithms ; Kluyveromyces/genetics ; Saccharomyces cerevisiae/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Yarrowia/genetics ; Rhodotorula/genetics ; Gene Library ; Saccharomycetales ; },
abstract = {Designing CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) single guide RNA (sgRNA) libraries targeting entire kingdoms of life will significantly advance genetic research in diverse and underexplored taxa. Current sgRNA design tools are often species-specific and fail to scale to large, phylogenetically diverse datasets, limiting their applicability to comparative genomics, evolutionary studies, and biotechnology. Here, we introduce ALLEGRO, a combinatorial optimization algorithm designed to compose minimal, yet highly effective sgRNA libraries targeting thousands of species at the same time. Leveraging integer linear programming, ALLEGRO identified compact sgRNA sets simultaneously targeting multiple genes of interest for over 2000 species across the fungal kingdom. We experimentally validated sgRNAs designed by ALLEGRO in Kluyveromyces marxianus, Komagataella phaffii, Yarrowia lipolytica, and Saccharomyces cerevisiae, confirming successful genome edits. Additionally, we employed a generalized Cas9-ribonucleoprotein delivery system to apply ALLEGRO's sgRNA libraries to untested fungal genomes, such as Rhodotorula araucariae. Our experimental findings, together with cross-validation, demonstrate that ALLEGRO facilitates efficient CRISPR genome editing, enabling the development of universal sgRNA libraries applicable to entire taxonomic groups.},
}
@article {pmid40792428,
year = {2025},
author = {Zhou, T and Yu, D and Wu, L and Tan, Y and Li, X and Sun, Z and Sun, X and Chen, J and Duan, M and Yuan, D},
title = {Improvement of Rice Seed Storability by Regulating Lipid Metabolism Using a CRISPR/Cas9 System.},
journal = {Journal of agricultural and food chemistry},
volume = {73},
number = {33},
pages = {20972-20983},
doi = {10.1021/acs.jafc.5c06276},
pmid = {40792428},
issn = {1520-5118},
mesh = {*Oryza/genetics/metabolism/chemistry ; *Seeds/metabolism/genetics/chemistry ; *Lipid Metabolism ; CRISPR-Cas Systems ; *Plant Proteins/genetics/metabolism ; Reactive Oxygen Species/metabolism ; Food Storage ; Plants, Genetically Modified/metabolism/genetics ; Fatty Acids/metabolism ; Gene Expression Regulation, Plant ; },
abstract = {Long-term storage of rice grain is critical for global food security, yet rice is inherently susceptible to deterioration during storage. Herein, rice seed storability was improved by targeting three key enzyme genes in the lipid metabolism pathway via CRISPR/Cas9 technology, and the mechanism underlying this was analyzed by an untargeted lipidomic approach. Our findings demonstrate that the significantly inferior seed storability in the Yu-Zhen-Xiang (YZX) cultivar compared with the Xi-Li-Gong-Mi (XLGM) cultivar arises from accelerated lipid catabolism and reactive oxygen species (ROS) overproduction. Moreover, a fad2-1/lox3/pldα1 triple mutant in the YZX background was rapidly generated by FMPKC systems, and the mutant exhibited lower fatty acid accumulation and reduced ROS content, along with improved grain quality and nutritional value after accelerated aging. Lipidomic analysis indicated that diminished lipid hydrolysis and peroxidation collectively accounted for enhanced storability of the flp mutant. Collectively, this study establishes a robust strategy for rapidly and significantly improving rice aging tolerance, with potential applicability to other cereal crops for addressing critical challenges of grain storage.},
}
@article {pmid40780629,
year = {2025},
author = {Du, X and Quinn, A and Menzies, M and Porto-Neto, LR},
title = {Optimizing the delivery of CRISPR/Cas9 ribonucleoproteins for efficient genome editing in bovine embryos.},
journal = {Gene},
volume = {966},
number = {},
pages = {149715},
doi = {10.1016/j.gene.2025.149715},
pmid = {40780629},
issn = {1879-0038},
mesh = {Animals ; *Gene Editing/methods ; Cattle ; *CRISPR-Cas Systems/genetics ; *Ribonucleoproteins/genetics ; Electroporation/methods ; Blastocyst/metabolism ; Receptors, Prolactin/genetics ; Female ; Transfection/methods ; RNA, Guide, CRISPR-Cas Systems/genetics ; },
abstract = {To assist in establishing a streamlined and efficient workflow for generating gene-edited bovine embryos, we evaluated three transfection approaches for the delivery of CRISPR Cas9-sgRNA ribonucleoproteins into bovine zygotes: lipofection with Lipofectamine CRISPRMAX, and electroporation using either Neon or NEPA21 electroporation systems. Bos taurus prolactin receptor (PRLR) was used as the target gene. The PRLR editing outcomes were analysed by PCR genotyping and Sanger sequencing of individual embryos at day 8 post-fertilization. CRISPRMAX transfection generated up to 30 % PRLR-edited blastocysts (8 % homozygous deletion), without affecting the embryo cleavage (93 %) and blastocyst rate (39 %) relative to non-transfected controls. For both NEPA21 and Neon electroporation, we found that increasing the voltage, length and number of pulses resulted in enhanced gene editing efficiency but compromised embryo cleavage and blastocyst rates. NEPA21 electroporation with a commercial electroporation enhancer reagent produced up to 47.6 % transfected embryos with the PRLR deletion, but with decreased embryo cleavage (62 %) and blastocyst (18 %) rates. Combining NEPA21 electroporation with CRISPRMAX lipofection enhanced the gene editing efficiency to 50 % (23 % homozygous editing), with 64 % embryo cleavage rate and 18 % blastocyst rate. Notably, Neon electroporation resulted in 65.2 % PRLR-edited blastocysts with 21 % homozygous editing (50% cleavage rate and 10 % blastocyst rate). Additional studies may be necessary to further optimize electroporation parameters to achieve an optimal balance between embryo viability and gene editing efficiency. These outcomes will provide valuable insights for improving gene editing workflows for bovines and may help to promote and accelerate the widespread implementation of genome editing technology in livestock.},
}
@article {pmid40538131,
year = {2025},
author = {Li, Z and Khan, WU and Bai, G and Dong, C and Wang, J and Zhang, Y and Wang, C and Zhang, H and Wang, W and Luo, M and Chen, F},
title = {From Code to Life: The AI-Driven Revolution in Genome Editing.},
journal = {Advanced science (Weinheim, Baden-Wurttemberg, Germany)},
volume = {12},
number = {30},
pages = {e17029},
doi = {10.1002/advs.202417029},
pmid = {40538131},
issn = {2198-3844},
support = {32172614//National Natural Science Foundation of China/ ; ZDYF2023XDNY050//Hainan Province Science and Technology Special Fund/ ; 324RC452//Hainan Provincial Natural Science Foundation of China/ ; 325QN234//Hainan Provincial Natural Science Foundation of China/ ; NKLTCB202337//Project of National Key Laboratory for Tropical Crop Breeding/ ; Y2021094//South China Botanical Garden, Chinese Academy of Sciences/ ; 2022B1515020026//Guangdong Natural Science Funds for Distinguished Young Scholars/ ; Y2021094//Youth Innovation Promotion Association, Chinese Academy of Sciences/ ; },
mesh = {*Gene Editing/methods/trends ; *Artificial Intelligence/trends ; Humans ; CRISPR-Cas Systems/genetics ; },
abstract = {Genome editing has revolutionized modern biotechnology, enabling precise modifications to DNA sequences with far-reaching applications in medicine, agriculture, and synthetic biology. Recent advancements in artificial intelligence (AI) have significantly enhanced genome editing by improving target selection, minimizing off-target effects, and optimizing CRISPR-associated systems. AI-driven models, such as deep learning-based predictors and protein language models, enable more accurate sgRNA design, novel Cas protein discovery, and enhanced gene regulatory network analysis. Additionally, AI-powered tools facilitate large-scale data integration, accelerating functional genomics and therapeutic genome editing. This review explores the intersection of AI and genome editing, highlighting key innovations, challenges, and future prospects. Despite its transformative potential, AI-driven genome editing raises ethical concerns regarding data bias, algorithmic transparency, and unintended genetic modifications. Addressing these challenges requires interdisciplinary collaboration between AI researchers, molecular biologists, and policymakers. As AI continues to evolve, its integration with genome editing will pave the way for groundbreaking advancements in precision medicine, genetic disease treatment, and sustainable agriculture.},
}
@article {pmid40369100,
year = {2025},
author = {Correia, L and Shalygin, A and Erbacher, A and Zaisserer, J and Gudermann, T and Chubanov, V},
title = {TRPM7 underlies cadmium cytotoxicity in pulmonary cells.},
journal = {Archives of toxicology},
volume = {99},
number = {8},
pages = {3269-3281},
pmid = {40369100},
issn = {1432-0738},
support = {TRR 152 (P15)//Deutsche Forschungsgemeinschaft (DFG)/ ; Research Training Group 2338 (P10)//Deutsche Forschungsgemeinschaft (DFG)/ ; },
mesh = {*TRPM Cation Channels/genetics/metabolism/antagonists &amp; inhibitors ; Humans ; Animals ; A549 Cells ; Mice ; *Cadmium/toxicity ; Cell Survival/drug effects ; *Lung/drug effects/cytology/metabolism ; *Protein Serine-Threonine Kinases/genetics/metabolism ; Mice, Knockout ; CRISPR-Cas Systems ; Gene Knockout Techniques ; },
abstract = {TRPM7 is a kinase-coupled ion channel that exhibits high activity in the immune and epithelial cells of different organs, including the lung. Electrophysiological studies have established that the TRPM7 channel displays high permeability to Mg[2+], Zn[2+], and Ca[2+], as well as trace metal cations. While the critical role of TRPM7 in the cellular balance of Mg[2+], Zn[2+], and Ca[2+] is well-documented, its contribution to the cellular uptake of trace metal cations, frequent respiratory pollutants, remains unclear. Here, we performed an electrophysiological assessment of pulmonary A549 cells revealing endogenous TRPM7 currents, which were eliminated by knockout (KO) of the TRPM7 gene using the CRISPR/Cas9 approach or by administration of NS8593 and VER155008, two structurally unrelated inhibitors of the TRPM7 channel. Unlike prior studies with various cell lines showing that TRPM7 KO mutation induces cell growth arrest, we observed that A549 cells maintained normal viability after genetic and pharmacological inactivation of TRPM7. Consequently, we used A549 cells to examine the impact of Cd[2+] on cell viability and found that TRPM7 KO mutation and both pharmacological agents mitigated the Cd[2+] cytotoxicity. Analogous to A549 cells, electrophysiological analysis of mouse primary alveolar type 2 (ATII) cells revealed endogenous TRPM7 currents and Cd[2+] exposure reduced the cell viability of ATII cells in a TRPM7-dependent fashion. Hence, the TRPM7 channel contributes to Cd[2+] cytotoxicity in pulmonary cells and can serve as a therapeutic target to alleviate the toxic effects of trace metal exposure.},
}
@article {pmid37407027,
year = {2025},
author = {Cutter, AD},
title = {Guerrilla eugenics: gene drives in heritable human genome editing.},
journal = {Journal of medical ethics},
volume = {51},
number = {9},
pages = {627-635},
doi = {10.1136/jme-2023-109061},
pmid = {37407027},
issn = {1473-4257},
mesh = {Humans ; *Gene Editing/ethics ; *Eugenics ; *CRISPR-Cas Systems ; *Genome, Human ; *Gene Drive Technology/ethics ; },
abstract = {CRISPR-Cas9 genome editing can and has altered human genomes, bringing bioethical debates about this capability to the forefront of philosophical and policy considerations. Here, I consider the underexplored implications of CRISPR-Cas9 gene drives for heritable human genome editing. Modification gene drives applied to heritable human genome editing would introduce a novel form of involuntary eugenic practice that I term guerrilla eugenics. Once introduced into a genome, stealth genetic editing by a gene drive genetic element would occur each subsequent generation irrespective of whether reproductive partners consent to it and irrespective of whether the genetic change confers any benefit. By overriding the ability to 'opt in' to genome editing, gene drives compromise the autonomy of carrier individuals and their reproductive partners to choose to use or avoid genome editing and impose additional burdens on those who hope to 'opt out' of further genome editing. High incidence of an initially rare gene drive in small human communities could occur within 200 years, with evolutionary fixation globally in a timeframe that is thousands of times sooner than achievable by non-drive germline editing. Following any introduction of heritable gene drives into human genomes, practices intended for surveillance or reversal also create fundamental ethical problems. Current policy guidelines do not comment explicitly on gene drives in humans. These considerations motivate an explicit moratorium as being warranted on gene drive development in heritable human genome editing.},
}
@article {pmid40829593,
year = {2025},
author = {Margolis, SR and Meeske, AJ},
title = {Crosstalk between three CRISPR-Cas types enables primed type VI-A adaptation in Listeria seeligeri.},
journal = {Cell host &amp; microbe},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.chom.2025.05.020},
pmid = {40829593},
issn = {1934-6069},
abstract = {CRISPR-Cas systems confer adaptive immunity to their prokaryotic hosts through the process of adaptation, where sequences are captured from foreign nucleic acids and integrated as spacers in the CRISPR array, thereby enabling crRNA-guided interference against new threats. While the Cas1-2 integrase is critical for adaptation, it is absent from many CRISPR-Cas loci, rendering the mechanism of spacer acquisition unclear for these systems. In this study, we show that the RNA-targeting type VI-A CRISPR system of Listeria seeligeri acquires spacers from DNA substrates through the action of a promiscuous Cas1-2 integrase encoded by a co-occurring type II-C system, in a transcription-independent manner. We further demonstrate that the type II-C integration complex is strongly stimulated by preexisting spacers in a third CRISPR system (type I-B), which imperfectly match phage targets and prime type VI-A adaptation. Altogether, our results reveal an unprecedented degree of communication among CRISPR-Cas loci encoded by a single organism.},
}
@article {pmid40829592,
year = {2025},
author = {Smith, LM and Fineran, PC},
title = {Type I CRISPR-Cas immunity primes type III spacer acquisition.},
journal = {Cell host &amp; microbe},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.chom.2025.07.021},
pmid = {40829592},
issn = {1934-6069},
abstract = {CRISPR-Cas systems are diverse, with microbes harboring multiple classes and subtypes. Type I DNA-targeting and type III RNA-targeting systems often co-occur, but their interactions remain unclear. Prodigiosinella has three CRISPR-Cas systems (I-E, I-F, and III-A) with independent adaptation machinery. Type III systems can trigger cell death, yet it is unknown how functional spacers are acquired. We found that type I interference generates substrates acquired by the type III adaptation machinery. Despite reducing type I interference efficiency, type III contributed to plasmid loss and provided an advantage when DNA-targeting systems failed. Type I priming influenced type III spacer length and source, with more spacers acquired near the type I target site. Invader DNA clearance by type I interference enabled retention of cytotoxic type III spacers that would otherwise be lost. This study reveals how RNA-targeting CRISPR-Cas systems function as a backup in multi-system hosts, bolstering population-level protection.},
}
@article {pmid40823824,
year = {2025},
author = {Moreno-Rodríguez, A and Rubio, A and Garzón, A and Smani, Y and Pérez-Pulido, AJ},
title = {The evolutionary replacement of restriction-modification by Ssp antiviral systems is associated with the distribution of prophages in the major clonal group of Acinetobacter baumannii.},
journal = {mBio},
volume = {},
number = {},
pages = {e0213525},
doi = {10.1128/mbio.02135-25},
pmid = {40823824},
issn = {2150-7511},
abstract = {Bacteriophages kill almost half of the world's bacterial cells every day. In response, bacteria have developed anti-phage defense mechanisms that number in the dozens. Individual defense systems are gained and lost by genomes of the same species, depending on their fitness advantage. Thus, the combination of defense systems presented by the individual genomes may differ, and the collection of all of them is what is known as the pan-immune system of the species. Here, we have analyzed thousands of genomes of the bacterium Acinetobacter baumannii, an opportunistic pathogen of humans of great clinical concern, and we have found 81 different defense systems. By analyzing how these systems combine, we have found that more than half of the genomes lack the universal DNA-methylating restriction-modification systems and harbor an alternative innate Ssp system that performs DNA phosphorothioate modification. The presence of one or the other innate system may modulate the evolution of the genomes of this species, causing them to present a different profile of phages integrated into the bacterial genome. In fact, the most widespread strain of this bacterium worldwide, global clone 2, showed an Ssp system as its distinctive feature. We have also observed that the presence of many other defense systems is associated with the presence of a higher number of prophages, which could be because the prophage carries the system, or because the bacterium would not need these systems in environments where the phage is absent.IMPORTANCEAcinetobacter baumannii is a bacterium of great concern in clinical contexts due to the plasticity of its genome and its resistance to antibiotics. Its cells are infected by a multitude of bacteriophages, and the bacterium defends itself with dozens of different defense systems. Here, we have analyzed the complete defensome of thousands of genomes of the species and found that more than half of the genomes do not have universal restriction-modification systems, which are replaced by another innate labeling and restriction system. Furthermore, these genomes belong to the international clone of the bacterium that causes the most concern in hospitals.},
}
@article {pmid40822281,
year = {2025},
author = {Teichmann, B and Melchior, F and Beyreuther, K and Chorianopoulou, MK},
title = {Moral judgment of genetic technologies: validation of the genetic technologies questionnaire in the German-speaking population.},
journal = {Frontiers in genetics},
volume = {16},
number = {},
pages = {1620962},
pmid = {40822281},
issn = {1664-8021},
abstract = {INTRODUCTION: The development of modern life sciences has expanded our biomedical capabilities to an unprecedented degree. For example, genetic testing can be used to predict hereditary predisposition or susceptibility to certain diseases. The development of gene scissors such as CRISPR/Cas makes it possible to repair the disease gene or introduce a protective gene in somatic cells but also in germline cells, leading to permanent changes of the genome. But is everything we "can" do morally justifiable? To what extent does the moral status of the living being, autonomy, and privacy influence the decision of whether something is morally "good" or "bad"? There is a lack of valid instruments to study the moral judgment of genetic technologies. Therefore, the aim of this study is to translate and validate the "Genetic Technologies Questionnaire" (GTQ) and the short version of the "Conventional Technologies Questionnaire" (CTQ5) into German.<br><br>METHODS: Convenience sampling (N = 317) was used to conduct a cross-sectional online study. Analyses included internal consistency, structural validity, known group construct validity, tests for floor and ceiling effects, and retest reliability with a subset of n = 69. Correlational analyses were conducted with education, age, prior knowledge of genetics, religiosity, conventional technologies, and prior genetic testing. This study used the STROBE checklist for reporting.<br><br>RESULTS: The GTQ30 (Cronbach's &#x3b1; = 0.938) and GTQ20 (&#x3b1; = 0.940) are reliable and stable instruments for testing the moral judgment of lay people, while the GTQ5 (&#x3b1; = 0.857) and CTQ5 (&#x3b1; = 0.697) showed some weaknesses. Conventional technologies were judged morally better than genetic technologies, and genetic testing considered better than genome editing. Two additional versions were validated: the GTQ-Human (GTQ-H), using all items relating to humans, and the GTQ-Moral Status (GTQ-MS), including one item per different group of living beings for genetic testing and one for genome editing.<br><br>CONCLUSION: The GTQ is a valid instrument that is now available in shorter versions for different areas of research: the GTQ-MS for philosophical questions addressing moral status and the GTQ-H for biomedical and psychological questions related to research, prognosis, diagnosis, and therapy in humans.},
}
@article {pmid40820704,
year = {2025},
author = {Chen, F and Chen, D and Deng, T and Li, J and Jiang, JH},
title = {CRISPR-Cas-Mediated Ultrasensitive Detection of Viral Nucleic Acids via Singlet Oxygen-Activated Chemiluminescence.},
journal = {Analytical chemistry},
volume = {},
number = {},
pages = {},
doi = {10.1021/acs.analchem.5c03994},
pmid = {40820704},
issn = {1520-6882},
abstract = {Herein, we report a novel clustered regularly interspaced short palindromic repeats (CRISPR)/Cas-mediated chemiluminescence assay (CRISPR-Cas-CLA) for the ultrasensitive detection of viral nucleic acids of HPV18 and HPV16. The CRISPR-Cas-CLA comprises a CRISPR/Cas12a system that specifically recognizes the target nucleic acid, a signal-conducting nanoconjugate (MB-ssDNA-PSNP) formed by coupling singlet-oxygen ([1]O2)-generating photosensitive nanoparticles (PSNPs) to magnetic beads (MBs) via a single-stranded DNA (ssDNA) linker, and [1]O2-activated chemiluminescence nanoparticles (CLNPs). In the presence of the target nucleic acid, the ssDNA linker of the nanoconjugate is cleaved by the target-activated CRISPR/Cas12a system, and the PSNPs are dissociated from the MBs. The PSNP-containing supernatant obtained by magnetic separation is added to the CLNP-coated detection plate. Upon light irradiation of the CLNP-PSNP mixture in the well, strong chemiluminescence is generated with the subsequent addition of hydrogen peroxide, enabling the detection of the target nucleic acids. The proposed CRISPR-Cas-CLA system offers ultrahigh sensitivity (∼1.04 aM), simple operation, and low cost, providing a new direction for the development of PCR-free detection strategies for ultralow abundance nucleic acids.},
}
@article {pmid40819925,
year = {2025},
author = {Zhou, X and Zhou, SJ and Liu, J and Wang, YX},
title = {CRISPR/Cas system targeting RNA and its derivative technology.},
journal = {Yi chuan = Hereditas},
volume = {47},
number = {8},
pages = {842-860},
doi = {10.16288/j.yczz.24-311},
pmid = {40819925},
issn = {0253-9772},
abstract = {RNA editing is one of the important research directions in the field of epigenetics. With further research, scientists have discovered that the CRISPR/Cas system can target not only DNA but also RNA, thereby achieving precise gene editing at the transcriptional level. Moreover, using the CRISPR/Cas system for RNA editing can also avoid damage to genome. At present, a variety of derivative technologies based on RNA-targeting CRISPR systems have been developed, including RNA knockdown and editing, nucleic acid detection and imaging, and RNA tracking. The emergence of these derivative technologies provides powerful tools for deciphering biological genetic mechanisms and disease treatment. In this review, we summarize the structure, function, mechanisms, and derived technologies of RNA-targeting CRISPR/Cas systems, aiming to enrich people's understanding of CRISPR/Cas-mediated RNA editing.},
}
@article {pmid40818496,
year = {2025},
author = {Masurkar, P and Meher, J and Thapa, S and Singh, RK and Bag, MK and Gautam, V and Maurya, S},
title = {Exploring Ustilaginoidea virens, the Causal Agent of False Smut of Rice Disease: A Comprehensive Study of Infection Dynamics, Effectors, and Genetic Structure.},
journal = {Microbial pathogenesis},
volume = {},
number = {},
pages = {107987},
doi = {10.1016/j.micpath.2025.107987},
pmid = {40818496},
issn = {1096-1208},
abstract = {Villosiclava virens (Anamorph: Ustilaginoidea virens) is an important and enigmatic pathogen that causes rice false smut. Some similarities between Claviceps and Ustilaginoidea genera have been found, but according to recent genomic sequence comparison research, they have different sequences. U. virens secretes mycotoxins, which make the infected grains unfavorable for human consumption. The transcriptomic analysis and genome sequencing of U. virens showed 52,554,142 clean reads assembled into 36,496 transcripts, representing 18,534 unigenes. U. virens also contains the UvNLP protein as an NLP-specific NPP1 domain that belongs to a MAMPs class protein that acts as an elicitor for defence responses in resistant plants. Unique proteins, UvCGBP1, UvPRO1, and UvBI-1, are associated with the virulence, growth, and sporulation of U. virens. Several host QTLs- qFsr1, qFsr2, qFsr4, qFsr8, qFsr10, qFsr11 and qFsr12 have been validated in diverse background (IR28, Tequing NILs, MR183-2), and are crucial for resistance breeding. The genetic diversity of U. virens was measured using molecular markers, including RAPD, AFLP, SSR, ISSR, and, more recently, SNPs. The genetic diversity of U. virens isolates was higher among the isolates than in the geographical population. This review integrates advances in molecular biology, genomics, and host-pathogen interactions to inform sustainable management strategies. Further research is needed in early detection, chlamydospore germination, targeted fungicides, and resistant hybrid rice development.},
}
@article {pmid40817028,
year = {2025},
author = {Bouzetos, E and Ganar, KA and van der Oost, J and Deshpande, S},
title = {Cell-free screening of CRISPR-Cas activity by microfluidics-assisted in vitro compartmentalization.},
journal = {Trends in biotechnology},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.tibtech.2025.07.007},
pmid = {40817028},
issn = {1879-3096},
abstract = {CRISPR-Cas systems are responsible for antiviral immunity of prokaryotic cells and have been repurposed as powerful genome-editing tools. Cell-free gene expression has been applied for the rapid characterization of CRISPR-Cas systems in microtiter plates. In vitro compartmentalization makes use of artificial microcompartments that individually act as bioreactors. Here, we performed cell-free reactions of CRISPR-Cas activity into microtiter plates, which we proceeded to encapsulate into double emulsion (DE) droplets generated by on-chip microfluidics. Emulsion droplets were screened for CRISPR-Cas activity based on relative fluorescence levels using a common cell sorter, and enrichment for the expected guide (g)RNA genotype was observed. Encapsulation of single gene copies per droplet is an important prerequisite for applying this technique to complex gene libraries. We show a proof-of-principle assay for efficient, compartmentalized gene amplification using magnetic microbeads. In conclusion, we demonstrate the feasibility of microfluidics-based, high-throughput, cell-free screening of CRISPR-Cas activity.},
}
@article {pmid40816052,
year = {2025},
author = {Ma, W and Liu, Q and Zhou, N and Wang, LJ and Jiao, Z and Wang, T and Zhang, CY},
title = {Engineering of a multi-modular CRISPR biomachine for ultrasensitive monitoring of β-glucosyltransferase activity.},
journal = {Biosensors &amp; bioelectronics},
volume = {289},
number = {},
pages = {117878},
doi = {10.1016/j.bios.2025.117878},
pmid = {40816052},
issn = {1873-4235},
abstract = {β: [note: ":" is changed to "-"] Glucosyltransferase (β-GT) is a pivotal enzymatic tool for 5-hydroxymethylcytosine (5-hmC) detection, and it can specifically catalyze the glycosylation of 5-hmC. This enzymatic reaction plays a crucial role in modulating bacteriophage-specific gene expression and facilitating the survival of bacteriophages and parasites within host cells. Herein, we engineer a multi-modular and structurally ordered CRISPR/Cas-based biomachine by integrating 5-hmC glycosylation-triggered palindrome-primed hyperbranched rolling circle amplification (PP-HRCA) for ultrasensitive analysis of exogenous β-GT activity. This biomachine integrates multiple functional modules including a β-GT-catalyzed protection module, a palindrome primer-initiated amplification module, and a CRISPR system-mediated cleavage module to improve the assay sensitivity and specificity toward exogenous β-GT. β-GT-catalyzed 5-hmC glycosylation effectively protects the dumbbell probe from MspI endonuclease-mediated digestion. The intact dumbbell probe subsequently serves as a template to initiate PP-HRCA, generating abundant long-tandem double-stranded DNA products. The amplification products can activate CRISPR/Cas-mediated cleavage of signal probes, releasing numerous Cy5 molecules that can be accurately quantified by both fluorescence spectroscopy and single-molecule imaging. This biomachine enables sensitive and zero-background detection of β-GT with a detection limit (LOD) of 2.75 × 10[-6] U/mL and a broad linear range spanning seven orders of magnitude. Moreover, it can analyze β-GT kinetic parameters and screen β-GT inhibitors, providing a robust platform for in-depth exploration of β-GT biological mechanisms and study of β-GT-related epigenetics.},
}
@article {pmid40814131,
year = {2025},
author = {Liu, P and Li, S and Zeng, J and Wang, M and Du, J and Wang, HH and Xiong, E and Yang, R},
title = {Creating a Modular Activatable CRISPR-Cas12a System by Engineering crRNA Scaffold with a Steric Hindrance Effector.},
journal = {Journal of the American Chemical Society},
volume = {},
number = {},
pages = {},
doi = {10.1021/jacs.5c11227},
pmid = {40814131},
issn = {1520-5126},
abstract = {CRISPR-Cas systems inherently lack spatiotemporal control over cleavage events, compromising their accuracy and efficiency in biotechnological applications. Although advances have been made in regulating these systems, developing modular activatable platforms adaptable to diverse stimuli remains challenging. To address this limitation, we developed an innovative modular activatable CRISPR-Cas12a system by engineering a crRNA scaffold with a steric hindrance effector (SHE) motif at its 3'-terminus, attached via a stimulus-cleavable linker. Through systematic evaluation of SHEs with varied sizes and structures─including linear, duplex, hairpin, and triplex─we identified their steric hindrance-dependent inhibitory effects on CRISPR-Cas12a functionality. Notably, the effective SHEs capable of completely inhibiting CRISPR-Cas12a functionality were successfully released upon ultraviolet light exposure, restoring system functionality. Furthermore, the crRNA scaffold-engineering strategy proved applicable to other type V-A Cas12a orthologs. Importantly, the system was adapted to respond to enzymatic stimuli (such as human apurinic/apyrimidinic endonuclease 1 [APE1]) and chemical stimuli (such as glutathione [GSH]). Finally, the modular activatable CRISPR-Cas12a system enabled light-activatable, one-pot nucleic acid diagnostics and APE1-activatable, tumor cell-selective microRNA imaging. Our study highlights the innovative use of steric hindrance to manipulate CRISPR-Cas12a functionality and provides valuable insights for the rational design of modular activatable CRISPR-Cas systems.},
}
@article {pmid40813265,
year = {2025},
author = {Zhuang, S and Bai, B and Liu, Y},
title = {CRISPR-based SNP detection technologies advance from classical methods to cutting-edge innovations.},
journal = {Chemical communications (Cambridge, England)},
volume = {},
number = {},
pages = {},
doi = {10.1039/d5cc03655k},
pmid = {40813265},
issn = {1364-548X},
abstract = {Single nucleotide polymorphisms (SNPs) constitute the most prevalent form of genetic variations, critically influencing human disease susceptibility, drug response, and pathogen evolution. Conventional SNP detection methods, however, face significant limitations: they often lack the necessary balance of precision, speed, and deployability required for diverse applications, ranging from point-of-care clinical diagnostics to rapid pathogen surveillance. The advent of CRISPR/Cas systems, particularly the discovery of the trans-cleavage activity of Cas nucleases, has revolutionized this field by offering unparalleled single-nucleotide specificity, isothermal operation, and signal amplification capabilities. In this review, we first systematically examine the foundational CRISPR-based SNP detection platforms, with a focused analysis of pioneering systems including SHERLOCK, HOLMES, and Cas14-DETECTR. Subsequently, we delve into the transformative technical advancements that have propelled these platforms towards cutting-edge innovations, emphasizing three critical pathways: (1) novel strategies for achieving ultra-high specificity in single-nucleotide discrimination, (2) breakthroughs in overcoming protospacer adjacent motif (PAM) sequence constraints, and (3) innovative approaches for optimizing sensitivity to meet stringent clinical detection thresholds. Finally, we critically evaluate the persistent challenges hindering the widespread adoption of current CRISPR-based SNP detection frameworks and propose actionable research trajectories aimed at advancing CRISPR technologies for high-precision SNP genotyping. This review provides a comprehensive overview of the remarkable evolution of CRISPR-based SNP detection, from its classical origins to its current status as a frontier innovation. It also sheds light on future directions essential for realizing the full potential of CRISPR as a transformative tool in precision medicine and global health surveillance.},
}
@article {pmid40812128,
year = {2025},
author = {Liu, J and Gao, Y},
title = {Hybrid strand displacement circuit-controlled CRISPR-Cas13a activation for one-pot sensing of Non-RNA analytes.},
journal = {Biosensors &amp; bioelectronics},
volume = {289},
number = {},
pages = {117865},
doi = {10.1016/j.bios.2025.117865},
pmid = {40812128},
issn = {1873-4235},
abstract = {The exceptionally sensitive and specific CRISPR-Cas systems have revolutionized molecular diagnostics, but their application remains largely confined to nucleic acid targets. To overcome this limitation and enable one-pot, mix-and-read detection of non-nucleic acid analytes, we present a generalizable CRISPR-Cas13a sensing platform that utilizes a hybrid DNA:RNA strand displacement-based switch to conditionally activate Cas13a. The switch leverages Cas13a's strict requirement for single-stranded RNA activators to mediate non-RNA target-triggered activation while protecting functional DNA elements from collateral cleavage. By integrating the modular Cas13a switch with various functional DNAs, including DNAzyme, aptazyme, and duplexed aptamer, we constructed a suite of one-pot sensors capable of detecting non-RNA targets such as lead ion, ATP, and thrombin. These CRISPR-Cas13a sensors demonstrate enhanced sensitivity, high selectivity, and robust performance in complex biological matrices. The high modularity of the CRISPR-Cas13a sensor allows rapid reconfiguration for new targets by simply altering the output sequence of functional DNA without extensive re-engineering. This versatile and robust sensing strategy expands the application landscape of CRISPR-Cas systems and lays the foundation for flexible, sensitive, and one-pot detection of a wide array of clinically and environmentally relevant targets.},
}
@article {pmid40777486,
year = {2025},
author = {Taboada, VP and Wu, Y and Cassidy, R and Medvedev, KE and Loeff, L and Nemudraia, A and Nemudryi, A},
title = {Bacterial Schlafens mediate anti-phage defense.},
journal = {bioRxiv : the preprint server for biology},
volume = {},
number = {},
pages = {},
pmid = {40777486},
issn = {2692-8205},
abstract = {Human Schlafen proteins restrict viral replication by cleaving tRNA, thereby suppressing protein synthesis. Although the ribonuclease domain of Schlafen proteins is conserved across all domains of life, its function in prokaryotes has remained unclear. Here, we show that prokaryotic Schlafen nucleases (pSlfns) are widespread antiviral effectors that protect bacteria from phages. These nucleases are fused to diverse protein domains that sense phage infection. We focus on a system where Schlafen nuclease is fused to a previously unknown immunoglobulin-like sensor domain and demonstrate that it recognizes T5-like phage tail assembly chaperones and cleaves both bacterial and viral tRNA, triggering abortive infection. Our findings redefine Schlafens as an ancient, mechanistically conserved family of immune effectors, revealing the deep evolutionary origin of tRNA-targeting antiviral immunity in humans.},
}
@article {pmid40811506,
year = {2025},
author = {Pernomian, L and Parente, JM and McCarthy, CG and Wenceslau, CF},
title = {Orphan Under Pressure: GPR146 as a Mechanotransduction Modulator.},
journal = {Circulation research},
volume = {137},
number = {5},
pages = {625-627},
doi = {10.1161/CIRCRESAHA.125.327056},
pmid = {40811506},
issn = {1524-4571},
}
@article {pmid40810510,
year = {2025},
author = {Chen, DF and Roe, LT and Yuping, L and Borges, AL and Zhang, JY and Babbar, P and Maji, S and Stevens, MGV and Correy, GJ and Diolaiti, ME and Smith, DH and Ashworth, A and Stroud, RM and Kelly, MJS and Bondy-Denomy, J and Fraser, JS},
title = {AcrIF11 is a potent CRISPR-specific ADP-ribosyltransferase encoded by phage and plasmid.},
journal = {mBio},
volume = {},
number = {},
pages = {e0169825},
doi = {10.1128/mbio.01698-25},
pmid = {40810510},
issn = {2150-7511},
abstract = {Phage-encoded anti-CRISPR (Acr) proteins inhibit CRISPR-Cas systems, allowing phage replication and lysogeny maintenance. Most of the Acrs characterized to date are stable stoichiometric inhibitors. While enzymatic Acrs have been characterized biochemically, little is known about their potency, specificity, and reversibility. Here, we examine AcrIF11, a widespread phage and plasmid-encoded ADP-ribosyltransferase (ART) that inhibits the Type I-F CRISPR-Cas system. We present a nuclear magnetic resonance (NMR) structure of an AcrIF11 homolog that reveals chemical shift perturbations consistent with NAD (cofactor) binding. In experiments that model both lytic phage replication and MGE/lysogen stability under high targeting pressure, AcrIF11 is a highly potent CRISPR-Cas inhibitor and more robust to Cas protein-level fluctuations than stoichiometric inhibitors. Furthermore, we demonstrate that AcrIF11 is remarkably specific, predominantly ADP-ribosylating Csy1 when expressed in P. aeruginosa. Given the reversible nature of ADP-ribosylation, we hypothesized that ADPr eraser enzymes (macrodomains) could remove ADPr from Csy1, a potential limitation of PTM-based CRISPR inhibition. We demonstrate that a human macrodomain can indeed remove the modification from Csy1 in P. aeruginosa lysate. Together, these experiments connect the in vitro observations of AcrIF11's enzymatic activity to its potent and specific effects in vivo, clarifying the advantages and drawbacks of enzymatic Acrs in the evolutionary arms race between phages and bacteria.IMPORTANCEBacteria have evolved diverse immune systems to prevent phage infection, and, consequently, phages have evolved diverse methods of evading bacterial immune systems. To evade the bacterial CRISPR-Cas immune system, phages encode anti-CRISPR proteins (Acrs). Acrs disable CRISPR-Cas by either stably binding to the CRISPR-Cas complex or by enzymatic modification. However, Acr enzymes have not been characterized in vivo during lytic infection or lysogenic maintenance. Here, we report the benefits and drawbacks of enzymatic inhibition with AcrIF11, an ADP-ribosyltransferase. Under "high pressure" scenarios such as high CRISPR targeting or CRISPR-Cas overexpression, AcrIF11 outperforms a strong, stable binding Acr by very specifically modifying the Cas8 protein, but nothing else in the cell. AcrIF11 additionally stabilizes lysogeny effectively, but the ADP-ribose modification can potentially be removed by macrodomains, which are ADP-ribose eraser enzymes. AcrIF11 is therefore a potent and widespread plasmid/phage-encoded inhibitor of Type I-F CRISPR-Cas systems with catalytic activity.},
}
@article {pmid40808298,
year = {2025},
author = {Andersen, S and Wolff, JH and Skov, TW and Janns, JH and Davis, LJ and Haldrup, JH and Haslund, D and Revenfeld, AL and Relkovic, D and Møller, BK and Lund, J and Bak, RO and Thomsen, EA and Mikkelsen, JG},
title = {Gene editing in hematopoietic stem cells by co-delivery of Cas9/sgRNA ribonucleoprotein and templates for homology-directed repair in 'all-in-one' lentivirus-derived nanoparticles.},
journal = {Nucleic acids research},
volume = {53},
number = {15},
pages = {},
doi = {10.1093/nar/gkaf767},
pmid = {40808298},
issn = {1362-4962},
support = {//Innovation Fund Denmark/ ; //Novo Nordisk A/S/ ; 8056-00010A//Innovation Fund Denmark/ ; NNF22OC0080684//Novo Nordisk Foundation/ ; 4285-00102B//Independent Research Fund Denmark/ ; CF21-0363//Carlsberg Foundation/ ; },
abstract = {Repair of double-strand DNA breaks generated by site-directed endonucleases, like Cas9, is the hallmark of gene editing based on homology-directed repair (HDR). HDR uses an exogenous DNA template to restore the cleaved DNA sequence and can facilitate specific gene corrections as well as insertion of genes or partial complementary DNA (cDNA) sequences. For CRISPR/Cas-directed gene editing, co-administration of the Cas9/single guide RNA (sgRNA) ribonucleoprotein (RNP) complex and a DNA template typically involves two different delivery strategies or different types of vehicles. This requires exquisite timing of delivery and may potentially challenge safety and therapeutic applicability. There is a need therefore for technologies that can ferry complete editing tool kits into cells. Here, we demonstrate the use of lentivirus-derived nanoparticles (LVNPs) to transport both RNP complexes and vector RNA, which upon reverse transcription serves as a repair template for HDR-directed gene editing. Such 'all-in-one' LVNPs support targeted gene insertion with reduced off-target effects relative to nucleofection procedures. We show potent editing in the HBB gene in human erythroid progenitor cells as well as HDR-directed editing in hematopoietic stem and progenitor cells. Our findings mark a first step toward using a single virus-derived vehicle for delivering a full HDR gene editing kit.},
}
@article {pmid40807307,
year = {2025},
author = {Alessa, O and Aiba, Y and Arbaah, M and Hidaka, Y and Watanabe, S and Miyanaga, K and Wannigama, DL and Cui, L},
title = {Synthetic and Functional Engineering of Bacteriophages: Approaches for Tailored Bactericidal, Diagnostic, and Delivery Platforms.},
journal = {Molecules (Basel, Switzerland)},
volume = {30},
number = {15},
pages = {},
doi = {10.3390/molecules30153132},
pmid = {40807307},
issn = {1420-3049},
support = {(grant No. JP25ae0121045, JP25gm1610002, and JP25fk0108698), (Grant No. JP24H00662, JP24K22027, JP25K13511, and JP25K19273) and (JPJ009237).//This research was funded by the Japan Agency for Medical Research and Development JSPS KAKENHI and partially by Moonshot R&amp;D Program for Agriculture, Forestry, Fisheries/ ; },
abstract = {Bacteriophages (phages), the most abundant biological entities on Earth, have long served as both model systems and therapeutic tools. Recent advances in synthetic biology and genetic engineering have revolutionized the capacity to tailor phages with enhanced functionality beyond their natural capabilities. This review outlines the current landscape of synthetic and functional engineering of phages, encompassing both in-vivo and in-vitro strategies. We describe in-vivo approaches such as phage recombineering systems, CRISPR-Cas-assisted editing, and bacterial retron-based methods, as well as synthetic assembly platforms including yeast-based artificial chromosomes, Gibson, Golden Gate, and iPac assemblies. In addition, we explore in-vitro rebooting using TXTL (transcription-translation) systems, which offer a flexible alternative to cell-based rebooting but are less effective for large genomes or structurally complex phages. Special focus is given to the design of customized phages for targeted applications, including host range expansion via receptor-binding protein modifications, delivery of antimicrobial proteins or CRISPR payloads, and the construction of biocontained, non-replicative capsid systems for safe clinical use. Through illustrative examples, we highlight how these technologies enable the transformation of phages into programmable bactericidal agents, precision diagnostic tools, and drug delivery vehicles. Together, these advances establish a powerful foundation for next-generation antimicrobial platforms and synthetic microbiology.},
}
@article {pmid40806631,
year = {2025},
author = {Sapakhova, Z and Kanat, R and Choi, K and Daurov, D and Daurova, A and Zhambakin, K and Shamekova, M},
title = {CRISPR-Cas Gene Editing Technology in Potato.},
journal = {International journal of molecular sciences},
volume = {26},
number = {15},
pages = {},
doi = {10.3390/ijms26157496},
pmid = {40806631},
issn = {1422-0067},
support = {BR21882269//The Science Committee of the Ministry of Science and Higher Education, Republic of Kazakhstan/ ; },
abstract = {Potato (Solanum tuberosum L.) is one of the most important food crops in the world, ranking fourth after rice, maize, and wheat. Potatoes are exposed to biotic and abiotic environmental factors, which lead to economic losses and increase the possibility of food security threats in many countries. Traditional potato breeding faces several challenges, primarily due to its genetic complexity and the time-consuming nature of the process. Therefore, gene editing-CRISPR-Cas technology-allows for more precise and rapid changes to the potato genome, which can speed up the breeding process and lead to more effective varieties. In this review, we consider CRISPR-Cas technology as a potential tool for plant breeding strategies to ensure global food security. This review summarizes in detail current and potential technological breakthroughs that open new opportunities for the use of CRISPR-Cas technology for potato breeding, as well as for increasing resistance to abiotic and biotic stresses, and improving potato tuber quality. In addition, the review discusses the challenges and future perspectives of the CRISPR-Cas system in the prospects of the development of potato production and the regulation of gene-edited crops in different countries around the world.},
}
@article {pmid40806610,
year = {2025},
author = {Shaposhnikov, LA and Rozanov, AS and Sazonov, AE},
title = {Genome-Editing Tools for Lactic Acid Bacteria: Past Achievements, Current Platforms, and Future Directions.},
journal = {International journal of molecular sciences},
volume = {26},
number = {15},
pages = {},
doi = {10.3390/ijms26157483},
pmid = {40806610},
issn = {1422-0067},
support = {&#x2116;18-03//Sirius University of Science and Technology/ ; },
abstract = {Lactic acid bacteria (LAB) are central to food, feed, and health biotechnology, yet their genomes have long resisted rapid, precise manipulation. This review charts the evolution of LAB genome-editing strategies from labor-intensive RecA-dependent double-crossovers to state-of-the-art CRISPR and CRISPR-associated transposase systems. Native homologous recombination, transposon mutagenesis, and phage-derived recombineering opened the door to targeted gene disruption, but low efficiencies and marker footprints limited throughput. Recent phage RecT/RecE-mediated recombineering and CRISPR/Cas counter-selection now enable scar-less point edits, seamless deletions, and multi-kilobase insertions at efficiencies approaching model organisms. Endogenous Cas9 systems, dCas-based CRISPR interference, and CRISPR-guided transposases further extend the toolbox, allowing multiplex knockouts, precise single-base mutations, conditional knockdowns, and payloads up to 10 kb. The remaining hurdles include strain-specific barriers, reliance on selection markers for large edits, and the limited host-range of recombinases. Nevertheless, convergence of phage enzymes, CRISPR counter-selection and high-throughput oligo recombineering is rapidly transforming LAB into versatile chassis for cell-factory and therapeutic applications.},
}
@article {pmid40806599,
year = {2025},
author = {Yang, L and Lu, Q},
title = {Beyond Cutting: CRISPR-Driven Synthetic Biology Toolkit for Next-Generation Microalgal Metabolic Engineering.},
journal = {International journal of molecular sciences},
volume = {26},
number = {15},
pages = {},
doi = {10.3390/ijms26157470},
pmid = {40806599},
issn = {1422-0067},
abstract = {Microalgae, with their unparalleled capabilities for sunlight-driven growth, CO2 fixation, and synthesis of diverse high-value compounds, represent sustainable cell factories for a circular bioeconomy. However, industrial deployment has been hindered by biological constraints and the inadequacy of conventional genetic tools. The advent of CRISPR-Cas systems initially provided precise gene editing via targeted DNA cleavage. This review argues that the true transformative potential lies in moving decisively beyond cutting to harness CRISPR as a versatile synthetic biology "Swiss Army Knife". We synthesize the rapid evolution of CRISPR-derived tools-including transcriptional modulators (CRISPRa/i), epigenome editors, base/prime editors, multiplexed systems, and biosensor-integrated logic gates-and their revolutionary applications in microalgal engineering. These tools enable tunable gene expression, stable epigenetic reprogramming, DSB-free nucleotide-level precision editing, coordinated rewiring of complex metabolic networks, and dynamic, autonomous control in response to environmental cues. We critically evaluate their deployment to enhance photosynthesis, boost lipid/biofuel production, engineer high-value compound pathways (carotenoids, PUFAs, proteins), improve stress resilience, and optimize carbon utilization. Persistent challenges-species-specific tool optimization, delivery efficiency, genetic stability, scalability, and biosafety-are analyzed, alongside emerging solutions and future directions integrating AI, automation, and multi-omics. The strategic integration of this CRISPR toolkit unlocks the potential to engineer robust, high-productivity microalgal cell factories, finally realizing their promise as sustainable platforms for next-generation biomanufacturing.},
}
@article {pmid40804951,
year = {2025},
author = {Li, G and Zhou, X and Zhu, G and Pan, Y and Yan, J and Meng, J and Ye, T and Cheng, Y and Liu, C and Gu, Z},
title = {Macrobrachium rosenbergii Genome Editing Breeding with CRISPR-Cas Nucleases, Base Editors, and Prime Editors.},
journal = {Animals : an open access journal from MDPI},
volume = {15},
number = {15},
pages = {},
doi = {10.3390/ani15152161},
pmid = {40804951},
issn = {2076-2615},
support = {2024SSYS0101//"Pioneer" and "Leading Goose" R&amp;D Program of Zhejiang/ ; 32401241//National Natural Science Foundation of China/ ; },
abstract = {This review focuses on CRISPR genome editing technology, particularly its application in the study of Macrobrachium rosenbergii (M. rosenbergii). It first elaborates on the basic principles and mechanisms of CRISPR-Cas9 technology, base editors, and prime editors. Then, it explores the application of this technology in M. rosenbergii breeding, including improving growth rate, enhancing disease resistance, and sex control. Additionally, it introduces the progress of genome editing technology in M. rosenbergii, epidemiology and pathogenesis, diagnostic techniques, analyzes the opportunities and challenges it faces, reviews the historical evolution, and looks ahead to future development directions. CRISPR technology has brought new opportunities to the research and industrial development of M. rosenbergii, but it also needs to address numerous technical and safety challenges.},
}
@article {pmid40804123,
year = {2025},
author = {Yu, M and Ai, L and Wang, B and Lian, S and Ip, L and Liu, J and Li, L and Tsai, SQ and Kleinstiver, BP and Zheng, Z},
title = {GenomePAM directs PAM characterization and engineering of CRISPR-Cas nucleases using mammalian genome repeats.},
journal = {Nature biomedical engineering},
volume = {},
number = {},
pages = {},
pmid = {40804123},
issn = {2157-846X},
support = {202001418//Vetenskapsr&#xe5;det (Swedish Research Council)/ ; 11103024//Research Grants Council, University Grants Committee (RGC, UGC)/ ; T12-101/23-N//Research Grants Council, University Grants Committee (RGC, UGC)/ ; DP2-CA281401//Foundation for the National Institutes of Health (Foundation for the National Institutes of Health, Inc.)/ ; },
abstract = {Characterizing the protospacer adjacent motif (PAM) requirements of different Cas enzymes is a bottleneck in the discovery of Cas proteins and their engineered variants in mammalian cell contexts. Here, to overcome this challenge and to enable more scalable characterization of PAM preferences, we develop a method named GenomePAM that allows for direct PAM characterization in mammalian cells. GenomePAM leverages genomic repetitive sequences as target sites and does not require protein purification or synthetic oligos. GenomePAM uses a 20-nt protospacer that occurs ~16,942 times in every human diploid cell and is flanked by nearly random sequences. We demonstrate that GenomePAM can accurately characterize the PAM requirement of type II and type V nucleases, including the minimal PAM requirement of the near-PAMless SpRY and extended PAM for CjCas9. Beyond PAM characterization, GenomePAM allows for simultaneous comparison of activities and fidelities among different Cas nucleases on thousands of match and mismatch sites across the genome using a single gRNA and provides insight into the genome-wide chromatin accessibility profiles in different cell types.},
}
@article {pmid40809473,
year = {2023},
author = {Mecacci, S and Torregrosa-Barragán, L and Asin-Garcia, E and Smith, RW},
title = {Multilayered safety framework for living diagnostics in the colon.},
journal = {Frontiers in systems biology},
volume = {3},
number = {},
pages = {1240040},
pmid = {40809473},
issn = {2674-0702},
abstract = {Introduction: Colorectal cancer is the second most deadly cancer worldwide. Current screening methods have low detection rates and frequently provide false positive results, leading to missed diagnoses or unnecessary colonoscopies. To tackle this issue, the Wageningen UR iGEM team from 2022 developed "Colourectal", a living diagnostic tool for colorectal cancer. Following a synthetic biology approach, the project used an engineered Escherichia coli Nissle 1917 strain capable of binding to tumour cells that detects two distinct cancer biomarkers, and secretes a coloured protein observable in stool. Due to the utilization of genetically modified bacteria in vivo, precautionary biosafety measures were included within a three level safe-by-design strategy. Results: The first genetic safeguard ensured confinement of the living diagnostic to the colon environment by implementing auxotrophy to mucin that is abundant in the colon lining. For this, a synthetic chimeric receptor was generated to ensure expression of essential genes in the presence of mucin. The second strategy limited the viability of the engineered bacteria to the human body, preventing proliferation in open environments. The use of a temperature sensitive kill switch induced bacterial cell death at temperatures below 37°C. The third biocontainment strategy was installed as an emergency kill switch to stop the Colourectal test at any point. By inducing a highly genotoxic response through CRISPR-Cas-mediated DNA degradation, cell death of E. coli Nissle is triggered. Discussion: While the use of engineered microorganisms in human applications is not yet a reality, the safety considerations of our multi-layered strategy provide a framework for the development of future living diagnostic tools.},
}
@article {pmid40803977,
year = {2025},
author = {Li, L and Zhang, Z and Zhang, B},
title = {CRISPR meets AlphaFold: guiding SWEET10-enhanced oil production.},
journal = {Trends in plant science},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.tplants.2025.08.001},
pmid = {40803977},
issn = {1878-4372},
abstract = {Enhancing seed oil content significantly benefits both human welfare and environmental sustainability. Clustered regularly interspaced short palindromic repeats/CRISPR-associated protein (CRISPR/Cas) and artificial intelligence (AI) are transformative tools for crop trait improvement. A recent study by Wang and colleagues reported that AlphaFold-guided CRISPR genome editing of SWEET10 boosts oil contents, highlighting a breakthrough in precision crop engineering.},
}
@article {pmid40803657,
year = {2025},
author = {Ceasar, SA and Ebeed, HT and Ramakrishnan, M and García-Caparrós, P and Ignacimuthu, S},
title = {Understanding low-phosphate stress responses in plants: Opportunities for genome editing to improve phosphorous use efficiency (PUE).},
journal = {Biotechnology advances},
volume = {},
number = {},
pages = {108686},
doi = {10.1016/j.biotechadv.2025.108686},
pmid = {40803657},
issn = {1873-1899},
abstract = {Phosphorus (P) is a critical macronutrient essential for plant growth, yet its availability in soil is often limited due to poor mobility and fixation with metal ions due to acidic or alkaline soils. Plants have evolved complex adaptive responses to overcome phosphate (Pi) deficiency. Recent advancements in genome editing, particularly CRISPR/Cas tools, offer opportunities to enhance these adaptive traits for sustainable agriculture. This review consolidates current understanding of low Pi stress signaling pathways, including morphological (root architecture changes), biochemical (hormone regulation, lipid modification, organic acid exudation), and molecular (transcription factors (TFs), phosphate transporters, and microRNAs), and identifies prime candidate genes for genome editing applications. Key regulators such as phosphate transporter (PHT, PHO1), TFs (PHR1, WRKYs) and microRNAs (miR399/827) manage Pi uptake, redistribution, and signaling. Genome editing strategies targeting root-specific traits, hormonal integration, lipid remodeling, and transcriptional regulation are discussed as viable ways for improving phosphorous use efficiency (PUE). Harnessing CRISPR/Cas tools can lead to the development of crops with optimized PUE, reduced dependency on synthetic fertilizers, and improved adaptability to Pi-deficient soils. The review provides a comprehensive roadmap for researchers and breeders to apply CRISPR/Cas technology toward building next-generation crops capable of thriving under low Pi conditions.},
}
@article {pmid40803478,
year = {2025},
author = {Jian, A and Zhao, G and Wang, Y and Wang, S and Li, N},
title = {Watershed Year of Cell and Gene Therapy (CGT): A Review of 2024 CGT Approvals.},
journal = {Cancer letters},
volume = {},
number = {},
pages = {217980},
doi = {10.1016/j.canlet.2025.217980},
pmid = {40803478},
issn = {1872-7980},
abstract = {The year 2024 is a pivotal year for therapeutic breakthroughs in human diseases, alongside with an uprising growth in precision medicine, especially in cell and gene therapies (CGTs), marked by an unprecedented approval number of 13 novel CGTs authorized by the U.S. FDA, China's NMPA, E.U. EMA, and Japan's PMDA. 2024 is also a year of many firsts: the first CRISPR therapy, the first MRI-guided intracranial AAV delivery gene therapy, and the first tumor infiltration lymphocyte therapy, opening a whole new chapter of clinical translation of innovations in gene-editing and cell technologies. CGTs represent an emerging translational modality in precision medicine through utilization of cellular or genetic materials to treat or prevent disease, offering curative potential for previously refractory diseases. Despite thriving in CGTs' development, comprehensive analyses of 2024 approvals remain absent. This review employs a quadruple axle comparative framework to analyze 2024-approved CGTs in China and USA, integrating mechanistic innovation with gradually improving regulatory advancements. Raw data was extracted from official agency databases, pivotal trials, and manufacturing reports. This work further delineates how mechanistic diversity converges with regulatory agility to redefine therapeutic development.},
}
@article {pmid40803134,
year = {2025},
author = {Wu, F and Campbell, BC and Greenfield, P and Hose, GC and Midgley, DJ and George, SC},
title = {There and back again: Genomic insights into microbial life in a recirculating petroleum refinery wastewater biotreatment system.},
journal = {Microbiological research},
volume = {301},
number = {},
pages = {128299},
doi = {10.1016/j.micres.2025.128299},
pmid = {40803134},
issn = {1618-0623},
abstract = {Petroleum refinery wastewater biotreatment relies on microbes to remediate carbon, nitrogen, and sulfur compounds, yet their life strategies and ecological roles remain unclear. This study characterises the ecological functions of 20 metagenome-assembled genomes (MAGs) from a full-scale petroleum refinery wastewater treatment plant in southern China. The taxonomic identity, nutrient metabolism genes (including C/N/S cycling), carbohydrate-active enzymes, and CRISPR-Cas systems of these MAGs were analysed. The recovered MAGs represented bacteria primarily from the Pseudomonadota and Bacteroidota phyla. The major carbon sources for the represented organisms are likely aromatic and aliphatic compounds, as well as carbohydrates including peptidoglycan, chitin, and starch. Almost all MAGs contained genes for nitrate or nitrite reduction, while metabolic pathways for sulfur metabolism were generally less prevalent. Meiothermus sp. bin.89 was the most metabolically versatile MAG. This organism possessed genes that allowed it to recycle biomass, break down aliphatic and monoaromatic compounds, and perform anaerobic respiration using nitrate. However, it was likely the most susceptible to viral predation, as indicated by the high abundance of CRISPR spacers. Overall, the results revealed that stress-tolerant ecological traits were common among organisms in this microbiome, showcasing the ability of the microbes to obtain carbon from aromatic and aliphatic compounds. This study provides a substantial contribution towards future efforts in optimising microbiome stability for pollutant removal in petroleum refinery wastewater biotreatment systems.},
}
@article {pmid40802883,
year = {2025},
author = {Song, N and Wang, L and Zhang, L and Tian, G and Yao, C and Yang, D},
title = {Precision Delivery of CRISPR/Cas Systems via DNA Nanostructures for Gene Therapy and Intracellular Detection.},
journal = {Chembiochem : a European journal of chemical biology},
volume = {},
number = {},
pages = {e202500357},
doi = {10.1002/cbic.202500357},
pmid = {40802883},
issn = {1439-7633},
support = {22225505//National Natural Science Foundation of China/ ; 22322407//National Natural Science Foundation of China/ ; 22174097//National Natural Science Foundation of China/ ; 224B2505//National Natural Science Foundation of China/ ; },
abstract = {The CRISPR/Cas system represents a transformative breakthrough in genome editing technology, featuring three principal effector proteins with distinct functionalities: Cas9, which induces site-specific double-strand breaks guided by a single guide RNA, enabling precise gene knockout and knock-in modifications; Cas12, which mediates targeted DNA cleavage through cis-activity while exhibiting nonspecific trans-cleavage of single-stranded DNA, a property exploited for ultrasensitive nucleic acid detection in molecular diagnostics; and Cas13, an RNA-guided RNase that specifically degrades complementary RNA transcripts, demonstrating significant potential for antiviral therapies and transcriptome regulation. Despite these advances, the clinical translation of CRISPR/Cas systems faces substantial challenges, particularly in achieving efficient and controllable delivery. This reviewsystematically examines current delivery modalities for CRISPR/Cas systems, with particular emphasis on the implementation of DNA-based functional materials as advanced delivery vehicles. The integration of multifunctional DNA nanostructures with diverse CRISPR/Cas systems may facilitate the development of integrated theranostic platforms, thereby advancing precision medicine through synergistic bioengineering approaches.},
}
@article {pmid40801580,
year = {2025},
author = {Leal, AF and Prieto, LE and Pachajoa, H},
title = {CRISPR/Cas-Based Ex Vivo Gene Therapy and Lysosomal Storage Disorders: A Perspective Beyond Cas9.},
journal = {Cells},
volume = {14},
number = {15},
pages = {},
doi = {10.3390/cells14151147},
pmid = {40801580},
issn = {2073-4409},
abstract = {Lysosomal storage disorders (LSDs) are inherited metabolic conditions characterized by lysosomal enzyme deficiencies leading to substrate accumulation. As genetic diseases, LSDs can be treated with gene therapies (GT), including the CRISPR/Cas systems. The CRISPR/Cas systems enable precise and programmable genome editing, leading to targeted modifications at specific genomic loci. While the classical CRISPR/Cas9 system has been extensively used to generate LSD disease models and correct disease-associated genetic alterations through homologous recombination (HR), recently described Cas proteins as well as CRISPR/Cas9-derived strategies such as base editing, prime editing, and homology-independent targeted integration (HITI) offer a novel way to develop innovative treatments for LSDs. The direct administration of the CRISPR/Cas9 system remains the primary strategy evaluated in several LSDs; nevertheless, the ex vivo CRISPR/Cas9-based approach has been recently explored, primarily in central nervous system-affecting LSDs. Ex vivo approaches involve genetically modifying, in theory, any patient cells in the laboratory and reintroducing them into the patient to provide a therapeutic effect. This manuscript reviews the molecular aspects of the CRISPR/Cas technology and its implementation in ex vivo strategies for LSDs while discussing novel approaches beyond the classical CRISPR/Cas9 system.},
}
@article {pmid40801057,
year = {2025},
author = {Talibli, F and Voß, B},
title = {Metagenomic CRISPR Array Analysis Tool: a novel graph-based approach to finding CRISPR arrays in metagenomic datasets.},
journal = {microLife},
volume = {6},
number = {},
pages = {uqaf016},
pmid = {40801057},
issn = {2633-6693},
abstract = {Clustered Regularly Interspersed Short Palindromic Repeats and CRISPR-associated genes (CRISPR-Cas) is a bacterial immune system also famous for its use in genome editing. The diversity of known systems could be significantly increased by metagenomic data. Here we present the Metagenomic CRISPR Array Analysis Tool (MCAAT), a highly sensitive algorithm for finding CRISPR arrays in unassembled metagenomic data. It takes advantage of the properties of CRISPR arrays that form multicycles in de Bruijn graphs. We show that MCAAT reliably predicts CRISPR arrays in bacterial genome sequences and that its assembly-free graph-based strategy outperforms assembly-based workflows and other assembly-free methods on synthetic and real metagenomes. Our new approach will help to increase the diversity of known CRISPR-Cas systems and enable studies of spacer evolution within metagenomic data sets.},
}
@article {pmid40796771,
year = {2025},
author = {Wolff, JH and Skov, TW and Haslund, D and Dorset, SR and Revenfeld, ALS and Aussel, C and Jørgensen, SE and Holm, M and Thomsen, MK and Ammann, S and Cathomen, T and Mogensen, TH and Møller, BK and Bak, RO and Mikkelsen, JG},
title = {Targeted gene editing and near-universal cDNA insertion of CYBA and CYBB as a treatment for chronic granulomatous disease.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {7475},
pmid = {40796771},
issn = {2041-1723},
support = {8056-00010A//Innovationsfonden (Innovation Fund Denmark)/ ; NNF220C0080684//Novo Nordisk Fonden (Novo Nordisk Foundation)/ ; CF21-0363//Carlsbergfondet (Carlsberg Foundation)/ ; CA 311/4-1 &amp; FANEDIT/EJPRD20-209//Deutsche Forschungsgemeinschaft (German Research Foundation)/ ; },
abstract = {Chronic granulomatous disease (CGD) is a severe inborn error of immunity caused by NADPH oxidase defects. Here, we develop CRISPR/Cas9-based gene editing strategies for correction of variants in the CYBA and CYBB genes causing CGD. For X-linked CGD, we also develop a near-universal gene editing strategy by targeted integration of a truncated CYBB cDNA in CD34[+] hematopoietic stem and progenitor cells (HSPCs). Throughout, off-target editing and chromosomal translocations are evident, which negatively impact the ability of gene-edited HSPCs to engraft in immunodeficient mice. However, by employing a high-fidelity Cas9 to minimize off-target editing, we demonstrate restoration of the multilineage engraftment potential of gene-edited HSPCs. Moreover, to further improve safety, we develop a D10A Cas9n editing approach with no detectable off-target activity or chromosomal translocations. Collectively, through risk assessments of different gene editing approaches, we present a D10A Cas9n-based strategy with improved safety, offering a potentially curative treatment for CGD patients.},
}
@article {pmid40796304,
year = {2025},
author = {Qing, Y and Liao, Z and An, D and Zeng, Y and Zhu, Q and Zhang, X},
title = {Comparative genomics reveals the genetic diversity and plasticity of Clostridium tertium.},
journal = {Journal of applied microbiology},
volume = {},
number = {},
pages = {},
doi = {10.1093/jambio/lxaf201},
pmid = {40796304},
issn = {1365-2672},
abstract = {AIMS: Clostridium tertium, increasingly recognized as the emerging human pathogen frequently isolated from environmental and clinical specimens, remains genetically underexplored despite its clinical relevance. This study aims to explore the genetic characteristics of C. tertium by genomic analysis.<br><br>METHODS AND RESULTS: This study presented a comprehensive genomic investigation of 45 C. tertium strains from the GenBank database. Genome sizes (3.27-4.55 Mbp) and coding gene counts varied markedly across strains. Phylogenetic analyses based on 16S rRNA gene and core genome uncovered distinct intra-species lineages, including evolutionarily divergent clusters likely shaped by niche specialization. Pan-genomic analysis confirmed an open genome, with accessory and strain-specific genes enriched in functions related to environmental adaptation and regulation. Functional annotation further identified diverse virulence factor genes (e.g. clpP, nagK) and antibiotic resistance genes (e.g. vatB, tetA(P)) co-occurring with mobile genetic elements (MGEs), suggesting that horizontal gene transfer (HGT) may be a key driver of genome plasticity in C. tertium. Notably, one-third of the strains carried CRISPR-Cas systems, indicating the defense potential against exogenous genetic elements.<br><br>CONCLUSIONS: C. tertium exhibited extensive genetic diversity and genome plasticity, probably driven by MGE-mediated HGT, defense mechanisms of CRISPR-Cas systems, and functional adaptation related to virulence and resistance. These traits may underlie its ability to colonize diverse environments and acquire pathogenicity and resistance.},
}
@article {pmid40794864,
year = {2025},
author = {Wang, F and Zhao, P and Bi, X and Zheng, R and Tian, X and Xu, J and Jiang, S and Li, G and Shen, Y and She, Q},
title = {Cyclic tetraadenylate binding induces dimerization of protein dimers to activate a CRISPR-associated PIN nuclease.},
journal = {Nucleic acids research},
volume = {53},
number = {14},
pages = {},
doi = {10.1093/nar/gkaf744},
pmid = {40794864},
issn = {1362-4962},
support = {2020YFA0906800//National Key R&amp;D Program of China/ ; 31771380//National Natural Science Foundation of China/ ; 32270040//National Natural Science Foundation of China/ ; 32393972//National Natural Science Foundation of China/ ; SKLMTFCP-2023-05//Frontiers and Challenges/ ; ZR2024QC307//State Key Laboratory of Microbial Technology/ ; //Shandong Provincial Natural Science Foundation/ ; },
mesh = {*CRISPR-Cas Systems ; Protein Multimerization ; *Archaeal Proteins/chemistry/metabolism/genetics ; *CRISPR-Associated Proteins/metabolism/chemistry/genetics ; Protein Binding ; *Adenine Nucleotides/metabolism/chemistry ; Models, Molecular ; *Endonucleases/metabolism/chemistry/genetics ; Crystallography, X-Ray ; Protein Domains ; },
abstract = {Type III CRISPR-Cas systems synthesize cyclic oligoadenylates (cOAs), the second messengers that bind to the CARF (CRISPR-associated Rossman fold) sensor domain and allosterically activate the effector domain of CRISPR ancillary effectors to mediate antiviral defense. An arsenal of such effectors has been identified, but only a minority of them have been characterized thus far. Here, CaPN (a CRISPR-associated PIN domain nuclease), a novel effector protein encoded by Saccharolobus islandicus, was characterized. Biochemical characterization of CaPN revealed that the CARF domain senses cA4 (cyclic tetraadenylate), and its binding to the CARF domain activates the PIN domain for robust RNA cleavage. Genetic assay showed that CaPN mediates growth arrest/cell death to its archaeal host upon cA4 sensing. Determination of the crystal structures of CaPN in apo and in the cA4-bound form revealed that cA4-CARF interactions trigger the conformational changes, leading to the dimerization of the CaPN dimers. These structural changes reposition D296, one of the active site residues in the catalytic pocket, to yield an active PIN domain nuclease. Together, these results unveil a novel molecular mechanism for the activation of cOA-activated Cas ancillary RNases in the CRISPR signaling pathway.},
}
@article {pmid40794863,
year = {2025},
author = {Brenker, L and Aschenbrenner, S and Bubeck, F and Staykov, K and Gebhardt, C and Wolf, B and Jendrusch, M and Kröll, AS and Becker, J and Ambiel, I and Fackler, OT and Grimm, D and Mathony, J and Niopek, D},
title = {A versatile anti-CRISPR platform for opto- and chemogenetic control of CRISPR-Cas9 and Cas12 across a wide range of orthologs.},
journal = {Nucleic acids research},
volume = {53},
number = {14},
pages = {},
doi = {10.1093/nar/gkaf752},
pmid = {40794863},
issn = {1362-4962},
support = {453202693//German Research Foundation/ ; //Aventis foundation/ ; //Rolf. M. Schwiete Stiftung/ ; //Ministry of Science, Research and Culture Baden-W&#xfc;rttemberg/ ; },
mesh = {*CRISPR-Cas Systems/genetics/drug effects ; *Gene Editing/methods ; Humans ; *Optogenetics/methods ; *CRISPR-Associated Proteins/antagonists &amp; inhibitors/genetics/metabolism ; CRISPR-Associated Protein 9/antagonists &amp; inhibitors/genetics ; Ligands ; Bacterial Proteins/genetics/metabolism ; HEK293 Cells ; Chemogenetics ; },
abstract = {CRISPR-Cas technologies have revolutionized life sciences by enabling programmable genome editing across diverse organisms. Achieving dynamic and precise control over CRISPR-Cas activity with exogenous triggers, such as light or chemical ligands, remains an important need. Existing tools for CRISPR-Cas control are often limited to specific Cas orthologs or selected applications, restricting their versatility. Anti-CRISPR (Acr) proteins are natural inhibitors of CRISPR-Cas systems and provide a flexible regulatory layer but are constitutively active in their native forms. In this study, we built on our previously reported concept for optogenetic CRISPR-Cas control with engineered, light-switchable anti-CRISPR proteins and expanded it from ortholog-specific Acrs towards AcrIIA5 and AcrVA1, broad-spectrum inhibitors of CRISPR-Cas9 and CRISPR-Cas12a, respectively. We then conceived and implemented a novel, chemogenetic anti-CRISPR platform based on engineered, circularly permuted ligand receptor domains, that together respond to six clinically relevant drugs. The resulting toolbox achieves both optogenetic and chemogenetic control of genome editing in human cells with a wide range of CRISPR-Cas effectors, including type II-A and II-C CRISPR-Cas9s, and CRISPR-Cas12a. In sum, this work establishes a versatile platform for the multidimensional control of CRISPR-Cas systems, with immediate applications in basic research and biotechnology, and with the potential for therapeutic use in the future.},
}
@article {pmid40794700,
year = {2025},
author = {Tagliaferri, TL and Krüttgen, A and Mendes, TAO and Gonçalves Dos Santos, S and Horz, HP},
title = {CRISPR-Cas9 targeting the blaKPC gene in a clinical isolate of Klebsiella michiganensis: Reduction of imipenem resistance and changes in genomic carbapenem resistance determinants.},
journal = {PloS one},
volume = {20},
number = {8},
pages = {e0328521},
doi = {10.1371/journal.pone.0328521},
pmid = {40794700},
issn = {1932-6203},
mesh = {*CRISPR-Cas Systems/genetics ; *Klebsiella/genetics/drug effects/isolation &amp; purification ; *Bacterial Proteins/genetics ; *Imipenem/pharmacology ; *beta-Lactamases/genetics ; Anti-Bacterial Agents/pharmacology ; Microbial Sensitivity Tests ; Humans ; Escherichia coli/genetics/drug effects ; Plasmids/genetics ; *Drug Resistance, Bacterial/genetics ; Porins/genetics ; Klebsiella Infections/microbiology/drug therapy ; Carbapenems/pharmacology ; },
abstract = {The CRISPR-Cas technology can be used to disable drug resistance genes. Since carbapenem resistance can be mediated by multiple resistance determinants, we here investigated the extent of re-sensizitation when targeting the blaKPC carbapenemase gene and assessed possible effects on porins and efflux pumps. While full re-sensitization was achieved in a laboratory strain of Escherichia coli solely equipped with blaKPC, resistance reduction in a clinical isolate of Klebsiella michiganensis was achieved in 63% of analyzed transformants, which was a consequence of plasmid copy number reduction and decreased blaKPC gene expression. Damages in the Cas9, as well as alterations in carbapenem-resistance promoting genes including ompK36 downregulation and mutations in the acrB gene were found, likely preventing more efficient re-sensitization. Hence, interference with a single resistance gene promoted the emergence of clonal variants that exhibit alterations in outer membrane proteins. Those bacterial countermeasures, however, were not sufficient to restore the original carbapenem-resistant phenotype.},
}
@article {pmid40794116,
year = {2025},
author = {Cai, R and Chai, N and Zhang, J and Tan, J and Liu, YG and Zhu, Q and Zeng, D},
title = {CRISPR/Cas system-mediated transgene-free or DNA-free genome editing in plants.},
journal = {TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik},
volume = {138},
number = {9},
pages = {210},
pmid = {40794116},
issn = {1432-2242},
support = {32272120//National Natural Science Foundation of China/ ; 2022YFF1002800//National Key Research and Development Program of China/ ; 2024GXNSFBA010386//Guangxi Natural Science Foundation Youth Science Fund Project/ ; 2024KY0071//The Project for Enhancing Young and Middle-aged Teacher's Research Basis Ability in Colleges of Guangxi/ ; },
mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems ; *Plants, Genetically Modified/genetics ; *Genome, Plant ; Transgenes ; *Crops, Agricultural/genetics ; Plant Breeding ; },
abstract = {CRISPR/Cas-based genome-editing technology serves as a powerful and versatile tool for genome modification. It has been broadly utilized in crop breeding to enhance traits such as yield, various quality attributes, and biotic and abiotic stress tolerance. Because of public safety concerns over genetically modified organisms (GMOs), many countries have established stringent regulatory policies for genetically modified plants, dramatically limiting the application of related products. However, genome editing in stably transformed plants can result in transgene-free progeny through self-pollination or hybridization or yield DNA-free gene-edited plants via transient transformation. These edited plants materially differ from GMOs and are referred to as genome-edited organisms (GEOs). GEOs have the potential to alleviate regulatory burdens and aid in commercialization. Various methods have been developed to expedite the creation of transgene-free or DNA-free GEOs. This review summarizes the various strategies for creating these types of GEOs based on the CRISPR/Cas systems. It also covers the advantages and drawbacks of these strategies. Additionally, we examine off-target effects and mitigation strategies for plant genome editing and outline regulatory policies for gene-edited crops in selected countries and regions. We hope this review offers valuable references for the advancement of transgene-free and DNA-free GEOs.},
}
@article {pmid40793766,
year = {2025},
author = {Xu, H-W and Wang, X-Y and Wei, Y and Cao, Y and Wang, S-G and Xia, P-F},
title = {Pathway crosstalk enables degradation of aromatic compounds in marine Roseobacter clade bacteria.},
journal = {Applied and environmental microbiology},
volume = {},
number = {},
pages = {e0097825},
doi = {10.1128/aem.00978-25},
pmid = {40793766},
issn = {1098-5336},
abstract = {Aromatic compounds are essential raw materials for almost all sectors of human societies but also persistent environmental pollutants recalcitrant to biodegradation. The ocean serves as a significant sink for these compounds, while their biological conversion routes remain poorly understood, hindering a comprehensive understanding of the marine carbon cycle and advancements in bioremediation and biological carbon upcycling. Here, we report the degradation pathway of aromatic molecules in the marine Roseobacter clade bacteria through multi-omics analysis and CRISPR-Cas-based genome editing. Using Roseovarius nubinhibens and 4-hydroxybenzoate (4HB) as representatives, we identified the transport of 4HB via TRAP, ABC, and MFS transporters. Then, we deciphered the integral β-ketoadipate pathway responsible for aromatic degradation. Next, we discovered a distinct pathway crosstalk at the final thiolation step, which serves as an intersection node of different pathways catalyzed by the 3-oxoadipyl-CoA thiolase from the β-ketoadipate pathway and the acetyl-CoA C-acetyltransferase and acetyl-CoA C-acyltransferase from the β-oxidation pathway. Finally, we proposed R. nubinhibens as a novel marine platform for systems-level interrogation and bioprospecting. Our study provides a foundation for leveraging Roseobacter clade bacteria as novel chassis for environmental and industrial innovations.IMPORTANCEAromatic compounds lie in an essential node of carbon cycling in both natural and engineered systems. Marine bacteria orchestrate the cycling of aromatic compounds in the ocean and, as emerging chassis, have shown unusual potentials in the degradation and valorization of aromatics. However, the corresponding metabolic pathway in marine bacteria remains poorly interpreted over decades, hindering further scientific interrogation and engineering practices. Here, we deciphered the complete degradation pathway of aromatic compounds in the marine Roseobacter clade bacteria and established a marine platform for systems and synthetic biology. Our study provides a paradigm for biological interrogation with combined multi-omics and the cutting-edge CRISPR-Cas approaches, laying a foundation for biological innovations with marine bacteria.},
}
@article {pmid40792102,
year = {2025},
author = {Chen, X and Xu, L and Luo, Z and Wang, L and Wang, Z and Li, Y and Jiao, X and Li, Q},
title = {Prevalence and genomic insights into type III-A CRISPR-Cas system acquisition in global Staphylococcus argenteus strains.},
journal = {Frontiers in cellular and infection microbiology},
volume = {15},
number = {},
pages = {1644286},
pmid = {40792102},
issn = {2235-2988},
mesh = {*CRISPR-Cas Systems/genetics ; *Staphylococcus/genetics/classification/isolation &amp; purification ; Humans ; *Genome, Bacterial ; *Staphylococcal Infections/microbiology/epidemiology/veterinary ; Animals ; Genomics ; DNA Transposable Elements ; Prevalence ; Food Microbiology ; Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {INTRODUCTION: The CRISPR-Cas system serves as a defense mechanism in bacteria and archaea, protecting them against the invasion of mobile genetic elements. Staphylococcus argenteus, a Gram-positive bacterium that diverged from Staphylococcus aureus, is characterized by the rare presence of the CRISPR-Cas system in only a few isolates.<br><br>METHODS: In this study, we analyzed the prevalence of the type III-A CRISPR-Cas system in 368 S. argenteus genome sequences from animals, food sources, and humans across 26 countries, available in public database.<br><br>RESULTS: Our findings revealed that 44.0% of these strains carry this immune system, with 98.1% of them belonging to the sequence type 2250 (ST2250). Genomic localization analysis indicated that the CRISPR-Cas is closely associated with SCCmec (mecA-&#x394;mecR1-IS1272-ccrB2-ccrA2) or Insertion sequence 1272 (IS1272) transposase. Further analysis identified a common IS1272 target inverted repeats (IR) sequence in ST2250 strains, providing insights into why these strains are more likely to acquire the CRISPR-Cas system. CRISPR typing identified 41 sequences types, classifying these strains into two clusters, with Cluster II being the predominant one. Homology analysis of spacers revealed that all the identified 15 spacers exhibited homology to sequences from plasmids, lytic phages, or prophages.<br><br>CONCLUSION: This study suggests that the acquisition of the CRISPR-Cas system in S. argenteus enhances its resistance to phage attacks and plasmid invasions in environmental settings, potentially posing significant challenges for clinical treatment of infections caused by these strains and hindering efforts to control their spread in food products using phage-based interventions.},
}
@article {pmid40790872,
year = {2025},
author = {Kale, SM and Paina, C and Füchtbauer, WS and Bjarup, P and Sarup, PM and Schatz-Jakobsen, JA and Orabi, J and Borum, F and Jahoor, A and Brinch-Pedersen, H},
title = {An introgression from Triticum timopheevii reduces grain protein content in winter wheat populations.},
journal = {The plant genome},
volume = {18},
number = {3},
pages = {e70090},
doi = {10.1002/tpg2.70090},
pmid = {40790872},
issn = {1940-3372},
support = {Halm til det hele//Promilleafgiftsfonden for Landbrugs/ ; },
mesh = {*Triticum/genetics/metabolism ; Genome-Wide Association Study ; *Grain Proteins/metabolism ; Phenotype ; Quantitative Trait Loci ; Plant Breeding ; *Genetic Introgression ; Edible Grain/genetics ; Plant Proteins/genetics/metabolism ; Chromosomes, Plant/genetics ; },
abstract = {Improving grain protein content (GPC) in wheat (Triticum aestivum L.) is crucial for enhancing end-use quality and ensuring efficient nitrogen (N) utilization, thereby reducing environmental damage caused by excess N. However, progress in increasing GPC has been limited because of the strong negative correlation between GPC and grain yield (GY), as well as the scarcity of multi-location, multi-year phenotypic data. In this study, we analyzed the variation in GPC, GY, and grain protein deviation (GPD) using multi-location, multi-year phenotypic data from winter wheat varieties in Scandinavian regions. As reported previously, we observed a negative correlation between GY and GPC, with recent cultivars showing higher GY but lower GPC. Additionally, a genome-wide association study (GWAS) in two independent populations identified significant marker-trait associations (MTAs) for GPC and GPD, with key MTAs located on chromosome 2B (chr2B), highlighting its central role in the regulation of these traits. Interestingly, the MTA for GPD on chr2B coincided with an introgression from Triticum timopheevii, which was associated with reduced GPC and GPD in elite lines carrying this region. This introgression, which contains a powdery mildew resistance gene (Pm6), appears to negatively affect GPC, likely due to linkage drag. These findings emphasize the importance of chr2B in wheat breeding and suggest that advanced genomic techniques, such as mutagenesis and CRISPR-Cas, could be employed to mitigate negative pleiotropic effects and improve GPC and GPD. Overall, this study provides valuable insights into the genetic architecture underlying GPC in wheat and offers directions for future breeding strategies aimed at enhancing protein content.},
}
@article {pmid40790091,
year = {2025},
author = {Kocsy, K and Wilkinson, H and Felix-Ilemhenbhio, F and Bax, B and Van Agtmael, T and Azzouz, M and Majid, A},
title = {Gene editing for collagen disorders: current advances and future perspectives.},
journal = {Gene therapy},
volume = {},
number = {},
pages = {},
pmid = {40790091},
issn = {1476-5462},
abstract = {Collagen disorders encompass a wide range of genetic conditions caused by pathogenic variants in collagen genes for which there is an unmet need for treatments. They present various clinical features, ranging from localised tissue abnormalities to severe systemic complications. Symptoms differ significantly and depend on the pathogenic variant, which can affect various systems, including the musculoskeletal, cardiovascular, and respiratory systems, highlighting the complex implications of collagen gene pathogenic variants and the wide range of expression patterns among different collagen types. Gene-editing technologies, particularly Clustered Regularly Interspaced Palindromic Repeats (CRISPR)-Cas systems, have emerged as promising therapeutic options for these disorders, representing a putative one-for-all treatment strategy. This review provides an overview of current gene-editing strategies aimed at collagen-related diseases, including osteogenesis imperfecta, Alport syndrome, and dystrophic epidermolysis bullosa. We explore the application of CRISPR-Cas9, which facilitates targeted DNA modifications, base editing (BE), and prime editing (PE), enabling precise single-nucleotide alterations without double-strand breaks (DSB). Preclinical and clinical studies have shown the potential of gene therapy to enhance collagen production, restore tissue integrity, and alleviate symptoms. However, challenges persist, including the lack of recurring mutations, the need for improved delivery methods, the reduction of off-target effects, and the development of novel therapies. Despite these challenges, advancements in gene editing techniques appear promising in enhancing editing efficiency while minimising unintended mutations, paving the way for more precise and safer genetic interventions for collagen disorders. Gene editing is fundamentally transforming medicine and biotechnology. Its applications encompass advanced diagnostics, tailored therapeutic strategies, and solutions for rare genetic disorders. By enabling precise genetic modifications, gene editing is paving the way for treatments of previously untreatable diseases, including those linked to collagen pathogenic variants. This review discusses the latest advancements in gene therapy techniques targeting collagen-related disorders. It explores innovative approaches like CRISPR-Cas9-mediated gene editing and highlights emerging strategies, such as allele-specific inactivation and base editing (BE). By examining these cutting-edge therapies and their potential clinical applications, this review highlights the transformative impact of gene editing in treating collagen-related conditions, while also identifying critical challenges and future directions for research.},
}
@article {pmid40696172,
year = {2025},
author = {Xu, Z and Wang, G and Zhu, X and Wang, R and Zhu, L and Tu, L and Liu, Y and Peng, R and Lindsey, K and Wang, M and Zhang, X and Jin, S},
title = {Genome assembly of two allotetraploid cotton germplasms reveals mechanisms of somatic embryogenesis and enables precise genome editing.},
journal = {Nature genetics},
volume = {57},
number = {8},
pages = {2028-2039},
pmid = {40696172},
issn = {1546-1718},
support = {32325039//China National Funds for Distinguished Young Scientists/ ; 32201856//National Science Foundation of China | Young Scientists Fund/ ; },
mesh = {*Gossypium/genetics ; *Genome, Plant/genetics ; *Gene Editing/methods ; Tetraploidy ; *Plant Somatic Embryogenesis Techniques/methods ; Retroelements/genetics ; CRISPR-Cas Systems ; },
abstract = {Somatic embryogenesis is crucial for plant genetic engineering, yet the underlying mechanisms in cotton remain poorly understood. Here we present a telomere-to-telomere assembly of Jin668 and a high-quality assembly of YZ1, two highly regenerative allotetraploid cotton germplasms. The completion of the Jin668 genome enables characterization of ~30.1 Mb of centromeric regions invaded by centromeric retrotransposon of maize and Tekay retrotransposons, an ~8.1 Mb 5S rDNA array containing 25,190 copies and a ~75.1 Mb major 45S rDNA array with 8,131 copies. Comparative analyses of regenerative and recalcitrant genotypes reveal dynamic transcriptional patterns and chromatin accessibility during the initial regeneration process. A hierarchical gene regulatory network identifies AGL15 as a contributor to regeneration. Additionally, we demonstrate that genetic variation affects sgRNA target sites, while the Jin668 genome assembly reduces the risk of off-target effects in CRISPR-based genome editing. Together, the complete Jin668 genome reveals the complexity of genomic regions and cotton regeneration, and improves the precision of genome editing.},
}
@article {pmid40607979,
year = {2025},
author = {Cohen, O and Maru, P and Liang, Q and Saeij, JPJ},
title = {Surface antigen SAG1 mediates Toxoplasma gondii fitness and host cell attachment in IFNγ-stimulated cells.},
journal = {Infection and immunity},
volume = {93},
number = {8},
pages = {e0001025},
doi = {10.1128/iai.00010-25},
pmid = {40607979},
issn = {1098-5522},
support = {//Societe de Pathologie Infectieuse de Langue Francaise/ ; R01AI173803//National Institute of Allergy and Infectious Diseases/ ; },
mesh = {*Toxoplasma/physiology/genetics/immunology ; *Interferon-gamma/immunology/pharmacology ; *Antigens, Protozoan/genetics/metabolism/immunology ; *Protozoan Proteins/genetics/metabolism/immunology ; *Host-Parasite Interactions ; Animals ; Humans ; Toxoplasmosis/parasitology/immunology ; Cell Adhesion ; CRISPR-Cas Systems ; Mice ; },
abstract = {Toxoplasma gondii is an obligate intracellular protozoan parasite that can establish lifelong infections and cause severe disease in immunocompromised individuals. Interferon gamma (IFNγ) is a key host defense cytokine that induces a variety of toxoplasmacidal mechanisms. Recent CRISPR/Cas9 loss-of-function screens identified multiple Toxoplasma genes important for fitness in IFNγ-stimulated cells. One consistent hit in several screens was the parasite surface antigen, SAG1. Here, we used CRISPR/Cas9 to generate a SAG1 knockout strain and found that SAG1 is important for parasite fitness specifically in IFNγ-stimulated cells. Mechanistic studies revealed that host surface sialic acids are important for parasite attachment, especially in IFNγ-stimulated cells. SAG1-deficient parasites had reduced attachment efficiency, which was exacerbated in IFNγ-treated cells. These findings highlight the role of SAG1 in mediating robust parasite attachment, especially in the context of immune pressure.},
}
@article {pmid40579538,
year = {2025},
author = {Hanlon, VCT and Cagan, A and Eves-van den Akker, S},
title = {How and when organisms edit their own genomes.},
journal = {Nature genetics},
volume = {57},
number = {8},
pages = {1823-1834},
pmid = {40579538},
issn = {1546-1718},
support = {BB/S006397/1//RCUK | Biotechnology and Biological Sciences Research Council (BBSRC)/ ; BB/X006352/1//RCUK | Biotechnology and Biological Sciences Research Council (BBSRC)/ ; BB/Y513246/1//RCUK | Biotechnology and Biological Sciences Research Council (BBSRC)/ ; RPG-2023-001//Leverhulme Trust/ ; R01AG087974//U.S. Department of Health &amp; Human Services | National Institutes of Health (NIH)/ ; },
mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; Animals ; Humans ; *Genome/genetics ; Bacteria/genetics ; Host-Pathogen Interactions/genetics ; },
abstract = {Mutations are often thought of as untargeted and non-adaptive, but in rare cases, organisms perform programmed, targeted and adaptive rearrangements of their own DNA sequences. Notable examples include the somatic diversification of immunoglobulin genes, which is the foundation of the vertebrate immune system, and natural CRISPR spacer arrays in bacteria, which recognize and cleave foreign DNA. These systems, along with a dozen known analogs scattered across the tree of life, often underlie critical biological functions, particularly in host-pathogen conflicts. In this Review, we compare the mechanisms by which organisms edit their own genomes. We show that superficially dissimilar editing systems often rely on surprisingly similar genetic mechanisms, regardless of function or taxon. Finally, we argue that the recurrence of editing in host-pathogen conflicts and the bias to a handful of well-studied organisms strongly suggest that new editing systems will be found in understudied pathogens and their hosts.},
}
@article {pmid40519079,
year = {2025},
author = {Geronazzo, J and Heimerl, A and Lindell, L and McCrimmon, S and Stormer, C and Horvai, B and Johnson, IP and Peterson, TM and Zuckerman, J and Scott, AI and Course, MM},
title = {Characterizing fatty acid oxidation genes in Drosophila.},
journal = {G3 (Bethesda, Md.)},
volume = {15},
number = {8},
pages = {},
doi = {10.1093/g3journal/jkaf139},
pmid = {40519079},
issn = {2160-1836},
support = {//CC Natural Sciences Executive Committee Research and Development/ ; //CC Student Collaborative Research/ ; //Seattle Children's Research Institute Center for Clinical and Translational Research/ ; //AIS/ ; },
mesh = {Animals ; *Fatty Acids/metabolism ; Oxidation-Reduction ; *Drosophila Proteins/genetics/metabolism ; Phenotype ; *Drosophila/genetics/metabolism ; Humans ; CRISPR-Cas Systems ; Mutation ; *Drosophila melanogaster/genetics/metabolism ; },
abstract = {In this study, we leverage the power and tractability of Drosophila genetics to better understand the molecular mechanisms underlying a group of rare genetic diseases known as fatty acid oxidation disorders. We use CRISPR-Cas9 to generate mutations in 6 putative fatty acid oxidation genes in Drosophila, then analyze the phenotypes and acylcarnitine profiles of these flies. We find that while Arc42 and CG4860 are both predicted orthologs of human ACADS, only Arc42 loss of function mirrors the acylcarnitine profile of ACADS loss of function. Acylcarnitine profiles also support our previous identification of Mcad as the likely ACADM ortholog, and reveal the deleterious effects of a single codon deletion in Mtpα (the predicted human HADHA ortholog). Finally, we observe that loss of function in Etf-QO and in CG7834-predicted orthologs of human ETFDH and ETFB, respectively-is homozygous lethal in flies. Producing animal models like these will enable new approaches to studying fatty acid oxidation disease progression, symptomatic variability, and therapeutic intervention.},
}
@article {pmid40476353,
year = {2025},
author = {Weisman, AS and Fisher, NM and Hunter, CP},
title = {Efficient iterative CRISPR/Cas9 editing using sid-1 co-conversion and feeding RNAi in Caenorhabditis elegans.},
journal = {G3 (Bethesda, Md.)},
volume = {15},
number = {8},
pages = {},
doi = {10.1093/g3journal/jkaf128},
pmid = {40476353},
issn = {2160-1836},
support = {GM089795/NH/NIH HHS/United States ; 62579/NH/NIH HHS/United States ; },
mesh = {Animals ; *Caenorhabditis elegans/genetics ; *CRISPR-Cas Systems ; *Gene Editing/methods ; *Caenorhabditis elegans Proteins/genetics ; *RNA Interference ; Phenotype ; Membrane Proteins ; },
abstract = {We present a sid-1 loss-of-function and restoration-of-function CRISPR/Cas9 co-conversion protocol in Caenorhabditis elegans. Introducing CRISPR reagents that induce sid-1 loss-of-function can produce survivors on lethal RNAi foods while reagents that induce sid-1 restoration-of-function can be screened for restoration of visible RNAi phenotypes. Both methods efficiently reduce the pool of candidates from hundreds or thousands of F1 progeny to tens with minimal experimenter effort. Furthermore, our optimized sid-1 CRISPR design allows a high ratio of CRISPR reagents targeting the gene of interest, maximizing successful co-conversion events. The interconvertibility of the sid-1 locus readily enables this strategy to be leveraged to iteratively create complex strains with multiple gene edits.},
}
@article {pmid40460280,
year = {2025},
author = {Nalley, MJ and Banerjee, S and Huang, MY and Madhani, HD},
title = {Near 100% efficient homology-dependent genome engineering in the human fungal pathogen Cryptococcus neoformans.},
journal = {G3 (Bethesda, Md.)},
volume = {15},
number = {8},
pages = {},
doi = {10.1093/g3journal/jkaf118},
pmid = {40460280},
issn = {2160-1836},
support = {//R01 AI000272/ ; },
mesh = {*Cryptococcus neoformans/genetics ; *Genome, Fungal ; CRISPR-Cas Systems ; *Gene Editing/methods ; Humans ; DNA End-Joining Repair ; *Genetic Engineering/methods ; RNA, Guide, CRISPR-Cas Systems/genetics ; },
abstract = {We recently described CRISPR/Cas9-based short homology-dependent genome engineering in the human fungal pathogen Cryptococcus neoformans, a haploid budding yeast that is the most common cause of fungal meningitis and an emerging model organism. This was achieved by electroporation of strains stably expressing a codon-optimized Cas9 with 2 separate DNA molecules, one encoding a selectable marker flanked by short homology arms and a second encoding a sgRNA under the control of the U6 snRNA promoter. However, the efficiency of desired homology-dependent repair relative to undesired non-homologous end-joining (NHEJ) events can be low and variable. Here, we describe methods and strains enabling extremely efficient (∼99%) homology-dependent genome editing in C. neoformans. This high-efficiency method requires 2 manipulations. First, we placed the sgRNA-expressing segment into the marker-containing DNA flanked by targeting homology; thus, only a single DNA molecule is introduced into cells. Second, we used a strain mutant for the non-homologous end-joining factor Ku80 (encoded by YKU80). We also report the engineering of a yku80::amdS mutant strain harboring an insertion mutation that can be removed scarlessly via recombination between direct repeats. This enables the functional restoration of YKU80 after homology-dependent genome editing after selection against the amdS marker using fluoroacetamide. This approach minimizes documented drawbacks of using Ku-defective strains in downstream experiments. Finally, we describe a plasmid series that enables rapid cloning of sgRNA-marker constructs for genomic manipulation of C. neoformans, including gene deletion and C-terminal tagging. These methods, strains, and plasmids accelerate the genomic manipulation of C. neoformans.},
}
@article {pmid40188316,
year = {2025},
author = {Teter, OM and McQuade, A and Hagan, V and Liang, W and Dräger, NM and Sattler, SM and Holmes, BB and Castillo, VC and Papakis, V and Leng, K and Boggess, S and Nowakowski, TJ and Wells, J and Kampmann, M},
title = {CRISPRi-based screen of autism spectrum disorder risk genes in microglia uncovers roles of ADNP in microglia endocytosis and synaptic pruning.},
journal = {Molecular psychiatry},
volume = {30},
number = {9},
pages = {4176-4193},
pmid = {40188316},
issn = {1476-5578},
support = {U01 MH115747/MH/NIMH NIH HHS/United States ; R01 MH125516/MH/NIMH NIH HHS/United States ; 2034836//National Science Foundation (NSF)/ ; AARF-22-973222//Alzheimer&apos;s Association/ ; 2022-A-016-FEL//Larry L. Hillblom Foundation (Larry L. Hillblom Foundation, Inc.)/ ; U01 MH115747/MH/NIMH NIH HHS/United States ; R01 MH125516/MH/NIMH NIH HHS/United States ; },
mesh = {*Microglia/metabolism ; *Autism Spectrum Disorder/genetics/metabolism ; Humans ; Endocytosis/genetics/physiology ; Phagocytosis/genetics ; Neurons/metabolism ; Induced Pluripotent Stem Cells/metabolism ; *Neuronal Plasticity/genetics/physiology ; CRISPR-Cas Systems/genetics ; Coculture Techniques ; *Nerve Tissue Proteins/genetics/metabolism ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Synapses/metabolism ; Genetic Predisposition to Disease/genetics ; Brain/metabolism ; },
abstract = {Autism Spectrum Disorders (ASD) are a set of neurodevelopmental disorders with complex biology. The identification of ASD risk genes from exome-wide association studies and de novo variation analyses has enabled mechanistic investigations into how ASD-risk genes alter development. Most functional genomics studies have focused on the role of these genes in neurons and neural progenitor cells. However, roles for ASD risk genes in other cell types are largely uncharacterized. There is evidence from postmortem tissue that microglia, the resident immune cells of the brain, appear activated in ASD. Here, we used CRISPRi-based functional genomics to systematically assess the impact of ASD risk gene knockdown on microglia activation and phagocytosis. We developed an iPSC-derived microglia-neuron coculture system and high-throughput flow cytometry readout for synaptic pruning to enable parallel CRISPRi-based screening of phagocytosis of beads, synaptosomes, and synaptic pruning. Our screen identified ADNP, a high-confidence ASD risk genes, as a modifier of microglial synaptic pruning. We found that microglia with ADNP loss have altered endocytic trafficking, remodeled proteomes, and increased motility in coculture.},
}
@article {pmid39394482,
year = {2025},
author = {Mantena, S and Pillai, PP and Petros, BA and Welch, NL and Myhrvold, C and Sabeti, PC and Metsky, HC},
title = {Model-directed generation of artificial CRISPR-Cas13a guide RNA sequences improves nucleic acid detection.},
journal = {Nature biotechnology},
volume = {43},
number = {8},
pages = {1266-1273},
pmid = {39394482},
issn = {1546-1696},
support = {U01AI151812//Division of Intramural Research, National Institute of Allergy and Infectious Diseases (Division of Intramural Research of the NIAID)/ ; T32 GM144273/GM/NIGMS NIH HHS/United States ; T32GM007753//U.S. Department of Health &amp; Human Services | NIH | National Institute of General Medical Sciences (NIGMS)/ ; T32GM144273//U.S. Department of Health &amp; Human Services | NIH | National Institute of General Medical Sciences (NIGMS)/ ; U19AI110818//Division of Intramural Research, National Institute of Allergy and Infectious Diseases (Division of Intramural Research of the NIAID)/ ; D18AC00006//United States Department of Defense | Defense Advanced Research Projects Agency (DARPA)/ ; 75D30122C15113//U.S. Department of Health &amp; Human Services | Centers for Disease Control and Prevention (CDC)/ ; K01AI163498//U.S. Department of Health &amp; Human Services | NIH | National Institute of Allergy and Infectious Diseases (NIAID)/ ; NU50CK000629//U.S. Department of Health &amp; Human Services | Centers for Disease Control and Prevention (CDC)/ ; INV-034761/GATES/Gates Foundation/United States ; INV-034761/GATES/Gates Foundation/United States ; },
mesh = {*CRISPR-Cas Systems/genetics ; *RNA, Guide, CRISPR-Cas Systems/genetics ; Algorithms ; Base Sequence ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; },
abstract = {CRISPR guide RNA sequences deriving exactly from natural sequences may not perform optimally in every application. Here we implement and evaluate algorithms for designing maximally fit, artificial CRISPR-Cas13a guides with multiple mismatches to natural sequences that are tailored for diagnostic applications. These guides offer more sensitive detection of diverse pathogens and discrimination of pathogen variants compared with guides derived directly from natural sequences and illuminate design principles that broaden Cas13a targeting.},
}
@article {pmid40790054,
year = {2025},
author = {Patel, MA and Boribong, BP and Sinha, H and Xiao, B and Xie, K and Vo, PQN and Chin, AB and Ellouzi, A and Little, SR and Shih, S and Wu, H and Muller, WJ and Hirukawa, A},
title = {Miniaturized scalable arrayed CRISPR screening in primary cells enables discovery at the single donor resolution.},
journal = {Scientific reports},
volume = {15},
number = {1},
pages = {29350},
pmid = {40790054},
issn = {2045-2322},
mesh = {Humans ; *CRISPR-Cas Systems ; Electroporation/methods ; *Gene Editing/methods ; Microfluidics/methods ; CD4-Positive T-Lymphocytes/metabolism ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Miniaturization ; },
abstract = {High-efficiency gene editing in primary human cells is critical for advancing therapeutic development and functional genomics, yet conventional electroporation platforms often require high cell input and are poorly suited to parallelized experiments. Here we introduce a next-generation digital microfluidics (DMF) electroporation platform that enables high-throughput, low-input genome engineering using discrete droplets manipulated on a planar electrode array. The system supports 48 independently programmable reaction sites and integrates seamlessly with laboratory automation, allowing efficient delivery of CRISPR-Cas9 RNPs and mRNA cargo into as few as 3,000 primary human cells per condition. The platform was validated across diverse primary human cell types and cargo modalities, demonstrating efficient delivery of various cargo, with high rates of transfection, gene knockout via non-homologous end joining, and precise knock-in through homology-directed repair. To showcase its utility in functional genomics, we applied the platform to an arrayed CRISPR-Cas9 screen in chronically stimulated human CD4[+] T cells, identifying novel regulators of exhaustion, including epigenetic and transcriptional modulators. These findings establish our DMF-based electroporation platform as a powerful tool for miniaturized genome engineering in rare or precious cell populations and provide a scalable framework for high-content genetic screening in primary human cells.},
}
@article {pmid40790042,
year = {2025},
author = {Li, H and He, Y and Jiang, J and Liu, Z and Liu, Y and Shi, Q and Ding, J and Li, H and Sun, W and Hu, X and Chen, Z and He, X},
title = {CRISPR screening reveals that RNA helicase DDX41 triggers ribosome biogenesis and cancer progression through R-loop-mediated RPL/RPS transcription.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {7409},
pmid = {40790042},
issn = {2041-1723},
support = {82121004//National Natural Science Foundation of China (National Science Foundation of China)/ ; 82472626//National Natural Science Foundation of China (National Science Foundation of China)/ ; 82472641//National Natural Science Foundation of China (National Science Foundation of China)/ ; 82172937//National Natural Science Foundation of China (National Science Foundation of China)/ ; },
mesh = {*DEAD-box RNA Helicases/genetics/metabolism ; Humans ; *Ribosomes/metabolism/genetics ; Animals ; Mice ; *Liver Neoplasms/genetics/pathology/metabolism/drug therapy ; Cell Line, Tumor ; Gene Expression Regulation, Neoplastic ; *Ribosomal Proteins/genetics/metabolism ; Transcription, Genetic ; Disease Progression ; Histone Acetyltransferases/metabolism ; Promoter Regions, Genetic ; CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; Neoplasms/genetics/pathology ; Protein Biosynthesis ; },
abstract = {The RNA helicase DDX41 is a DEAD-box helicase that is well known as a virus sensor in dendritic cells and a tumor suppressor that is frequently mutated in myeloid neoplasms. However, the functions and relevance of DDX41 in solid tumors remain largely unexplored. In this study, through in vivo CRISPR screening, we demonstrate that DDX41 is highly expressed in various solid tumor types and promotes tumorigenicity in liver cancer. Mechanistically, DDX41 facilitates R-loop processing and accelerates the transcription of RPL/RPS genes, thereby promoting ribosome biogenesis and protein synthesis. Additionally, we show that the acetyltransferase KAT8 is required for H3K9ac modification of the DDX41 promoter and that NR2C1/NR2C2 are responsible for DDX41 expression. Moreover, elevated DDX41 levels increase liver cancer cell sensitivity to protein synthesis inhibitors; treatment with homoharringtonine (HHT), an approved drug, significantly inhibits tumor growth in DDX41-overexpressing liver cancer models. Taken together, the results of this study highlight that DDX41 acts as an oncogene in liver cancer and suggest that protein synthesis inhibition may be a promising therapy for liver cancers with high DDX41 expression.},
}
@article {pmid40790018,
year = {2025},
author = {Wang, K and Wang, J and Yang, X and Sun, W and Sheng, G and Wang, Y},
title = {Structural insights into Type II-D Cas9 and its robust cleavage activity.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {7396},
pmid = {40790018},
issn = {2041-1723},
support = {Z231100007223004//Beijing Municipal Science and Technology Commission/ ; 32330055, 31930065, 22121003, 32071198, 32071444//National Natural Science Foundation of China (National Science Foundation of China)/ ; 5232022//Natural Science Foundation of Beijing Municipality (Beijing Natural Science Foundation)/ ; XDB0570000, XDB37010202//Chinese Academy of Sciences (CAS)/ ; },
mesh = {*CRISPR-Associated Protein 9/metabolism/chemistry/genetics/ultrastructure ; Cryoelectron Microscopy ; DNA/metabolism/chemistry/genetics ; RNA, Guide, CRISPR-Cas Systems/metabolism/genetics/chemistry ; DNA Cleavage ; CRISPR-Cas Systems ; Gene Editing ; *Bacterial Proteins/metabolism/genetics/chemistry ; Catalytic Domain ; },
abstract = {Type II-D Cas9 proteins (Cas9d) are more compact than typical Type II-A/B/C Cas9s. Here, we demonstrate that NsCas9d from Nitrospirae bacterium RBG_13_39_12 derived from a metagenomic assembly exhibits robust dsDNA cleavage activity comparable to SpCas9 in vitro. Unlike typical Cas9 enzymes that generate blunt ends, NsCas9d produces 3-nucleotide staggered overhangs. Our high-resolution cryo-EM structure of the NsCas9d-sgRNA-dsDNA complex in its catalytic state reveals the target and non-target DNA strands positioned within the HNH and RuvC catalytic pockets, respectively. NsCas9d recognizes the 5'-NRG-3' protospacer adjacent motif (PAM), with 5'-NGG-3' showing the highest cleavage efficiency. Its sgRNA structure, resembling the 5' end of IscB ωRNA, along with structural features shared with other Cas9 variants, suggests that Cas9d are hypothesized to resemble evolutionary intermediates between other Cas9 sub-types and IscB. These findings deepen our understanding of Cas9 evolution and mechanisms, highlighting NsCas9d as a promising genome-editing tool due to its compact size, DNA cleavage pattern, and efficient PAM recognition.},
}
@article {pmid40789840,
year = {2025},
author = {Goyal, H and Kaur, J},
title = {Long non-coding RNAs and autophagy: dual drivers of Hepatocellular carcinoma progression.},
journal = {Cell death discovery},
volume = {11},
number = {1},
pages = {376},
pmid = {40789840},
issn = {2058-7716},
abstract = {Hepatocellular carcinoma (HCC), a leading cause of cancer-related mortality worldwide, is characterized by poor prognosis, high recurrence rates, and limited responsiveness to current therapies. Autophagy, a conserved catabolic pathway essential for cellular homeostasis, plays a paradoxical role in HCC, acting as a tumor suppressor during initiation but promoting survival and progression in advanced stages. Long non-coding RNAs (lncRNAs) have emerged as critical regulators of autophagy, influencing tumorigenesis, metastasis, and therapy resistance through mechanisms such as miRNA sponging, chromatin remodeling, and protein interactions. This review describes how autophagy contributes to HCC at different stages, outlines the dual functions of lncRNAs as oncogenic drivers or tumor suppressors, and illustrates their integration into key signaling networks of autophagy (e.g., PI3K/AKT/mTOR, AMPK, Beclin-1). LncRNAs have been shown to modulate drug resistance, including resistance to first-line agents, by altering autophagic flux and associated molecular pathways. We also explored emerging strategies for targeting the lncRNA-autophagy axis, such as siRNAs, antisense oligonucleotides, and CRISPR/Cas systems, that have shown promise in preclinical studies and may be adapted for HCC. Furthermore, autophagy-related lncRNAs hold potential as non-invasive diagnostic and prognostic biomarkers and as predictors of recurrence. Integrating multi-omics approaches to validate these candidates will be critical for translation into clinical practice. Collectively, this review highlights the lncRNA-autophagy network as a promising frontier of biomarker discovery for precision diagnostics and targets for innovative therapeutics. The regulatory role of lncRNAs in autophagy presents a paradigm shift, heralding new strategies for targeted treatment.},
}
@article {pmid40789802,
year = {2025},
author = {Noormohamadi, H and Soleimani Samarkhazan, H and Kargar, M and Maroufi, F and Servatian, N and Davami, F},
title = {CRISPR/Cas technologies in pancreatic cancer research and therapeutics: recent advances and future outlook.},
journal = {Discover oncology},
volume = {16},
number = {1},
pages = {1530},
pmid = {40789802},
issn = {2730-6011},
abstract = {Pancreatic cancer is marked by a poor prognosis and an exceptionally high mortality rate, with its aggressive nature contributing to its classification as a highly malignant disease. For effective therapeutic strategies, the development of sophisticated and regulated DNA manipulation methods is essential. Originally part of the prokaryotic immune system, CRISPR/Cas has emerged as a pivotal genome-editing tool with promising applications in pancreatic cancer research and therapy. This gene editing method is known for simplicity, rapid advancement, and superior precision compared to earlier techniques. Its adaptability allows precise gene editing for therapeutic purposes, including oncogene silencing and correction of pathogenic mutations. Additionally, CRISPR-driven gene editing has facilitated the development of pancreatic cancer models, which serve as valuable platforms for drug discovery and personalized treatment strategies, offering deeper insights into the genetic landscape of pancreatic tumors. This article provides an overview of the current applications of CRISPR technology in gene therapy and cancer research, particularly in the context of pancreatic cancer, and lays the foundation for future studies.},
}
@article {pmid40789027,
year = {2025},
author = {Huang, Y and Li, C and Desingu Rajan, AR and Bronner, ME},
title = {Sox11 genes affect neuronal differentiation in the developing zebrafish enteric nervous system.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {122},
number = {33},
pages = {e2510548122},
doi = {10.1073/pnas.2510548122},
pmid = {40789027},
issn = {1091-6490},
support = {HD105604//HHS | NIH | Eunice Kennedy Shriver National Institute of Child Health and Human Development (NICHD)/ ; DK133480//HHS | NIH | National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)/ ; },
mesh = {Animals ; *Zebrafish/genetics/embryology ; *Enteric Nervous System/embryology/metabolism/cytology ; *Zebrafish Proteins/genetics/metabolism ; *SOXC Transcription Factors/genetics/metabolism ; *Cell Differentiation/genetics ; Gene Expression Regulation, Developmental ; Neural Crest/metabolism/cytology ; *Neurons/metabolism/cytology ; CRISPR-Cas Systems ; Neurogenesis/genetics ; },
abstract = {The vertebrate enteric nervous system (ENS) is derived from vagal neural crest cells, which enter the foregut as progenitors that migrate from rostral to caudal to populate the entire length of the gut. Here, we show that transcription factors sox11a and sox11b, zebrafish orthologs of the human SOX11 gene, are highly expressed in neural crest cells transitioning from progenitors to differentiating neuronal subtypes. Accordingly, CRISPR-Cas9 depletion shows that loss of sox11 paralogs reduces the number of neurons that express the inhibitory motor neuron marker adcyap1b without affecting cell proliferation or death. Transcription factor footprinting analysis of open chromatin regions identified by ATAC-seq reveals Sox11 binding sites in the adcyap1b enhancer. Furthermore, mutational analysis shows these binding sites are required for mediating enhancer-driven reporter expression. Taken together, our results demonstrate an important and previously unrecognized role for sox11a and sox11b in neuronal subtype specification in the developing zebrafish ENS.},
}
@article {pmid40788907,
year = {2025},
author = {Zhang, Y and Fu, Z and Yang, B and Wang, J and Lu, T and Shi, DL and Shao, M},
title = {Generation of Maternal Mutants Using zpc:cas9 Knock-in Zebrafish.},
journal = {Journal of visualized experiments : JoVE},
volume = {},
number = {221},
pages = {},
doi = {10.3791/68642},
pmid = {40788907},
issn = {1940-087X},
mesh = {Animals ; *Zebrafish/genetics ; Female ; *Gene Knock-In Techniques/methods ; *CRISPR-Cas Systems ; Animals, Genetically Modified ; *RNA-Binding Proteins/genetics ; *Zebrafish Proteins/genetics ; },
abstract = {Oogenesis and early embryonic development are critically dependent on maternally derived mRNAs and proteins. Eliminating these maternal factors necessitates homozygous mutations in female zebrafish, often resulting in lethal or infertile phenotypes, which prevent the acquisition of maternal mutant embryos. Our previous work introduced a rapid approach to bypass zygotic lethality through oocyte-specific genome editing. However, the previously reported cas9 transgene exhibits instability and undergoes gradual silencing over successive generations. Furthermore, the presence of Tol2 transposable elements flanking the zpc:cas9 cassette in this line hinders the potential for further sgRNA transgenesis using Tol2 system, which is currently the most efficient transgenic system in zebrafish. Consequently, there is a critical need for a Tol2-free zebrafish line that ensures stable and robust oocyte-specific Cas9 expression. Here, we present a line with zpccas9 knock-in at the rbm24a locus that addresses this requirement. Using this enhanced tool, we provide a pipeline for the rapid generation of maternal mutants of genes with zygotically lethal mutant phenotypes within the zebrafish model.},
}
@article {pmid40788566,
year = {2025},
author = {Risse, J and Pietrek, L and Cantz, T and Krzemien, M and Schnalke, J and Eggenschwiler, R and Heinemann, T and Dederer, HG},
title = {"Snip, snip, cure"? Philosophical, legal and biomedical perspectives on novel somatic genomic therapies.},
journal = {Medicine, health care, and philosophy},
volume = {},
number = {},
pages = {},
pmid = {40788566},
issn = {1572-8633},
abstract = {The advent of innovative techniques, such as the CRISPR/Cas system, has opened up a new range of possibilities for modifying the genome, with the potential to address previously unmet therapeutic needs of patients with genetic diseases. These new possibilities have not only raised ethical concerns but also challenged existing classifications of genome modification techniques. While the legal status of some of these new therapies remains uncertain, there is an ongoing debate within philosophy of biology about the information-related metaphors adopted by scientists to describe and classify the genome and its therapeutic modification. Given the continuing advance of new genomic therapies, we show, employing an interdisciplinary approach, that a comprehensive framework for the classification of these technologies is needed to resolve legal and philosophical issues. The first section provides an analysis of the current state of novel genome-modifying techniques in medical genetics. In the second section, we assess the regulatory status of these techniques within the European regulatory framework for advanced therapy medicinal products (ATMPs). Drawing on these results, we argue in the third section from a philosophical perspective that metaphors, such as 'editing' the genome, which are based on a conception of the genome as linear information, cannot adequately capture the breadth of advanced genomic technologies. To accurately categorise these techniques in a manner that meets their diverse applications, we propose introducing the umbrella term 'somatic genomic therapies' (SGTs). Urging an integrative approach to defining and classifying new technologies in medical genetics, we advocate for the development of an integrative concept of SGTs.},
}
@article {pmid40787451,
year = {2025},
author = {Sprissler, J and Pannicke, U and Rump, EM and Schrezenmeier, H and Casadei, N and Pogoda, M and Kuhlburger, L and Oquendo, MB and Czemmel, S and Debatin, KM and Erlacher, M and Schwarz, K and Felgentreff, K},
title = {RAG recombinase expression discriminates the development of natural killer cells.},
journal = {Frontiers in immunology},
volume = {16},
number = {},
pages = {1607664},
pmid = {40787451},
issn = {1664-3224},
mesh = {*Killer Cells, Natural/immunology/cytology/metabolism ; Humans ; *Cell Differentiation/immunology/genetics ; Induced Pluripotent Stem Cells/immunology/metabolism/cytology ; *Homeodomain Proteins/genetics/metabolism ; V(D)J Recombination ; *DNA-Binding Proteins/genetics/metabolism ; CRISPR-Cas Systems ; },
abstract = {INTRODUCTION: V(D)J recombination, initiated by recombination-activating gene (RAG) endonucleases, is a crucial process for the generation of diversified antigen receptors of T and B lymphocytes but regarded dispensable for innate natural killer (NK) lymphocytes lacking clonotypic receptors.<br><br>METHODS: To explore the impact of potential rearrangements on NK cell maturation, RAG-fate mapping reporter human induced pluripotent stem cell (iPSC) lines were generated by introduction of RSS-invEGFP constructs into the AAVS1 locus using CRISPR/Cas9 and differentiated into NK cells in vitro.<br><br>RESULTS: GFP expression was observed in up to 14% of mature NK cells characterized by a CD45[dim] CD56[dim]CD57[+]NKG2C[+/-]KIR[+/-] phenotype and unproductive genetic rearrangements in the IGH locus. Advanced maturation was further revealed by transcriptomic studies using RNA sequencing. Despite their strong effector function, DNA damage response and survival to ionizing radiation were compromised.<br><br>DISCUSSION: These findings suggest a role of RAG expression in NK cell ontogeny supporting the development of a terminally differentiated effector population.},
}
@article {pmid40784966,
year = {2025},
author = {Wuttinontananchai, C and Yamamoto, J and Sakamoto, S and Yamaguchi, Y},
title = {Genome-wide CRISPR screen for human factors involved in alternative polyadenylation based on differential localization of CD47.},
journal = {Scientific reports},
volume = {15},
number = {1},
pages = {29269},
pmid = {40784966},
issn = {2045-2322},
support = {23K07807//Japan Society for the Promotion of Science/ ; 20H03182//Japan Society for the Promotion of Science/ ; },
mesh = {Humans ; *Polyadenylation/genetics ; *CD47 Antigen/metabolism/genetics ; *CRISPR-Cas Systems ; Genome, Human ; HEK293 Cells ; *Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {At least 70% of the human protein-coding genes contain multiple polyadenylation sites (PAS) and undergo alternative polyadenylation (APA), generating distinct transcripts from a single gene. While APA has been implicated in various physiological and pathological processes, its regulatory factors and cellular mechanisms remain incompletely understood. A previous study demonstrated that APA influences the localization of the cell surface marker CD47. Here, we present the results of a genome-wide CRISPR screen aimed at identifying APA regulators using CD47 as a reporter. Given that isoform-specific knockdown of CD47, as well as knockdown of core 3' end processing factors, alters CD47 localization, we developed an immunofluorescence-based method that simultaneously detects cell surface and intracellular CD47 protein, enabling the visualization of APA-dependent changes at the single-cell level. Leveraging this approach, we conducted a CRISPR screen and identified multiple genes affecting CD47 cell-surface expression. In addition to known membrane trafficking factors, we uncovered several nuclear factors, among which POLDIP2 emerged as a potential novel APA regulator with a global impact on APA. This study provides a foundation for further investigations into the molecular mechanisms governing APA.},
}
@article {pmid40752775,
year = {2025},
author = {Meshram, HK and Gupta, SK and Gupta, A and Nagori, K and Ajazuddin, },
title = {Next-generation CRISPR gene editing tools in the precision treatment of Alzheimer's and Parkinson's disease.},
journal = {Ageing research reviews},
volume = {111},
number = {},
pages = {102851},
doi = {10.1016/j.arr.2025.102851},
pmid = {40752775},
issn = {1872-9649},
mesh = {Humans ; *Gene Editing/methods ; *Parkinson Disease/therapy/genetics ; *Alzheimer Disease/therapy/genetics ; *CRISPR-Cas Systems/genetics ; Animals ; *Genetic Therapy/methods ; *Precision Medicine/methods ; },
abstract = {Emerging gene-editing technologies, such as the CRISPR system, represent a potential pathway for precision medicine targeting the genetic and molecular causes of diseases. Second-generation CRISPR technologies, including base editing, prime editing, and engineered Cas variants, have improved fidelity and offer alternative strategies for precise gene correction, transcriptional repression or activation, and modulation of pathological pathways in neurodegeneration. These tools can correct single-nucleotide mutations, reduce pathological protein accumulation, and modulate neuroinflammatory responses, all integral to the pathogenesis of Alzheimer's disease (AD) and Parkinson's disease (PD), both chronic, progressive neurodegenerative disorders. Unfortunately, currently available treatments are limited and primarily palliative. Preclinical studies have shown promising results, with improvements in cognitive and motor deficits in animal models. However, significant challenges must be addressed to ensure safe and effective delivery to the CNS, minimize off-target effects, and address ethical concerns. Current clinical investigations aim to translate these findings into available therapeutic options. This review also identifies the biological mechanisms, therapeutic use cases, and current limitations of next-generation CRISPR systems as tools in the context of AD and PD, providing both therapeutic and research capabilities through their unique strengths. Ultimately, the future of transactional neurogenomics will determine the clinical possibilities of CRISPR-based strategies for advancing neurodegenerative disease management beyond palliative and symptomatic treatment, toward a feasible mechanistic form of disease modification.},
}
@article {pmid40729503,
year = {2025},
author = {Bao, B and Li, L and Li, M and Long, Y and Zhu, Y and Yin, J and Zhang, X and Tang, Y},
title = {Biomimetic Composite Nanoparticles with Immune Modulation and CRISPR Gene Editing for Enhancing Mild Photothermal Therapy-Based Synergistic Antitumor Therapy.},
journal = {Biomacromolecules},
volume = {26},
number = {8},
pages = {5245-5257},
doi = {10.1021/acs.biomac.5c00735},
pmid = {40729503},
issn = {1526-4602},
mesh = {Animals ; *Nanoparticles/chemistry ; Mice ; *Photothermal Therapy/methods ; Female ; *Gene Editing/methods ; *Biomimetic Materials/chemistry/pharmacology ; Humans ; Cell Line, Tumor ; Mice, Inbred BALB C ; HSP90 Heat-Shock Proteins/genetics ; CRISPR-Cas Systems ; *Breast Neoplasms/therapy ; Tumor Microenvironment/drug effects ; RAW 264.7 Cells ; },
abstract = {Photothermal therapy (PTT) is a promising cancer treatment. However, the high temperature generated during therapy may harm normal tissues, and the immunosuppressive microenvironment induced by tumor-associated macrophages (TAMs) hinders immune clearance of residual tumors after PTT. Therefore, developing mild PTT and remodeling the immunosuppressive microenvironment are crucial to improve antitumor efficacy and irradiation safety. Herein, we developed a biomimetic composite nanoparticle based on ribonucleoprotein (RNP), tetra-methylphenidine (TMP195), and a designed amphiphilic NIR-II conjugated polymer, PCQ-PEG-NH2. PCQ-PEG-NH2 exhibits a high photothermal conversion efficiency (58.1%), enabling PTT under safe laser intensity. The loaded RNP specifically knocks down the HSP90α gene, reducing tumor thermotolerance to enhance mild PTT efficiency. TMP195 reprograms TAMs from M2 to M1 phenotype, alleviating immunosuppression. Additionally, macrophage membrane modification endows nanoparticles with excellent biocompatibility and active tumor-targeting ability. In a breast cancer mouse model, this synergistic strategy outperformed traditional PTT, providing a promising strategy for mild-PTT tumor therapy with high efficacy.},
}
@article {pmid40651704,
year = {2025},
author = {Zhao, B and Shi, J and Zhao, R and Gao, S and Li, Y and Zhang, Y and Wei, Y and Guo, Y},
title = {Constructing CRISPR-Cas9 system for metabolic reprogramming and cordycepin biomanufacturing in Pichia pastoris.},
journal = {Bioresource technology},
volume = {436},
number = {},
pages = {132967},
doi = {10.1016/j.biortech.2025.132967},
pmid = {40651704},
issn = {1873-2976},
mesh = {*CRISPR-Cas Systems/genetics ; *Deoxyadenosines/biosynthesis/metabolism ; *Metabolic Engineering/methods ; Fermentation ; Bioreactors ; *Saccharomycetales/metabolism/genetics ; Metabolic Reprogramming ; },
abstract = {Cordycepin, a nucleoside analog mainly produced byCordyceps militaris, is widely used in food, medicine, and feed industries.Conventional microbial engineering faces challenges from antibiotic resistance genes, which increase environmental risks.Here, we engineeredPichia pastorisusing an optimized CRISPR-Cas9 system with gRNA-tRNA array and Brex27-enhanced homologous recombination, achieving antibiotic marker-free cordycepin biosynthesis. Through modular metabolic engineering strategies that optimized promoter combinations, gene copy numbers, methanol assimilation, precursor supply, and ATP/NADPH balance, strain PC19 achieved 2509.7 mg/L cordycepin in shake-flask fermentation. In fed-batch fermentation, PC19 achieved the highest production of 18.3 g/L (3.05 g/L/d and 122.2 mg/g DCW) to date in a 10 L bioreactor, and the CO2-eq emissions were 3.3-57.6 times lower than C. militaris and other microbial cell factories. This CRISPR-Cas9 system lays the foundation for low-carbon and efficient biosynthesis of cordycepin and other nucleoside analogs inP. pastoris.},
}
@article {pmid40627934,
year = {2026},
author = {Guo, A and Wu, Y and Xie, Y and Guo, W and Guo, Y and Zhang, X and Zhang, W and Zou, X and Sun, Z},
title = {CRISPR-based fluorescent aptasensor combined with smartphone for on-site visual detection of DEHP in packaged foods.},
journal = {Spectrochimica acta. Part A, Molecular and biomolecular spectroscopy},
volume = {344},
number = {Pt 1},
pages = {126649},
doi = {10.1016/j.saa.2025.126649},
pmid = {40627934},
issn = {1873-3557},
mesh = {*Smartphone ; *Diethylhexyl Phthalate/analysis ; *Aptamers, Nucleotide/chemistry/genetics ; *Food Contamination/analysis ; *Biosensing Techniques/methods ; Limit of Detection ; *CRISPR-Cas Systems ; *Food Packaging ; Spectrometry, Fluorescence/methods ; Fluorescent Dyes/chemistry ; *Food Analysis/methods ; },
abstract = {Di-(2-ethylhexyl) phthalate (DEHP) can leach into food and the environment, posing health and ecological risks. This paper introduces a novel CRISPR-based fluorescent aptasensor for on-site DEHP detection. The aptasensor selectively binds to DEHP, triggering a competitive displacement reaction that releases an Aptamer-dsDNA probe. After magnetic separation, the supernatant is analyzed via a Cas12a reporter system, where Cas12a activation cleaves a FAM-BHQ reporter, generating fluorescence. Combined with smartphone imaging, this method enables rapid result acquisition. The aptasensor shows high selectivity and sensitivity, detecting DEHP from 1 pg/mL to 1 μg/mL, with a lower limit of 0.15 pg/mL. It effectively detects DEHP in various real samples, offering reliable visual monitoring. This method offers a rapid and effective on-site detection strategy for food safety and environmental pollution monitoring.},
}
@article {pmid40783509,
year = {2025},
author = {Luo, X and Dou, Y and Lang, Y and Zhao, H and Liu, X and Zhang, X and Li, Y and Liang, D and Xia, H},
title = {CRISPR/Cas9-mediated editing of carotenoid biosynthesis genes alters carotenoid concentrations in kiwifruit.},
journal = {BMC plant biology},
volume = {25},
number = {1},
pages = {1056},
pmid = {40783509},
issn = {1471-2229},
support = {32472679//National Natural Science Foundation of China/ ; 2024-YF05-00408-SN//Chengdu Science and Technology Department/ ; 2023YFN0095, 2024JDRC0011, 2025ZNSFSC0190//Sichuan Province Science and Technology Department Projects/ ; },
mesh = {*CRISPR-Cas Systems ; *Gene Editing/methods ; *Actinidia/genetics/metabolism ; *Carotenoids/metabolism ; Plants, Genetically Modified/genetics/metabolism ; Oxidoreductases/genetics/metabolism ; Fruit/genetics/metabolism ; Genes, Plant ; Agrobacterium/genetics ; },
abstract = {BACKGROUND: CRISPR/Cas9 technology has garnered increasing attention for its simplicity and precision in genome editing, making it an indispensable tool for gene function research and crop genetic improvement. However, the inefficiency and time-consuming nature of genetic transformation continue to pose substantial challenges to its widespread application in woody plants.<br><br>RESULTS: In this study, we developed a rapid and efficient Agrobacterium-mediated transformation system using petioles as explants for kiwifruit. Positive resistant seedlings were obtained within three months by inoculating on MS medium supplemented with 2.0 mg&#xb7;L[-1] 6-benzylaminopurine (6-BA), 0.2 mg&#xb7;L[-1] naphthaleneacetic acid (NAA), and 10 mg&#xb7;L[-1] hygromycin, which was faster than using leaves as explants. Using this system, CRISPR/Cas9-mediated editing of phytoene desaturase (AcPDS) and &#x3b6;-carotene desaturase (AcZDS) achieved an editing efficiency of 20%. Transgenic kiwifruit lines with edited AcZDS exhibited a significant reduction in carotenoid content.<br><br>CONCLUSIONS: Overall, we established an efficient Agrobacterium-mediated transformation system using petioles as explants, which is applicable for CRISPR/Cas9-mediated gene editing in kiwifruit, thereby facilitating functional gene studies and genetic improvement.},
}
@article {pmid40779487,
year = {2025},
author = {Polinski, NK and Puoliväli, J and Rauhala, L and Stenius, TK and Bragge, T and Parkkari, T and Penttinen, AM and Chen, Y and Mabrouk, OS and Glajch, KE and Hirst, WD and Perkinton, M and Martinez, TN and Frasier, MA},
title = {Expression of human A53T alpha-synuclein without endogenous rat alpha-synuclein fails to elicit Parkinson's disease-related phenotypes in a novel humanized rat model.},
journal = {PloS one},
volume = {20},
number = {8},
pages = {e0329823},
pmid = {40779487},
issn = {1932-6203},
mesh = {Animals ; *alpha-Synuclein/genetics/metabolism ; Humans ; *Parkinson Disease/genetics/pathology/metabolism ; Rats ; Disease Models, Animal ; Phenotype ; Rats, Sprague-Dawley ; Gene Knockout Techniques ; CRISPR-Cas Systems ; Male ; Rats, Transgenic ; },
abstract = {Alpha-synuclein (aSyn) is linked to Parkinson's disease (PD) through SNCA genetic mutations, phosphorylated aSyn in Lewy bodies and Lewy neurites, and most recently through evidence of aSyn aggregation in patient spinal fluid using the aSyn seed amplification assay. Therefore, understanding the biology of this protein and developing therapeutic interventions targeting pathological processing of aSyn are a key area of focus for novel treatments to slow or stop PD. Reliable preclinical models are imperative for these efforts. To this end, we developed a novel model using CRISPR/Cas9 to humanize the regions surrounding the naturally occurring threonine 53 amino acid in the Sprague Dawley rat to generate a humanized A53T aSyn rat model (aSyn A53T KI). We also generated an Snca knockout (aSyn KO) line to pair with the humanized A53T aSyn rat line to confirm that phenotypes were not due to loss of endogenous rat aSyn protein. A systematic phenotyping study was performed on these lines, assessing PD-related pathology and phenotypes at multiple timepoints. The aSyn KO rat line was profiled at 6 and 12 months of age, revealing successful aSyn protein knockout. The aSyn A53T KI model was profiled at 4, 8, 12, and 18 months of age for motor and non-motor phenotypes, nigrostriatal degeneration, and brain pathology. We confirmed the aSyn A53T KI rat expresses human aSyn while lacking endogenous rat aSyn. Motor function and non-motor function remain largely unaffected in this model, and no overt nigrostriatal degeneration or brain pathology are observed up to 18 months of age. Although the aSyn A53T KI rat lacks the ability to model PD pathology and phenotypes at baseline, it is an ideal model for investigating the impact of exogenous synuclein aggregates or environmental triggers on human aSyn in an in vivo model system.},
}
@article {pmid40651547,
year = {2025},
author = {Lu, Y and Stoof, J and Tanoé, YR and Walsh, N and Bijlsma, MF and Lei, H and Chen, T and Grahovac, J and Grützmann, R and Pilarsky, C},
title = {CRISPR/Cas9 genome engineering in PDAC: From preclinical studies to translation and clinical research.},
journal = {Seminars in cancer biology},
volume = {114},
number = {},
pages = {242-255},
doi = {10.1016/j.semcancer.2025.07.004},
pmid = {40651547},
issn = {1096-3650},
mesh = {*CRISPR-Cas Systems ; Humans ; Animals ; *Carcinoma, Pancreatic Ductal/genetics/therapy/pathology ; *Pancreatic Neoplasms/genetics/therapy/pathology ; *Gene Editing/methods ; Translational Research, Biomedical ; Disease Models, Animal ; Genetic Engineering/methods ; Mice ; },
abstract = {CRISPR/Cas9 technology has emerged as a powerful tool in pancreatic ductal adenocarcinoma cancer (PDAC) research, facilitating the study of genes involved in cell signaling pathways, proliferation, migration, invasion, and chemotherapy resistance. In this review, we discuss the evolution of CRISPR technologies from sophisticated editing techniques to broad screening methods, examine the utility of isogenic models and genetically engineered mouse models (GEMMs). We also explore how CRISPR/Cas9 screens can reveal immune-tumor cell interactions, highlighting the multifaceted role of this technology in PDAC research. Moreover, we emphasize the use of CRISPR technology in diagnostics for CAR-T cell therapies, where CRISPR/Cas9 enhances the precision of targeting malignant cells while minimizing off-tumor effects.},
}
@article {pmid40527655,
year = {2025},
author = {Momokawa, N and Ikeda, T and Ishida, T and Nimura-Matsune, K and Funabashi, H and Watanabe, S and Kuroda, A and Hirota, R},
title = {Role of the Pho regulon and genetic reconstruction of a phosphite-dependent Escherichia coli.},
journal = {Journal of bioscience and bioengineering},
volume = {140},
number = {3},
pages = {117-122},
doi = {10.1016/j.jbiosc.2025.05.012},
pmid = {40527655},
issn = {1347-4421},
mesh = {*Regulon/genetics ; *Escherichia coli/genetics/metabolism ; *Escherichia coli Proteins/genetics/metabolism ; Gene Expression Regulation, Bacterial ; *Phosphites/metabolism ; CRISPR-Cas Systems ; Gene Editing ; Bacterial Proteins/genetics/metabolism ; },
abstract = {A phosphite (Pt)-dependent biological containment strategy, achieved by introducing a Pt-metabolic pathway and disrupting endogenous phosphate transporters, renders Escherichia coli growth strictly dependent on Pt, a compound rarely detected in natural environments, thereby preventing unintended environmental spread. In this study, we demonstrated that expression of phosphate regulon (Pho regulon) genes was markedly upregulated in a Pt-dependent E. coli strain due to the elimination of phoU, a negative regulator of the Pho regulon, along with the high-affinity phosphate transporter pstSCAB. However, further genetic modification of this strain for detailed analysis was hindered by the presence of multiple antibiotic resistance markers. To overcome this limitation, we reconstructed a Pt-dependent E. coli strain using CRISPR-Cas12a-mediated genome editing, enabling the removal of the antibiotic resistance markers and facilitating subsequent genetic manipulation. Using this strain, we disrupted the PhoBR two-component regulatory genes and found that deletion of phoBR alleviated the constitutive overexpression of Pho regulon genes and partially restored growth of the Pt-dependent strain. These findings provide mechanistic insights and technical advances for the refinement and practical application of Pt-dependent biocontainment strategy.},
}
@article {pmid40514427,
year = {2025},
author = {Liu, L and Chen, S and Lei, Y and Lin, Z and Zhou, R and Zeng, G and Zheng, Z and Liu, W and Zhou, Q and Chen, L},
title = {REPS2 attenuates cancer stemness through inhibiting Wnt signaling by autophagy mediated degradation of β-catenin.},
journal = {Oncogene},
volume = {44},
number = {33},
pages = {2942-2955},
pmid = {40514427},
issn = {1476-5594},
mesh = {Humans ; *Autophagy/genetics ; *beta Catenin/metabolism/genetics ; *Wnt Signaling Pathway/genetics ; *Neoplastic Stem Cells/metabolism/pathology ; Animals ; Mice ; *Lung Neoplasms/pathology/genetics/metabolism ; Cell Line, Tumor ; Proteolysis ; Xenograft Model Antitumor Assays ; CRISPR-Cas Systems ; },
abstract = {Tumor suppressor genes (TSGs) that regulate the stemness of lung cancer cells remain to be determined. We conducted a genome-wide CRISPR/Cas9-mediated screening and identified REPS2 as a potent TSG that negatively regulates the stemness of lung cancer cells. Its tumor suppressive function was confirmed both in vitro and in vivo. Mechanistically, P62 interacts simultaneously with both β-catenin and REPS2, leading to autophagy-lysosome-mediated degradation of β-catenin and attenuation of Wnt signaling. A β-catenin inhibitor synergizes with inhibitors for driver mutants to induce immunogenic cell death, which could be exploited for enhancing efficacy of tumor immunotherapy.},
}
@article {pmid40738806,
year = {2025},
author = {Patel, SJ and Chen, ZJ},
title = {VDAC2 brake release: unleashing inflammation via IFNγ.},
journal = {Trends in pharmacological sciences},
volume = {46},
number = {8},
pages = {695-696},
doi = {10.1016/j.tips.2025.07.001},
pmid = {40738806},
issn = {1873-3735},
mesh = {Humans ; *Interferon-gamma/metabolism ; *Voltage-Dependent Anion Channel 2/metabolism/genetics/antagonists &amp; inhibitors ; *Inflammation/metabolism/drug therapy ; Animals ; *Neoplasms/drug therapy/metabolism/genetics/immunology ; CRISPR-Cas Systems ; DNA, Mitochondrial/metabolism ; },
abstract = {Identification of therapeutic vulnerabilities in cancer remains a high priority for cancer research. A recent CRISPR/Cas9 screen identified that VDAC2 deletion in tumors enhanced their sensitivity to interferon-γ (IFNγ) through the release of mitochondrial DNA (mtDNA) and activation of the cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) pathway. These data suggest that VDAC2 inhibition could enhance antitumor therapies.},
}
@article {pmid40695277,
year = {2025},
author = {Sousa, AA and Terrey, M and Sakai, HA and Simmons, CQ and Arystarkhova, E and Morsci, NS and Anderson, LC and Xie, J and Suri-Payer, F and Laux, LC and Roze, E and Forlani, S and Gao, G and Frost, S and Frost, N and Sweadner, KJ and George, AL and Lutz, CM and Liu, DR},
title = {In vivo prime editing rescues alternating hemiplegia of childhood in mice.},
journal = {Cell},
volume = {188},
number = {16},
pages = {4275-4294.e23},
doi = {10.1016/j.cell.2025.06.038},
pmid = {40695277},
issn = {1097-4172},
mesh = {Animals ; *Sodium-Potassium-Exchanging ATPase/genetics/metabolism ; *Hemiplegia/genetics/therapy ; Humans ; Mice ; *Gene Editing/methods ; Disease Models, Animal ; Mutation ; Male ; CRISPR-Cas Systems ; Female ; Genetic Therapy/methods ; Mice, Inbred C57BL ; HEK293 Cells ; Brain/metabolism ; },
abstract = {Alternating hemiplegia of childhood (AHC) is a neurodevelopmental disorder with no disease-modifying treatment. Mutations in ATP1A3, encoding an Na[+]/K[+] ATPase subunit, cause 70% of AHC cases. Here, we present prime editing (PE) and base editing (BE) strategies to correct ATP1A3 and Atp1a3 mutations in human cells and in two AHC mouse models. We used PE and BE to correct five prevalent ATP1A3 mutations with 43%-90% efficiency. AAV9-mediated in vivo PE corrects Atp1a3 D801N and E815K in the CNS of two AHC mouse models, yielding up to 48% DNA correction and 73% mRNA correction in bulk brain cortex. In vivo PE rescued clinically relevant phenotypes, including restoration of ATPase activity; amelioration of paroxysmal spells, motor defects, and cognition deficits; and dramatic extension of animal lifespan. This work suggests a potential one-time PE treatment for AHC and establishes the ability of PE to rescue a neurological disease in animals.},
}
@article {pmid40779454,
year = {2025},
author = {Jain, PK and Mahanty, S and Chittora, H and Henriot, V and Janke, C and Sirajuddin, M and Dhandapany, PS},
title = {Tubulin tyrosine ligase variant perturbs microtubule tyrosination, causing hypertrophy in patient-specific and CRISPR gene-edited iPSC-cardiomyocytes.},
journal = {JCI insight},
volume = {10},
number = {15},
pages = {},
doi = {10.1172/jci.insight.187942},
pmid = {40779454},
issn = {2379-3708},
mesh = {Humans ; *Myocytes, Cardiac/metabolism/pathology ; Induced Pluripotent Stem Cells/metabolism ; Gene Editing ; *Cardiomyopathy, Hypertrophic/genetics/pathology ; *Microtubules/metabolism ; Mutation, Missense ; Tubulin/metabolism ; *Peptide Synthases/genetics/metabolism ; Male ; CRISPR-Cas Systems ; Female ; },
abstract = {Hypertrophic cardiomyopathy (HCM) is a hereditary heart condition characterized by either preserved or reduced ejection fraction without any underlying secondary causes. The primary cause of HCM is sarcomeric gene mutations, which account for only 40%-50% of the total cases. Here, we identified a pathogenic missense variant in tubulin tyrosine ligase (TTL p.G219S) in a patient with HCM. We used clinical, genetics, computational, and protein biochemistry approaches, as well as patient-specific and CRISPR gene-edited induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs), to demonstrate that the TTL pathogenic variant results in a reduced enzymatic activity and the accumulation of detyrosinated tubulin leading to the disruption of redox signaling, ultimately leading to HCM. Our findings highlight - for the first time to our knowledge - the crucial roles of the TTL variant in cardiac remodeling resulting in disease.},
}
@article {pmid40778611,
year = {2025},
author = {Brara, Z and Madani, K and Costa, J and Taouzinet, L and Djaoudene, O and Amrane-Abider, M and Bougherra, H and Mouhoubi, K and Brahimi, N and Bouiche, C and Meghlaoui, Z and Mafra, I},
title = {Current Progress on the Detection of Genetically Modified Organisms in Food: From Transgenic Towards Genome-Edited Crops.},
journal = {Comprehensive reviews in food science and food safety},
volume = {24},
number = {5},
pages = {e70243},
doi = {10.1111/1541-4337.70243},
pmid = {40778611},
issn = {1541-4337},
support = {2021.03583.CEECIND/CP1662/CT0012//Funda&#xe7;&#xe3;o para a Ci&#xea;ncia e a Tecnologia/ ; UIDB/50006/2020//Funda&#xe7;&#xe3;o para a Ci&#xea;ncia e a Tecnologia/ ; 2021.03670.CEECIND/CP1662/CT0011//Funda&#xe7;&#xe3;o para a Ci&#xea;ncia e a Tecnologia/ ; DGRSDT/MESRS-Algeria//General Directorate&#xa0;of Scientific Research and Technological Developmen/ ; },
mesh = {*Plants, Genetically Modified/genetics ; *Crops, Agricultural/genetics ; Gene Editing ; *Food, Genetically Modified ; CRISPR-Cas Systems ; },
abstract = {Progresses in biotechnology, particularly the introduction of genetically modified organisms (GMO) and, more recently, clustered regularly interspaced short palindromic repeats (CRISPR)/Cas-mediated genome editing, have revolutionized agriculture, prompting the need for robust food-labeling regulations. To meet the GMO legislation requirements, analytical methods for the reliable detection of GMO in food, based either on DNA or protein markers, have been constantly proposed. DNA-based methods relying on polymerase chain reaction (PCR) technologies are the most popular for this purpose, with real-time PCR being the gold standard for GMO quantification. Lately, digital PCR has proved to be a suitable alternative to real-time PCR. The development of rapid, low-cost, user-friendly, and field-deployable tools to quickly generate data on the presence of GMO is crucial, especially with the rapid increase in their complexity and the number of events entering the food market. In this context, alternative technologies based on isothermal amplification and genosensors have emerged. The immunochemical assays in the formats of microtiter plates, lateral flow devices, and immunosensors still occupy a relevant role in GMO detection. Finally, next generation sequencing technologies stand up as tools to face the challenges of detecting unauthorized GMO and genome-edited crops. This review intends to provide a comprehensive overview on the methodologies available for the detection, identification, and quantification of GMO, including gene-edited mutants in foods, while discussing their advantages and limitations, with focus on the latest advances.},
}
@article {pmid40777742,
year = {2025},
author = {Kalter, N and Fuster-García, C and Silva, A and Ronco-Díaz, V and Roncelli, S and Turchiano, G and Gorodkin, J and Cathomen, T and Benabdellah, K and Lee, C and Hendel, A},
title = {Off-target effects in CRISPR-Cas genome editing for human therapeutics: Progress and challenges.},
journal = {Molecular therapy. Nucleic acids},
volume = {36},
number = {3},
pages = {102636},
pmid = {40777742},
issn = {2162-2531},
abstract = {Targeted nucleases, primarily CRISPR-Cas-based systems, have revolutionized genome editing by enabling precise modification of target genes or transcripts. Many pre-clinical and clinical studies leverage this technology to develop treatments for human diseases; however, substantial off-target genotoxicity concerns delay its clinical translation. Despite the development of a wide array of tools, assays, and technologies aimed at identifying and quantifying off-target effects, the absence of standardized guidelines leads to inconsistent practices across studies. This review highlights the key challenges and potential solutions in ensuring the safety of gene editing studies for therapeutic applications, focusing on gRNA design, off-target sites prediction, and off-target activity measurement.},
}
@article {pmid40777740,
year = {2025},
author = {Park, SJ and Lee, GE and Cho, SM and Choi, EH},
title = {Recent applications, future perspectives, and limitations of the CRISPR-Cas system.},
journal = {Molecular therapy. Nucleic acids},
volume = {36},
number = {3},
pages = {102634},
pmid = {40777740},
issn = {2162-2531},
abstract = {The CRISPR-Cas system has transformed our ability to edit and modify genomes in eukaryotic cells, offering unmatched precision and broad applicability. By utilizing a programmable RNA protein complex to introduce targeted double-strand breaks, the CRISPR-Cas system enables the correction of pathogenic mutations and the modulation of gene function with unprecedented efficiency. Its broad applicability spans the correction of inherited genetic defects through homology-directed repair to the disruption of deleterious alleles via non-homologous end joining. In this review, we first outline the molecular architecture and mechanistic basis of CRISPR-Cas9 and then consider its latest applications in modeling, drug screening, small-molecule-mediated editing, and treating hereditary, autoimmune, and oncological diseases. Emphasis is placed on the generation of disease-relevant cellular and animal models and on the potential of CRISPR-Cas9-mediated gene therapy to address hitherto intractable disorders. Finally, we discuss current challenges including off-target activity, gene editing efficiency, delivery constraints, and immunogenicity and highlight emerging strategies to overcome these hurdles and broaden the clinical impact of CRISPR-Cas systems.},
}
@article {pmid40777476,
year = {2025},
author = {Walker, MWG and Richard, E and Wiegand, T and Wang, J and Yang, Z and Casas-Ciniglio, AA and Hoffmann, FT and Shahnawaz, H and Gaudet, RG and Arpaia, N and Fernández, IS and Sternberg, SH},
title = {Temperate phages enhance host fitness via RNA-guided flagellar remodeling.},
journal = {bioRxiv : the preprint server for biology},
volume = {},
number = {},
pages = {},
doi = {10.1101/2025.07.22.666180},
pmid = {40777476},
issn = {2692-8205},
abstract = {Bacterial flagella drive motility and chemotaxis while also playing critical roles in host-pathogen interactions, as their oligomeric subunit, flagellin, is specifically recognized by the mammalian immune system and flagellotropic bacteriophages. We recently discovered a family of phage-encoded, RNA-guided transcription factors known as TldR that regulate flagellin expression. However, the biological significance for this regulation, particularly in the context of host fitness, remained unknown. By focusing on a human clinical Enterobacter isolate that encodes a Flagellin Remodeling prophage (FRφ), here we show that FRφ exploits the combined action of TldR and its flagellin isoform to dramatically alter the flagellar composition and phenotypic properties of its host. This transformation has striking biological consequences, enhancing bacterial motility and mammalian immune evasion, and structural studies by cryo-EM of host- and prophage-encoded filaments reveal distinct architectures underlying these physiological changes. Moreover, we find that FRφ improves colonization in the murine gut, illustrating the beneficial effect of prophage-mediated flagellar remodeling in a host-associated environment. Remarkably, flagellin-regulating TldR homologs emerged multiple times independently, further highlighting the strong selective pressures that drove evolution of RNA-guided flagellin control. Collectively, our results reveal how RNA-guided transcription factors emerged in a parallel evolutionary path to CRISPR-Cas and were co-opted by phages to remodel the flagellar apparatus and enhance host fitness.},
}
@article {pmid40777396,
year = {2025},
author = {Zhou, S and Yang, S and Xu, J and Zhu, G},
title = {Engineering circular guide RNA and CRISPR-Cas13d-encoding mRNA for the RNA editing of Adar1 in triple-negative breast cancer immunotherapy.},
journal = {bioRxiv : the preprint server for biology},
volume = {},
number = {},
pages = {},
doi = {10.1101/2025.07.22.666181},
pmid = {40777396},
issn = {2692-8205},
abstract = {Clustered regularly interspaced short palindromic repeat Cas endonuclease (CRISPR-Cas) systems, such as RNA-editing CRISPR-Cas13d, are poised to advance the gene therapy of various diseases. However, their clinical development has been challenged by 1) the limited biostability of linear guide RNAs (lgRNAs) susceptible to degradation, 2) the immunogenicity of prokaryotic microorganism-derived Cas proteins in human that restrains their long-term therapeutic efficacy, and 3) off-targeting gene editing caused by the prolonged Cas expression from DNA vectors. Here, we report the development of highly stable circular gRNAs (cgRNAs) and transiently-expressing Cas13d-encoding mRNA for efficient CRISPR-Cas13d editing of target mRNA. We first optimized cgRNA for CRISPR-Cas13d editing of adenosine deaminase acting on RNA type I (Adar1) transcript for the combination immunotherapy of triple negative breast cancer (TNBC). cgRNAs were synthesized by enzymatic ligation of lgRNA precursors. cgRNAs enhanced biostability with comparable Cas13d-binding affinity relative to lgRNA. Next, using ionizable lipid nanoparticles (LNPs), we co-delivered the resulting Adar1 -targeting cgRNA with an mRNA encoding RfxCas13d (mRNA-RfxCas13d), a widely used Cas13d variant, to TNBC cells. As a result, relative to lgRNA, cgRNA significantly enhanced the efficiency of Adar1 knockdown with minimal collateral activity, which sensitized the cancer cells for cytokine-mediated cell apoptosis. In a 4T1 murine TNBC tumor model in syngeneic mice, Adar1 -targeting cgRNA outperformed lgRNA for tumor immunotherapy in combination with immune checkpoint blockade (ICB). Collectively, these results demonstrate the great potential of cgRNA and mRNA-RfxCas13d for RNA-targeted gene editing.},
}
@article {pmid40775081,
year = {2025},
author = {Chandrasekaran, SN and Alix, E and Arevalo, J and Borowa, A and Byrne, PJ and Charles, WG and Chen, ZS and Cimini, BA and Deng, B and Doench, JG and Ewald, JD and Fritchman, B and Fuller, CJ and Gaetz, J and Goodale, A and Haghighi, M and Han, Y and Hanifehlou, Z and Hennig, H and Hernandez, D and Jacob, CB and James, T and Jetka, T and Kalinin, AA and Komalo, B and Kost-Alimova, M and Krawiec, T and Marion, BA and Martin, G and McCarthy, NJ and Miller, L and Monsees, A and Moshkov, N and Muñoz, AF and Ogier, A and Otrocka, M and Rataj, K and Root, DE and Rubbo, F and Scrace, S and Selinger, DW and Senft, RA and Sommer, P and Thibaudeau, A and Trisorus, S and Valiya Veettil, R and Van Trump, WJ and Wang, S and Warchoł, M and Weisbart, E and Weiss, A and Wiest, M and Zaremba, A and Zinovyev, A and Singh, S and Carpenter, AE},
title = {Morphological map of under- and overexpression of genes in human cells.},
journal = {Nature methods},
volume = {22},
number = {8},
pages = {1742-1752},
pmid = {40775081},
issn = {1548-7105},
support = {MIRA R35 GM122547//U.S. Department of Health &amp; Human Services | NIH | Center for Information Technology (Center for Information Technology, National Institutes of Health)/ ; },
mesh = {Humans ; *Single-Cell Analysis/methods ; CRISPR-Cas Systems ; Cell Line, Tumor ; },
abstract = {Cell Painting images offer valuable insights into a cell's state and enable many biological applications, but publicly available arrayed datasets only include hundreds of genes perturbed. The JUMP Cell Painting Consortium perturbed roughly 75% of the protein-coding genome in human U-2 OS cells, generating a rich resource of single-cell images and extracted features. These profiles capture the phenotypic impacts of perturbing 15,243 human genes, including overexpressing 12,609 genes (using open reading frames) and knocking out 7,975 genes (using CRISPR-Cas9). Here we mitigated technical artifacts by rigorously evaluating data processing options and validated the dataset's robustness and biological relevance. Analysis of phenotypic profiles revealed previously undiscovered gene clusters and functional relationships, including those associated with mitochondrial function, cancer and neural processes. The JUMP Cell Painting genetic dataset is a valuable resource for exploring gene relationships and uncovering previously unknown functions.},
}
@article {pmid40457553,
year = {2025},
author = {Yuan, Y and Zhou, T and Yang, Z and Zhang, Y and Ye, Z and Liu, L and Zheng, L and Yan, Z and Hu, H and Han, M and Westhof, E and Dai, F and Tong, X},
title = {Doublesex gene in Bombyx mori: CRISPR-mediated female-to-male sex reversal through exon skipping.},
journal = {Pest management science},
volume = {81},
number = {9},
pages = {5917-5927},
doi = {10.1002/ps.8947},
pmid = {40457553},
issn = {1526-4998},
support = {2023YFD1600901//National key research and development program of China/ ; 2023YFF1103801//National key research and development program of China/ ; SWU-XJLJ202309//Fundamental Research Funds for the Central Universities/ ; SWU-XDPY22011//Fundamental Research Funds for the Central Universities/ ; CARS-18-ZJ0102//China Agriculture Research System of MOF and MARA/ ; CARS-18-ZJ0103//China Agriculture Research System of MOF and MARA/ ; cstc2021jcyj-cxtt0005//Natural Science Foundation of Chongqing, China/ ; },
mesh = {Animals ; *Bombyx/genetics/growth &amp; development ; Male ; Female ; *CRISPR-Cas Systems ; *Sex Determination Processes/genetics ; *Insect Proteins/genetics/metabolism ; Exons ; Gene Editing ; DNA-Binding Proteins ; },
abstract = {BACKGROUND: Sex control technology has great potential to improve the efficiency of economic insect utilization and integrated pest management by enabling targeted population suppression or elimination. The silkworm (Bombyx mori), a lepidopteran model, holds immense economic importance attributable to its unique silk production capabilities. Male silkworms favored over females as a result of their higher yield and quality silk. Doublesex (dsx) is a key gene for sex determination in insects. Previously, inactivation of Bmdsx, among other sex determination-related genes, resulted in intersex individuals rather than a complete female-to-male transformation.<br><br>RESULTS: We used CRISPR/Cas9 technology to edit the splice sites of Bmdsx at female-specific exons 3 and 4 to induce transformation of Bmdsx[F] to Bmdsx[M]. The edited genome produced only male-specific Bmdsx[M]. The results revealed that these genetic modifications had significant effects on genital development in female individuals. The edited genome led to the emergence of male-specific gonads in females, causing genital developmental defects. However, it is worth noting that males remained unaffected by these genetic alterations. Furthermore, we conducted a comprehensive analysis of dsx gene structure and explored the potential applicability of this method for inducing female-to-male transgenic transformation in other insects.<br><br>CONCLUSIONS: This study demonstrates that precision editing of dsx splice sites can enforce unidirectional sex conversion in insects, disrupting female reproductive capacity without affecting males. Overall, these findings provide valuable insights into our understanding of gonadal development and insect population management. &#xa9; 2025 Society of Chemical Industry.},
}
@article {pmid40457530,
year = {2025},
author = {Guo, D and Zhao, SY and Su, SC and Bu, LA and Sun, H and Gao, CF and Wu, SF},
title = {Doublesex regulates the development of sex-specific organs in rice stem borer, Chilo suppressalis.},
journal = {Pest management science},
volume = {81},
number = {9},
pages = {5876-5889},
doi = {10.1002/ps.8941},
pmid = {40457530},
issn = {1526-4998},
support = {//National Key Research and Development Program of China/ ; //National Natural Science Foundation of China/ ; //Fundamental Research Funds for the Central Universities/ ; },
mesh = {Animals ; *Moths/genetics/growth &amp; development ; Female ; Male ; *Insect Proteins/genetics/metabolism ; Phylogeny ; *Sex Determination Processes ; Oryza ; CRISPR-Cas Systems ; },
abstract = {BACKGROUND: The doublesex (dsx) gene is a crucial component of the insect sex determination pathway, playing a pivotal role in sexual differentiation and development. While the functions of dsx have been extensively characterized in several insect species, its molecular mechanisms and specific roles in sex determination remain largely unexplored in the lepidopteran rice stem borer, Chilo suppressalis (Walker), a significant agricultural pest.<br><br>RESULTS: In this study, we identified two male-specific and five female-specific Csdsx transcripts in C. suppressalis. Phylogenetic analyses and multiple sequence alignments revealed that Csdsx is highly conserved among lepidopterans. To investigate its functional roles, we employed CRISPR/Cas9-mediated mutagenesis to generate C. suppressalis mutants. Specifically, target sites were designed on exons 1, 3, 4, and 5 to disrupt the common region (exon 1), female-specific exons (exon 3 and exon 4), and male-specific regions of Csdsx (exon 5). As anticipated, mutants exhibited abnormal development of both external and internal genitalia during the pupal and adult stages. Furthermore, mutations induced sex-specific sterility. Notably, knockout of sex-specific Csdsx isoforms also resulted in abnormal wing patterns and antennal morphology.<br><br>CONCLUSION: This study demonstrates the conserved role of Csdsx in sexual differentiation in C. suppressalis and highlights its potential as a molecular target for environmentally friendly pest management strategies against this agricultural pest. &#xa9; 2025 Society of Chemical Industry.},
}
@article {pmid40417776,
year = {2025},
author = {Lin, M and Zhou, Z and Li, Z and Feng, W and Yang, C and Zhu, Z and Chen, Q},
title = {A dual-probe one-pot RPA-CRISPR/Cas12a: a highly sensitive and rapid method for detection of Phytophthora vignae.},
journal = {Pest management science},
volume = {81},
number = {9},
pages = {5784-5795},
doi = {10.1002/ps.8932},
pmid = {40417776},
issn = {1526-4998},
support = {2024YFD1400100//National Key R&amp;D Program of China/ ; ZDYF2024XDNY250//Science and Technology special fund of Hainan Province/ ; ZDYF2022XDNY241//Science and Technology special fund of Hainan Province/ ; KYQD(ZR)-20080//Scientific Research Foundation of Hainan University/ ; },
mesh = {*Phytophthora/isolation &amp; purification/genetics ; *Plant Diseases/microbiology/parasitology ; *Vigna/microbiology/parasitology ; *CRISPR-Cas Systems ; Sensitivity and Specificity ; *Nucleic Acid Amplification Techniques/methods ; },
abstract = {BACKGROUND: Phytophthora vignae is the primary pathogen affecting cowpea plants. Early on-site and accurate detection of the causal pathogen is critical for successful management. Therefore, it is both essential and urgent to create a rapid, sensitive, and accurate detection for P. vignae in cowpeas.<br><br>RESULTS: In this study, the one-pot RPA-CRISPR/Cas12a assay demonstrated the ability to effectively differentiate closely related oomycete species, exhibiting excellent specificity. The entire detection process can be completed within 15-20&#x2009;min, with results being visually determined using a lateral flow strip (LFS) or by fluorescence detection under blue or ultraviolet (UV) light. Additionally, we have added dual probes to the one-pot RPA-CRISPR/Cas12a assay detection system, this modification improved reaction efficiency and significantly enhanced sensitivity (200&#x2009;fg&#x2009;&#x3bc;L[-1]).<br><br>CONCLUSION: This study developed a dual-probe one-pot RPA-CRISPR/Cas12a detection platform that demonstrates rapid and highly sensitive identification of P. vignae. The proposed technology enables field-deployable detection without requiring specialized technical expertise, representing a promising approach with substantial potential for practical applications. &#xa9; 2025 Society of Chemical Industry.},
}
@article {pmid40415729,
year = {2025},
author = {Ban, Y and Zhang, Z and Wei, J and Xu, G and Ma, Y and Yin, S and Dong, L and Feng, Z},
title = {Establishment of a CRISPR-based system for rapidly detecting the target-site resistance of American sloughgrass (Beckmannia syzigachne) to Pinoxaden.},
journal = {Pest management science},
volume = {81},
number = {9},
pages = {5649-5658},
doi = {10.1002/ps.8919},
pmid = {40415729},
issn = {1526-4998},
support = {2023YFD1400501//National Key R&amp;D Program of China/ ; 32372567//National Natural Science Foundation of China/ ; KYCX24-0982//Postgraduate Research &amp; Practice Innovation Program of Jiangsu Province/ ; },
mesh = {*Herbicide Resistance/genetics ; *Herbicides/pharmacology ; *CRISPR-Cas Systems ; *Poaceae/genetics/drug effects ; *Oxazines/pharmacology ; *Plant Weeds/genetics/drug effects ; Acetyl-CoA Carboxylase/genetics/antagonists &amp; inhibitors ; Mutation ; Plant Proteins/genetics ; Weed Control ; Heterocyclic Compounds, 2-Ring ; },
abstract = {BACKGROUND: Weeds resistant to herbicides pose significant challenges in crop production, making early resistance monitoring crucial for timely control of resistant weeds. Prolonged use of ACCase-inhibiting herbicides, like pinoxaden, has led to the evolution of high-level resistance in weed populations over time. American sloughgrass (Beckmannia syzigachne), is a noxious grass weed, that severely impacts the yield and quality of wheat and rapeseed crops.<br><br>RESULTS: To accurately and rapidly detect the mutations in the target gene of B. syzigachne, we developed a novel rapid detection method based on the CRISPR-Cas12b/sgRNA system to evaluate the target mutation at amino acid position 1781 of the ACCase gene. By optimizing various reaction conditions, the novel detection system can assess target-site resistance of B. syzigachne to pinoxaden within 40&#x2009;min at a constant temperature of 54&#x2009;&#xb0;C. This novel system exhibits excellent specificity, high sensitivity, simplicity in procedure, also with time-efficient and high throughput.<br><br>CONCLUSION: This study presents an efficient method based on the CRISPR-Cas12b system for rapidly detecting the target-site resistance, which will facilitate the precise management of resistant weeds. &#xa9; 2025 Society of Chemical Industry.},
}
@article {pmid40396330,
year = {2025},
author = {Yin, H and Ge, Z and Yao, S and Chen, G and Yuan, S and Yang, Q and Deng, W},
title = {Resistance to ACCase-inhibiting herbicides conferred by the Ile-1781-Val mutation in Leptochloa fusca and visual detection of target mutations by RAA-CRISPR/Cas12a.},
journal = {Pest management science},
volume = {81},
number = {9},
pages = {5581-5588},
doi = {10.1002/ps.8911},
pmid = {40396330},
issn = {1526-4998},
support = {137050535//Qing Lan Project of Yangzhou University/ ; },
mesh = {*Herbicide Resistance/genetics ; *Herbicides/pharmacology ; CRISPR-Cas Systems ; *Acetyl-CoA Carboxylase/antagonists &amp; inhibitors/genetics ; Mutation ; *Poaceae/genetics/drug effects/enzymology ; *Plant Proteins/genetics/antagonists &amp; inhibitors/metabolism ; },
abstract = {BACKGROUND: Leptochloa fusca is a new dominant grass weed in paddy ecosystem of local region in China. The extensive herbicide application has selected the cyhalofop-butyl-resistant L. fusca populations. Here, we aimed to (i) characterize the target-site resistance mechanisms to ACCase inhibitors, and (ii) establish an efficient and rapid method for detecting mutations.<br><br>RESULTS: The L. fusca population (SFC-R) was resistant to cyhalofop-butyl (RF&#x2009;=&#x2009;5.4), metamifop (RF&#x2009;=&#x2009;5.5), and fenoxaprop-P-ethyl (RF&#x2009;=&#x2009;6.8). Gene sequencing revealed that the Ile-1781-Val mutation was the molecular basis of resistance. A recombinase-aided amplification (RAA) combined with CRISPR/Cas12a method was successfully developed, which could rapidly and visually detect the single nucleotide variation underling the Ile-1781-Val substitution in L. fusca.<br><br>CONCLUSION: This study confirmed that target-site mutation (Ile-1781-Val) endowed resistance to ACCase inhibitors in L. fusca, and represented the first report of applying the RAA-CRISPR/Cas12a assay for mutation detection in resistant weeds. Considering its simplicity and specificity, this technique may be used in monitoring and early warning of resistant weeds. &#xa9; 2025 Society of Chemical Industry.},
}
@article {pmid40304168,
year = {2025},
author = {Xie, QP and Wang, BY and Dou, W and Smagghe, G and Zhang, Q and Wang, JJ},
title = {CRISPR/Cas9-mediated vitellogenin receptor knockout impairs vitellogenin uptake and reproduction in Bactrocera dorsalis.},
journal = {Pest management science},
volume = {81},
number = {9},
pages = {5043-5051},
doi = {10.1002/ps.8857},
pmid = {40304168},
issn = {1526-4998},
support = {32302340//National Natural Science Foundation of China/ ; 32072422//National Natural Science Foundation of China/ ; SWU-KQ23010//Fundamental Research Funds for the Central Universities/ ; CARS-26//China Agricultural Research System/ ; },
mesh = {Animals ; *CRISPR-Cas Systems ; Female ; *Tephritidae/genetics/physiology/metabolism ; *Vitellogenins/metabolism ; *Insect Proteins/genetics/metabolism ; *Egg Proteins/genetics/metabolism ; Reproduction/genetics ; *Receptors, Cell Surface/genetics/metabolism ; Ovary/growth &amp; development/metabolism ; Gene Knockout Techniques ; Male ; },
abstract = {BACKGROUND: Reproduction is a critical process in the insect life cycle, with the ovary serving as the central organ responsible for population maintenance. Successful development of the ovary is dependent on vitellogenin (Vg) transport into oocytes via the vitellogenin receptor (VgR). Exploring the VgR function is crucial for understanding the physiological mechanisms of insect ovarian development. However, the functional role of VgR in Bactrocera dorsalis (Hendel), a notorious agricultural invasive pest with exceptional reproductive plasticity, remains unclear.<br><br>RESULTS: Here, we identified BdVgR, an ovary-specific receptor with 1903 amino acids, as a critical determinant of reproductive success. CRISPR/Cas9-mediated BdVgR knockout resulted in a 211-bp genomic deletion spanning exonic (126&#x2009;bp) and intronic (85&#x2009;bp) regions, leading to near-complete loss of VgR expression in female adults. Functional analyses revealed that BdVgR deficiency disrupted ovarian Vg (Vg1/Vg2/Vg3) accumulation, impaired ovary maturation, and thus caused severe reproductive defects, including a decrease in the size of the ovaries by 49%, mating rates by 45%, egg production by 38%, and hatching rate by 22%.<br><br>CONCLUSION: Collectively, these findings indicate that BdVgR plays a key role in the reproductive process in B. dorsalis, and that disrupting VgR function can inhibit egg production, leading to sterility, which highlights the potential that targeting VgR via CRISPR can create genetically sterile females. Data are discussed with regard to integration of a sterile insect technique approach in the design of novel, efficient and safe pest management tactics. &#xa9; 2025 Society of Chemical Industry.},
}
@article {pmid40256962,
year = {2025},
author = {Niu, RC and Zeng, QH and Wang, WJ and Wang, YJ and Liu, TX and Zhang, SZ},
title = {Knockout of silk fibroin genes in Plutella xylostella results in decreased fitness and performance.},
journal = {Pest management science},
volume = {81},
number = {9},
pages = {5010-5020},
doi = {10.1002/ps.8852},
pmid = {40256962},
issn = {1526-4998},
support = {//National Natural Science Foundation of China/ ; //Key R&amp;D Program Project of Ningxia Hui Autonomous Region/ ; },
mesh = {Animals ; *Fibroins/genetics/metabolism ; *Moths/genetics/growth &amp; development/physiology ; Gene Knockout Techniques ; Pupa/growth &amp; development/genetics ; Larva/growth &amp; development/genetics/physiology ; *Insect Proteins/genetics/metabolism ; CRISPR-Cas Systems ; Silk/genetics ; *Genetic Fitness ; },
abstract = {BACKGROUND: The ability of insects to spin silk is crucial for their survival, reproduction, and interactions with the environment. The diamondback moth (DBM), Plutella xylostella, a serious agricultural pest, relies on silk production, which influences its behavior and population dynamics.<br><br>RESULTS: This study employed CRISPR/Cas9 technology to knock out three genes, that is, silk fibroin heavy chain (PxFibH), silk fibroin light chain (PxFibL), and fibrohexamerin (PxP25), to investigate their roles in silk gland development and related biological traits. We successfully generated PxFibH, PxFibL, and PxP25 knockout mutants, which displayed defective cocoon formation and developed into naked pupae. Further analysis revealed significant alterations in silk gland structure and various biological parameters, including increased larval mortality, prolonged developmental time, reduced pupal weight, and shortened adult lifespan.<br><br>CONCLUSIONS: These findings highlight the importance of silk fibroin genes in silk production and growth development in P. xylostella, positioning them as potential targets for innovative pest control strategies. &#xa9; 2025 Society of Chemical Industry.},
}
@article {pmid40777797,
year = {2025},
author = {Zhao, F and Chen, F and Yu, H and Fan, S and Bai, M and Xue, J and Zhao, Y and Zuo, X and Fan, C and Zhao, Y},
title = {CRISPR/Cas system-guided plasmid mutagenesis without sequence restriction.},
journal = {Fundamental research},
volume = {5},
number = {4},
pages = {1481-1487},
pmid = {40777797},
issn = {2667-3258},
abstract = {Plasmid mutagenesis is an essential step to engineer protein variants with desired properties. Direct generation of mutations on plasmids is a promising method compared to PCR-based methods and subcloning, yet it suffers from the sequence restriction in the generation of single-stranded circular plasmids. Here we demonstrate the CRISPR/Cas system-guided plasmid mutagenesis, a method using gRNA/Cas9 nickase complex to generate single-stranded circular plasmids as polymerization templates for mutagenesis. This method can directly create user-defined mutation libraries on the plasmid. It offers broad sequence programmability and even covers methylated plasmids. We further combine this method with rational design to engineer genome-editing protein FnCpf1. The FnCpf1 variants with mutations in the PAM-binding groove were generated. Several variants expanded the PAM range and exhibited lower off-target effect, which can loosen PAM constraint and enhance target specificity for genome editing. This work presents an effective tool for plasmid mutagenesis and protein engineering studies.},
}
@article {pmid40774991,
year = {2025},
author = {Tabashiri, R and Mahmoodian, S and Pakdel, MH and Shariati, V and Meimandipour, A and Zamani, J},
title = {Comprehensive in vitro and whole-genome characterization of probiotic properties in Pediococcus acidilactici P10 isolated from Iranian broiler chicken.},
journal = {Scientific reports},
volume = {15},
number = {1},
pages = {28953},
pmid = {40774991},
issn = {2045-2322},
mesh = {Animals ; *Pediococcus acidilactici/genetics/isolation &amp; purification/physiology ; *Probiotics ; *Chickens/microbiology ; *Genome, Bacterial ; Whole Genome Sequencing ; Bacterial Adhesion/genetics ; Bacteriocins/genetics ; },
abstract = {This study presents a comprehensive characterization of Pediococcus acidilactici strain P10, a novel probiotic isolated from native broiler chickens, integrating in vitro analyses with whole-genome sequencing. P10 demonstrates promising probiotic attributes, supported by both phenotypic and genomic evidence. The strain was non-hemolytic and exhibited high survival rates under simulated gastrointestinal conditions (95-99% in acidic pH, 55% in bile salts), with genomic analysis confirming the presence of associated stress resistance genes. Importantly, P10 displayed potent broad-spectrum antimicrobial activity against key pathogens, underpinned by the identification of multiple putative bacteriocin-encoding genes. Furthermore, the strain showed strong adherence to intestinal epithelial cells, with corresponding adhesion genes identified in its genome. Beyond these phenotypic-genotypic correlations, P10's whole-genome sequencing revealed significant novel findings. The 1.84 Mb genome confirmed P10 as P. acidilactici and, most notably, identified a complete, functional Type II-A CRISPR-Cas system. This system, with 17 phage-matching spacers, represents a robust antiviral defense mechanism, a key and distinct feature for probiotic application. Additionally, pan-genomic analysis highlighted 59 genes unique to P10 not found in other P. acidilactici strains, suggesting novel metabolic and adaptive capabilities previously uncharacterized within the species. In summary, Pediococcus acidilactici strain P10 is a highly promising probiotic, combining confirmed resilience and antimicrobial action with unique genomic advantages such as its specialized CRISPR-Cas system and novel genetic elements, making it a valuable candidate for applications in animal health and functional foods.},
}
@article {pmid40774482,
year = {2025},
author = {Geerthana, S and Suresh, K and Prabhakar, N and Rani, BU and Thangaraj, K and Paramasivam, M and Kumar, A and Asokan, R and Karuppannasamy, A},
title = {Harnessing CRISPR/Cas-mediated genetic innovations in lepidopteran insects.},
journal = {International journal of biological macromolecules},
volume = {},
number = {},
pages = {146574},
doi = {10.1016/j.ijbiomac.2025.146574},
pmid = {40774482},
issn = {1879-0003},
abstract = {CRISPR/Cas-mediated genome editing of eukaryotic organisms gained immense popularity after the monumental publications made in 2012 and 2013 in heterologous editing of Escherichia coli and human kidney cells, respectively. In this regard, CRISPR/Cas has several important applications in agriculture, including gene-function validation and pest management. Recently researchers have contemplated employing this approach in achieving a chemical-free insect pest management strategy called 'genetic pest management'. In this approach both sex ratio and male sterility are achieved in one go by editing precisely the sex-determination genes and spermatogenesis-related genes. In vitro target gene validation is the key before taking up the final field application which is accomplished by employing ribonucleoprotein [RNP] complex combining guide RNA [gRNA] targeting specific regions and Cas protein which cuts the DNA at specific site/s followed by the error-prone repair by non-homologous end joining [NHEJ] resulting loss-of-function of the target gene. CRISPR/Cas-based genetic pest management possesses a lot of potential in Lepidoptera an important species order in Insecta and several globally significant pests. Therefore, the present review focuses on pertinent, up-to-date information on genome editing of the functional and phenotypic genes in Lepidoptera.},
}
@article {pmid40774041,
year = {2025},
author = {Long, J and Wu, J and Xi, Y and Zhang, J and Chen, S and Yang, H and Duan, G},
title = {Association between Type IV-A CRISPR/Cas system and plasmid-mediated transmission of carbapenemase genes in Klebsiella pneumoniae.},
journal = {Microbiological research},
volume = {301},
number = {},
pages = {128297},
doi = {10.1016/j.micres.2025.128297},
pmid = {40774041},
issn = {1618-0623},
abstract = {The global rise of carbapenem-producing K. pneumoniae is largely attributed to plasmid-mediated transmission of carbapenemase genes. Type IV-A CRISPR/Cas system is mainly located on plasmids in K. pneumoniae and involved in plasmid competition. However, the role of Type IV-A system in the dissemination of carbapenemase genes in K. pneumoniae remains unclear. Here, we comprehensively investigated the relationship between Type IV-A system and plasmid-mediated transmission of carbapenemase genes based on 152 K. pneumoniae clinical strains and 46226 K. pneumoniae public genomes available in NCBI database. We found that the presence of Type IV-A system was positively associated with blaNDM-1, blaNDM-5, blaOXA-48, and blaVIM-1 but negatively related to blaKPC-2, blaKPC-3,blaIMP and blaOXA-181. Additionally, plasmids carrying Type IV-A system were predominantly the vehicles of blaNDM-1 gene. Protospacer search revealed that Type IV-A system frequently matched conjugation transfer region of blaKPC-2-related IncF plasmids, especially IncFIB(K)/IncFII(K), IncFII(pHN7A8)/IncR, and IncFIB(pQil)/IncFII(K) plasmids. The prevalence of self-targeting event further highlighted the interference mechanism of transcriptional repression proposed by Type IV system. Despite frequent targeting of IncF plasmids by Type IV-A system, different types of IncF plasmids displayed varying distribution between CRISPR-positive and -negative genomes, thereby suggesting a differentiated response of Type IV-A system to IncF plasmids. Our results underscore complex interactions between Type IV-A system and plasmid-mediated carbapenemase genes, revealing its significant role in shaping the transmission dynamics of carbapenemase-encoding plasmids.},
}
@article {pmid40773238,
year = {2025},
author = {Pai, VJ and Shan, H and Donaldson, CJ and Vaughan, JM and De Souza, EV and O'Connor, C and Liem, M and Pinto, AFM and Diedrich, J and Saghatelian, A},
title = {CRISPR-Cas9 screening reveals microproteins regulating adipocyte proliferation and lipid metabolism.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {122},
number = {32},
pages = {e2506534122},
doi = {10.1073/pnas.2506534122},
pmid = {40773238},
issn = {1091-6490},
support = {F32 DK132927/DK/NIDDK NIH HHS/United States ; R01 GM102491/GM/NIGMS NIH HHS/United States ; RC2 DK129961/DK/NIDDK NIH HHS/United States ; Chair//Ferring Foundation/ ; },
mesh = {Animals ; Mice ; *CRISPR-Cas Systems/genetics ; *Cell Proliferation/genetics ; 3T3-L1 Cells ; *Lipid Metabolism/genetics ; *Adipocytes/metabolism/cytology ; Open Reading Frames/genetics ; Cell Differentiation/genetics ; Micropeptides ; },
abstract = {Small open reading frames (smORFs) encode microproteins that play crucial roles in various biological processes, yet their functions in adipocyte biology remain largely unexplored. In a previous study, we identified thousands of smORFs in white and brown adipocytes derived from the stromal vascular fraction of mice using ribosome profiling. Here, we expand on this work by identifying additional smORFs related to adipocytes using the in vitro 3T3-L1 preadipocyte model. To systematically investigate the functional relevance of these smORFs, we designed a custom CRISPR/Cas9 single guide RNA (sgRNA) library and screened for smORFs influencing adipocyte proliferation and differentiation. Through a dropout screen and fluorescence-assisted cell sorting of lipid droplets, we identified dozens of smORFs that regulate either cell proliferation or lipid accumulation. The smORFs on the 5'- and 3'-untranslated regions (i.e., upstream smORFs (uORFs) and downstream smORFs (dORFs)) of functional genes can exert activity through cis-regulatory effects of the main ORF on these messenger RNAs (mRNAs), such as uORFs of MDM2 that impact proliferation. However, other smORFs, especially those from mRNAs with no other ORFs, point to a functional microprotein. Indeed, we tested a candidate smORF 1183 from a long noncoding RNA 923011K14Rik and demonstrated that the microprotein regulates adipocyte differentiation. These findings highlight the potential of CRISPR/Cas9-based screening to uncover functional smORFs and provide a framework for further exploration of microproteins in adipocyte biology and metabolic regulation.},
}
@article {pmid40773236,
year = {2025},
author = {Ogawa, Y and Nguyen, DVM and Ogawa, A and Rasband, MN},
title = {Hide-and-Seek genome editing reveals that Gephyrin is required for axo-axonic synapse assembly.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {122},
number = {32},
pages = {e2500726122},
doi = {10.1073/pnas.2500726122},
pmid = {40773236},
issn = {1091-6490},
support = {NS122073//HHS | NIH | National Institute of Neurological Disorders and Stroke (NINDS)/ ; N/A//Dr. Miriam and Sheldon G. Adelson Medical Research Foundation (AMRF)/ ; },
mesh = {*Gene Editing/methods ; *Synapses/metabolism/genetics ; Animals ; *Membrane Proteins/genetics/metabolism ; CRISPR-Cas Systems ; *Axons/metabolism ; Mice ; Neurons/metabolism ; Dependovirus/genetics ; Mitochondria/metabolism ; *Carrier Proteins/genetics/metabolism ; },
abstract = {The visualization and manipulation of proteins in neurons is widely used to deduce their functions. While every experimental approach has limitations, the concurrent knock-in and knockout of two different proteins can be especially challenging. To this end, we developed Hide-and-Seek genome editing, which allows the simultaneous visualization and knockout of proteins in neurons using Adeno-associated viral vectors and the CRISPR/Cas9 system. We demonstrate the efficacy and flexibility of this method for rapid, efficient, and simultaneous knock-in and knockout of proteins in vitro and in vivo, at the synapse, axon initial segment (AIS), nucleus, and mitochondria. Using Hide-and-Seek, we show that the scaffolding protein Gephyrin is required for the proper assembly of axo-axonic synapses at the AIS.},
}
@article {pmid40773116,
year = {2025},
author = {Patil, C and Priyanka, R and Harshitha, BM and Oshik, S and Yashwanth, S and Darshan, BR and Patil, S and Prajwal, KA and Naik, P and Goudanavar, P and Mallamma, T},
title = {Advanced nanotheranostic approaches for targeted glioblastoma treatment: a synergistic fusion of CRISPR-Cas gene editing, AI-driven tumor profiling, and BBB-modulation.},
journal = {Medical oncology (Northwood, London, England)},
volume = {42},
number = {9},
pages = {413},
pmid = {40773116},
issn = {1559-131X},
mesh = {*Glioblastoma/genetics/therapy/pathology ; Humans ; *Blood-Brain Barrier/metabolism ; *Gene Editing/methods ; *CRISPR-Cas Systems ; *Brain Neoplasms/genetics/therapy/pathology ; *Theranostic Nanomedicine/methods ; Animals ; Nanoparticles ; },
abstract = {Glioblastoma (GBM) is the most aggressive primary brain tumor in adults. It is hard to treat because it is very invasive, has a lot of genetic variation, and the blood-brain barrier (BBB) limits its growth. Traditional GBM treatments, including surgery, radiation and chemotherapy have only marginally improved survival requiring a paradigm shift. This review starts a new way of thinking about how to treat GBM by combining multi-responsive nanotheranostics, next-generation genome engineering, and AI-enabled stratification. The study compares smart nanocarriers that can transport payloads accurately and only when needed. CRISPR/Cas systems are useful for directly editing oncogenes, controlling GBM signaling cascades, making resistant cells sensitive again, and changing the ways that cells avoid the immune system. Nanoparticle-mediated CRISPR delivery and BBB disruption approaches, including targeted ultrasound, receptor-mediated transcytosis, and exosome-based delivery, could help get around the neuroprotective shield without damaging it. This review investigates the molecular aspects of the BBB, highlighting its dual role as a metabolic and efflux transporter-mediated barrier, and assesses cutting-edge nanocarrier systems, in particular hybrid exosome-nanoparticle designs, that aim to circumvent these limitations. Additionally, it explores the current limitations and possible future applications of gene editing tools and AI models for navigating the genomic instability and ever-changing tumor microenvironment of GBM. This study also discusses the ethical and legal issues related to these developing technologies, therefore stressing the need for clinically feasible, ethically controlled innovation pipelines. With an emphasis on cutting-edge research, this review delves into emerging areas such as nanotheranostics, cellular composition of GBM, CRISPR-based gene editing, AI-enabled precision medicine, and targeted disruption of the blood-brain barrier.},
}
@article {pmid40772537,
year = {2025},
author = {Yin, Y and Li, Z and Liu, C and Zhang, XB and Ou, Q},
title = {CRISPR-Based Live Cell Biosensing: Innovations in Signal Labeling Strategies and Delivery Systems.},
journal = {ACS sensors},
volume = {},
number = {},
pages = {},
doi = {10.1021/acssensors.5c01973},
pmid = {40772537},
issn = {2379-3694},
abstract = {CRISPR technology provides unprecedented precision for molecular imaging and target detection at the cellular level. However, interested researchers inevitably encounter challenges, including weak labeling signals, low delivery efficiency, and off-target effects when establishing application frameworks. Therefore, discussing signal labeling strategies and delivery systems is crucial to further improving the performance of CRISPR-based live cell biosensing. In this review, we first focus on signal labeling strategies for CRISPR-based cellular imaging, including fluorescent protein fusion, fluorescent protein recruitment, RNA modification and hybridization, and irrelevant signal reporters. Depending on the effector proteins and application scenarios, selecting the appropriate signal labeling method can help improve imaging sensitivity and signal intensity. Second, we summarize materials that can be used for CRISPR intracellular delivery, such as nanoparticles, nanosheets, and other nanomaterials. Some nanomaterials have been shown to further enhance the activity of effector proteins, in addition to facilitating the cellular entry of active components. Furthermore, we discuss the challenges and future directions of CRISPR imaging technologies. By integrating multidisciplinary innovations, the CRISPR-based live cell biosensor holds promise as a next-generation visualization tool for life science research and precision medicine.},
}
@article {pmid40771633,
year = {2025},
author = {Sleckman, BP and Chen, BR},
title = {THE GID COMPLEX REGULATES DNA END PROCESSING DURING DNA DOUBLE-STRAND BREAK REPAIR BY NONHOMOLOGOUS END JOINING.},
journal = {Transactions of the American Clinical and Climatological Association},
volume = {135},
number = {},
pages = {281-293},
pmid = {40771633},
issn = {0065-7778},
mesh = {*DNA Breaks, Double-Stranded ; *DNA End-Joining Repair ; Humans ; Genomic Instability ; CRISPR-Cas Systems ; Animals ; *Ubiquitin-Protein Ligases/metabolism/genetics ; DNA Repair ; },
abstract = {Genome instability is a hallmark of cancer, allowing for clonal evolution and improved tumor fitness. The mis-repair of DNA double-strand breaks (DSBs) is a major source of genome instability. DNA DSBs are normally repaired by homologous recombination (HR) and nonhomologous end joining (NHEJ). The nucleolytic resection of broken DNA ends generates single-stranded DNA (ssDNA) overhangs that are required for HR, but inhibitory to NHEJ. DNA end resection must be prevented in nondividing cells where NHEJ is the only active DSB repair pathway. Using a novel whole genome gRNA CRISPR/Cas9 screen, we identified the GID complex as functioning to protect DNA ends from nucleolytic resection. The GID complex contains multiple E3 ubiquitin ligase subunits and regulates the expression and function of pro-resection machinery. Thus, by antagonizing DNA end resection GID may prevent homology-mediated joining leading to aberrant DSB repair and genome instability in normal and cancerous nondividing cells.},
}
@article {pmid40771623,
year = {2025},
author = {Sharpe, AH},
title = {THE BIOLOGY BEHIND PD-1 CHECKPOINT BLOCKADE.},
journal = {Transactions of the American Clinical and Climatological Association},
volume = {135},
number = {},
pages = {169-183},
pmid = {40771623},
issn = {0065-7778},
mesh = {Humans ; *Programmed Cell Death 1 Receptor/antagonists &amp; inhibitors/immunology/metabolism/genetics ; *Immune Checkpoint Inhibitors/therapeutic use ; *Neoplasms/immunology/genetics/drug therapy ; CRISPR-Cas Systems ; Animals ; Signal Transduction/drug effects ; Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {Programmed death 1 (PD-1) pathway inhibitors have transformed cancer therapy, leading to durable responses in some patients. However, many patients do not benefit from PD-1 blockade therapy, which highlights the critical need to identify new therapeutic targets to complement PD-1 pathway inhibitors. To address this need, we have developed an in vivo clustered regularly interspaced short palindromic repeats (CRISPR)-based screening platform to discover novel regulators of anti-tumor immunity. In this article, I will first discuss the biology of the PD-1 pathway and its role in regulating anti-tumor immunity. Next, I will introduce our innovative CRISPR-based platforms designed for conducting gene screens in mature immune cell lineages and for enabling gene perturbation without stimulating or manipulating immune cells, two approaches that can affect immune cell development and function. In addition, I will illustrate how these platforms facilitate discovery of new targets that can promote anti-tumor immunity and their potential to lead to more effective cancer therapies.},
}
@article {pmid40770768,
year = {2025},
author = {Wu, L and Zhu, X and Liu, Y and Zhao, D and Yu, BC and Wei, Z and Lin, X and Qi, LS},
title = {Identification of replicative aging and inflammatory aging signatures via whole-genome CRISPRi screens.},
journal = {Genome biology},
volume = {26},
number = {1},
pages = {233},
pmid = {40770768},
issn = {1474-760X},
support = {R21AG077193//NIH National Institute of Aging/ ; no. 2046650//the National Science Foundation CAREER award/ ; },
mesh = {Humans ; *Cellular Senescence/genetics ; *Inflammation/genetics ; *Aging/genetics ; Genome-Wide Association Study ; Mesenchymal Stem Cells/metabolism/cytology ; *CRISPR-Cas Systems ; Genomics ; Clustered Regularly Interspaced Short Palindromic Repeats ; Genome, Human ; },
abstract = {BACKGROUND: Aging is a major risk factor for chronic diseases and cancer. Cellular aging, particularly in adult stem cells, offers a high-throughput framework for dissecting the molecular mechanisms of aging.<br><br>RESULTS: We perform multiple genome-wide CRISPR interference (CRISPRi) screenings in human primary mesenchymal stem cells derived from adipose tissue during either replicative senescence or inflammation-induced senescence. These screens reveal distinct sets of potential novel regulators specific to each senescence pathway. Combining our perturbation-based functional genomic data with 405 genome-wide association study datasets, including 50 aging-related studies, we find that the inflammatory aging signatures identified from CRISPRi screenings are significantly associated with diverse aging processes, suggesting novel molecular signatures for analyzing and predicting aging status and aging-related disease.<br><br>CONCLUSIONS: The signatures verified through comprehensive functional genomics and genetic analyses may provide new targets for modulating the aging process and enhancing the quality of cell therapy products.},
}
@article {pmid40770375,
year = {2025},
author = {Aldiss, Z and Lam, Y and Robinson, H and Dixon, R and Steinhardt, L and Crisp, P and Godwin, I and Borrell, A and Hickey, L and Massel, K},
title = {The PIN2 ortholog in barley modifies root gravitropism and architecture.},
journal = {The plant genome},
volume = {18},
number = {3},
pages = {e70061},
doi = {10.1002/tpg2.70061},
pmid = {40770375},
issn = {1940-3372},
support = {DP190102185//Australian Research Council/ ; LP200200927//ARC Linkage project/ ; //Australian Government Research Training Program and InterGrain/ ; FT220100350//ARC Future Fellowship/ ; },
mesh = {*Hordeum/genetics/growth &amp; development/physiology ; *Plant Roots/genetics/growth &amp; development/anatomy &amp; histology/physiology ; *Gravitropism/genetics ; *Plant Proteins/genetics/metabolism ; CRISPR-Cas Systems ; },
abstract = {Roots provide the critical interface where plants acquire nutrients and water, but our limited understanding of the genetic controls modulating root system architecture (RSA) in crop species constrains opportunities to develop future cultivars with improved root systems. However, there is vast knowledge of root developmental genes in model plant species, which has the potential to accelerate progress in crops with more complex genomes, particularly given that genome editing protocols are now available for most species. PIN-FORMED2 (PIN2) encodes a root-specific polar auxin transporter, where its absence resulted in roots being unable to orient themselves using gravity, producing a significantly wider root system. To explore the role of PIN2 in a cereal crop, we used CRISPR/Cas9 (where CRISPR is clustered regularly interspaced short palindromic repeats) editing to knockout of PIN2 in barley (Hordeum vulgare). Like Arabidopsis, the roots of barley pin2 loss-of-function mutants displayed an agravitropic response at seedling growth stages, resulting in a significantly shallower and wider root system at later growth stages. Notably, despite the significant change in RSA, there was no change in shoot architecture or total shoot biomass, with an insensitivity to the effects of higher planting density. We discuss the future challenges and opportunities to harness the PIN2 pathway to optimize RSA in crops for a range of production scenarios without a shoot trade-off.},
}
@article {pmid40770192,
year = {2025},
author = {Erkek, F and Kizilkaya, R and Baybara, S and Gulden, G and Sert, B and Yazicilar, MS and Yilmaz, MC and Odabas, SP and Bal, E and Yelgen, G and Teymur, T and Ay, Y and Dogdu, S and Tiryaki, NN and Baran, B and Gelsin, B and Araz, H and Cavrar, I and Tastan, C},
title = {CRISPR.BOT an autonomous platform for streamlined genetic engineering and molecular biology applications.},
journal = {Scientific reports},
volume = {15},
number = {1},
pages = {28699},
pmid = {40770192},
issn = {2045-2322},
support = {&#xdc;&#xdc;BAP-YP-2020-005//Uskudar University Scientific Research Studies/ ; },
mesh = {Humans ; *CRISPR-Cas Systems ; *Gene Editing/methods ; *Genetic Engineering/methods ; Green Fluorescent Proteins/genetics ; Synthetic Biology/methods ; HEK293 Cells ; Plasmids/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; },
abstract = {In the realm of synthetic biology, the pursuit of streamlined experimental processes has given rise to innovative technologies poised to redefine research paradigms. Traditional workflows, laden with laborious and error-prone stages, incur prolonged timelines and escalating costs. Autonomous liquid processing systems emerge as promising tools, holding the potential to enhance precision and efficiency. Yet, the adoption of fully automated systems is impeded by prohibitive costs and maintenance expenses. This study introduces the CRISPR.BOT as a transformative solution, a pioneering autonomous genetic engineering platform designed to revolutionize molecular biology practices. The CRISPR.BOT demonstrates its prowess through a series of pivotal experiments, ranging from the efficient transfer of green fluorescent protein (GFP) encoding plasmid DNA into bacterial hosts to intricate manipulations involving lentiviral transduction and CRISPR-Cas9-mediated genetic editing in human cell lines. Notably, the CRISPR.BOT achieves exceptional results in single-cell subcloning, yielding GFP + CRISPR-gRNA + cells with unprecedented purity levels of 90-100%. Moreover, this study underscores the CRISPR.BOT's potential to facilitate safe genetic engineering practices, enabling researchers to work with pathogenic microorganisms like the SARS CoV-2 virus without direct human contact. An exploration into the cost-effectiveness of LEGO Mindstorms robots reveals their suitability for daily laboratory routines, presenting a cost reduction of up to tenfold compared to commercial alternatives. In conclusion, the CRISPR.BOT emerges as a transformative force in synthetic biology, offering a pathway to redefine experimental processes, enhance precision, and unlock new possibilities in genetic engineering. This research marks a paradigm shift where automation and innovation converge, empowering researchers to navigate uncharted territories with confidence and efficiency.},
}
@article {pmid40759748,
year = {2025},
author = {Wolf, B and Shehu, P and Brenker, L and von Bachmann, AL and Kroell, AS and Southern, N and Holderbach, S and Eigenmann, J and Aschenbrenner, S and Mathony, J and Niopek, D},
title = {Rational engineering of allosteric protein switches by in silico prediction of domain insertion sites.},
journal = {Nature methods},
volume = {22},
number = {8},
pages = {1698-1706},
pmid = {40759748},
issn = {1548-7105},
mesh = {*Protein Engineering/methods ; Humans ; Allosteric Regulation ; Machine Learning ; Escherichia coli/genetics/metabolism ; Allosteric Site ; Computer Simulation ; Protein Domains ; CRISPR-Cas Systems ; *Proteins/chemistry/genetics ; },
abstract = {Domain insertion engineering is a powerful approach to juxtapose otherwise separate biological functions, resulting in proteins with new-to-nature activities. A prominent example are switchable protein variants, created by receptor domain insertion into effector proteins. Identifying suitable, allosteric sites for domain insertion, however, typically requires extensive screening and optimization. We present ProDomino, a machine learning pipeline to rationalize domain recombination, trained on a semisynthetic protein sequence dataset derived from naturally occurring intradomain insertion events. ProDomino robustly identifies domain insertion sites in proteins of biotechnological relevance, which we experimentally validated in Escherichia coli and human cells. Finally, we used light- and chemically regulated receptor domains as inserts and demonstrate the rapid, model-guided creation of potent, single-component opto- and chemogenetic protein switches. These include novel CRISPR-Cas9 and -Cas12a variants for inducible genome engineering in human cells. Our work enables one-shot domain insertion engineering and substantially accelerates the design of customized allosteric proteins.},
}
@article {pmid40704969,
year = {2025},
author = {Li, J and Lau, CH and Wang, J and Wu, W and Huang, Z and Chen, X and Li, J and Huang, Y and Wang, T and Li, Y and Zhao, Z and Xu, M and Chen, G and Tong, S and Zhu, H},
title = {Rapid, multiplex, one-pot CRISPR/Dx system for detecting cancer fusion genes.},
journal = {Analytical methods : advancing methods and applications},
volume = {17},
number = {31},
pages = {6304-6316},
doi = {10.1039/d5ay00783f},
pmid = {40704969},
issn = {1759-9679},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Lung Neoplasms/genetics/diagnosis ; Proto-Oncogene Proteins/genetics ; *Oncogene Proteins, Fusion/genetics ; Protein-Tyrosine Kinases/genetics ; Gene Fusion ; },
abstract = {Targeted therapies directed at fusion genes have proven remarkably effective against cancers. Therefore, the rapid and reliable identification of cancer fusion genes can guide subsequent therapeutic treatment and predict prognosis. By integrating the RT-RPA and CRISPR/Cas12a approaches, we developed a one-pot CRISPR/Dx system for the rapid and multiplex detection of cancer fusion genes. A tube with unique assemblies was created using 3D printing technology to realize this application. As proof of principle, we demonstrated the feasibility of the one-pot CRISPR/Dx system in detecting lung cancer by targeting ROS1 fusions. The performance of the one-pot CRISPR/Dx detection system was comparable to a two-tube-based testing platform. When tested with synthetic RNA fusions, both approaches efficiently detected all 14 ROS1 fusions with an LOD in the range of 5-10 copies per μL, without generating a background signal, even in the presence of a large excess of wild-type RNA. The total reaction time for both approaches was 30 minutes. Notably, the one-pot CRISPR/Dx detection system minimized the operation steps and aerosol contamination without compromising detection sensitivity and specificity. Furthermore, its diagnostic power was validated using clinical samples. Thus, we successfully developed a rapid, multiplex, one-pot CRISPR/Dx detection system for detecting 14 clinically relevant ROS1 fusions with high sensitivity and specificity. It is also cost-effective and simple to operate, thereby realizing the ultimate goal of establishing CRISPR/Dx as the paragon of cancer diagnostics for home self-testing and point-of-care testing.},
}
@article {pmid40695391,
year = {2025},
author = {Bui, VKH and Trung, LG and Do, HH and Tran, NT and Nguyen, MK},
title = {Unlocking the algae toolbox: Cutting-edge tools for environmental and biotechnological solutions.},
journal = {Biotechnology advances},
volume = {83},
number = {},
pages = {108652},
doi = {10.1016/j.biotechadv.2025.108652},
pmid = {40695391},
issn = {1873-1899},
mesh = {*Biotechnology/methods ; *Microalgae/genetics ; CRISPR-Cas Systems ; Biofuels ; Genetic Engineering ; Ecosystem ; },
abstract = {Algae are highly adaptable photosynthetic organisms with growing relevance in biotechnology, environmental management, and sustainable industries. Recent advancements have expanded the "Algae Toolbox", integrating cutting-edge screening, monitoring, characterization, and large-scale production technologies. This current work critically examines the latest developments in algal research, including CRISPR-Cas9 genetic engineering, high-throughput omics, machine learning-driven modeling, and remote sensing-based ecosystem monitoring. Moreover, innovations in optical sensors, flow cytometry, biosensors, and environmental DNA have further improved precision in tracking algal dynamics. These advances support industrial scale-up, boosting productivity and sustainability in biofuels, pharmaceuticals, aquaculture, and bioremediation. However, large-scale production still faces challenges in economic viability and environmental sustainability. Future research must incorporate artificial intelligence, internet of things-enabled sensing, and synthetic biology for optimized cultivation and bioproduct harvesting. This review provides a comprehensive overview of emerging tools and methodologies, emphasizing their role in advancing algal biotechnology and global sustainability.},
}
@article {pmid40663809,
year = {2025},
author = {Wang, J and Tian, Y and Zhang, K and Zhao, Y},
title = {A high-sensitivity ECL biosensor for single-cell analysis: Integrating CRISPR/Cas12a and entropy-driven amplification.},
journal = {Bioelectrochemistry (Amsterdam, Netherlands)},
volume = {166},
number = {},
pages = {109045},
doi = {10.1016/j.bioelechem.2025.109045},
pmid = {40663809},
issn = {1878-562X},
mesh = {*Biosensing Techniques/methods ; Humans ; *Single-Cell Analysis/methods ; *CRISPR-Cas Systems ; HEK293 Cells ; *Entropy ; *Luminescent Measurements/methods ; *Nucleic Acid Amplification Techniques/methods ; *Electrochemical Techniques/methods ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {This study presents a homogeneous electrochemiluminescence (ECL) biosensor for ultrasensitive detection of HEK293 cells by targeting the hERG potassium channel at the single-cell level. The biosensor integrates multiple signal amplification steps, including photocleavable DNA-antibody conjugates, entropy-driven strand displacement, T7 RNA polymerase-mediated transcription, and CRISPR/Cas12a-mediated trans-cleavage. This cascade enables precise and robust signal enhancement. A key feature of the system is its ability to generate a clearly measurable ECL response from as little as a single HEK293 cell, without the need for signal averaging. Specificity was confirmed using unrelated cell lines and mismatched DNA sequences, with minimal background observed in negative controls. Optimization of key parameters-such as enzyme concentrations, reaction times, and duplex composition-ensured consistent and reproducible performance. ECL measurements were conducted under a defined voltage scan (0-1.3 V) in a three-electrode system, and emission signals were recorded using a photomultiplier tube. This biosensor demonstrates the feasibility of single-cell detection with high sensitivity and specificity, offering a promising platform for future applications in cell-based analysis and molecular diagnostics.},
}
@article {pmid40652737,
year = {2025},
author = {Shi, Z and Yan, Y and Zhu, R and Zhu, X and Hu, K and Yue, Y and Xu, W and Xuan, M and Gan, X and Yang, Z and Zhang, Y and Cao, Z},
title = {DUXA regulates the early development of porcine cloned embryos.},
journal = {Theriogenology},
volume = {247},
number = {},
pages = {117563},
doi = {10.1016/j.theriogenology.2025.117563},
pmid = {40652737},
issn = {1879-3231},
mesh = {Animals ; Swine/embryology/genetics ; *Cloning, Organism/veterinary ; *Embryonic Development/genetics/physiology ; Nuclear Transfer Techniques/veterinary ; *Gene Expression Regulation, Developmental/physiology ; *Homeodomain Proteins/genetics/metabolism ; *Transcription Factors/genetics/metabolism ; Embryo, Mammalian ; Blastocyst/physiology ; Female ; CRISPR-Cas Systems ; },
abstract = {Abnormal zygotic genome activation (ZGA) during the early development of somatic cell nuclear transfer (SCNT) embryos is one of the main reasons for the low cloning efficiency. The double homeobox (DUX) family, which includes important transcription factors in mammals, has been shown to play an important role in the ZGA process in mice. However, the role of DUXA, a member of the DUX family, in the early development of porcine somatic cloned embryos is unknown. Here, CRISPR/Cas9 gene editing and lentiviral infection technologies were used to construct stable DUXA knockout and overexpression cell lines for the production of SCNT embryos. Compared with that of wild-type (WT) SCNT embryos, the blastocyst rate of DUXA knockout embryos was significantly lower (P < 0.05), whereas the blastocyst rate of DUXA-overexpressing embryos was significantly greater (P < 0.05). Moreover, RT‒qPCR results revealed that DUXA knockout significantly reduced the expression levels of ZGA-related genes (TDG, SNAI1, RSRP1, TFAP2C, ZSCAN4, LEUTX, and KLF17) (P < 0.05). Additionally, in DUXA-overexpressing embryos, the mRNA levels of TDG, SNAI1, RSRP1, and TFAP2C significantly decreased (P < 0.05), whereas the ZSCAN4, LEUTX, and KLF17 mRNA levels increased (P < 0.05). These findings suggest that DUXA regulates the early development of porcine SCNT embryos by modulating the expression of ZGA-related genes. This research provides significant insights into the potential mechanisms of early embryo loss in porcine SCNT.},
}
@article {pmid40651075,
year = {2025},
author = {Xiao, X and Lu, Y and Zhang, J and Ni, W and Liu, J and Li, C and Yao, Q and Sun, Y and Zhang, GJ and Zhang, Y and Qin, Y and Zhou, Y},
title = {Rolling circle amplification and CRISPR/Cas14a with nanozyme for electrochemical detecting miRNA-205 in NPC-derived exosomes.},
journal = {Bioelectrochemistry (Amsterdam, Netherlands)},
volume = {166},
number = {},
pages = {109046},
doi = {10.1016/j.bioelechem.2025.109046},
pmid = {40651075},
issn = {1878-562X},
mesh = {*MicroRNAs/analysis/genetics ; Humans ; *Electrochemical Techniques/methods ; *Exosomes/chemistry/genetics ; *Nucleic Acid Amplification Techniques/methods ; *Biosensing Techniques/methods ; *CRISPR-Cas Systems ; *Nasopharyngeal Carcinoma/genetics/diagnosis ; Limit of Detection ; *Nasopharyngeal Neoplasms/genetics/diagnosis ; Nanowires/chemistry ; Platinum/chemistry ; },
abstract = {Nasopharyngeal carcinoma (NPC) is a highly malignant tumor, and early detection of biomarkers like miRNA-205 (miR-205) is critical for improving prognosis. However, accurate detection of miR-205 remains challenging due to its low abundance and matrix interference. Herein, an ultrasensitive electrochemical biosensor integrating Pt nanowires/MXene (PtNWs/MXene), rolling circle amplification (RCA), and CRISPR/Cas14a was developed for detecting exosomal miR-205. The dual characteristics of PtNWs/MXene (differential adsorption capacity for intact and cleaved DNA; HRP-like nanozyme activity) enable the conversion of the miR-205-triggered RCA-Cas14a cascade reaction into significant electrochemical signal changes. This biosensor eliminates the requirement for signal probe labeling of the electrode-modified DNA. Moreover, the enzyme-mimicking catalytic activity of PtNWs/MXene enables the catalysis of numerous 3,3',5,5'-tetramethylbenzidine (TMB) molecules, realizing a "one-to-many" signal amplification effect that significantly improves detection sensitivity. The biosensor achieves a detection limit of 4.6 aM (50 aM-10 pM linear range) and distinguishes single-base mismatches. Clinical validation confirmed its ability to differentiate NPC patients from healthy individuals, aligning with qRT-PCR results. By adjusting the RCA template, this strategy can be adapted for diverse RNA/DNA targets, offering a versatile platform for early disease diagnosis.},
}
@article {pmid40604285,
year = {2025},
author = {Chen, AXY and Yap, KM and Kim, JS and Sek, K and Huang, YK and Dunbar, PA and Wiebking, V and Armitage, JD and Munoz, I and Todd, KL and Derrick, EB and Nguyen, D and Tong, J and Chan, CW and Hoang, TX and Audsley, KM and van Elsas, MJ and Middelburg, J and Lee, JN and de Menezes, MN and Cole, TJ and Li, J and Scheffler, C and Scott, AM and Mackay, LK and Waithman, J and Oliaro, J and Harrison, SJ and Parish, IA and Lai, J and Porteus, MH and House, IG and Darcy, PK and Beavis, PA},
title = {Rewiring endogenous genes in CAR T cells for tumour-restricted payload delivery.},
journal = {Nature},
volume = {644},
number = {8075},
pages = {241-251},
pmid = {40604285},
issn = {1476-4687},
mesh = {Animals ; Mice ; Humans ; *Receptors, Chimeric Antigen/genetics/immunology/metabolism ; *T-Lymphocytes/immunology/metabolism ; Female ; *Immunotherapy, Adoptive/methods ; *Neoplasms/therapy/immunology/genetics/pathology ; Interleukin-2/genetics/immunology/metabolism/administration &amp; dosage ; Gene Knock-In Techniques ; Promoter Regions, Genetic/genetics ; Interleukin-12/genetics/immunology/metabolism ; CRISPR-Cas Systems/genetics ; Male ; Transgenes/genetics ; Cell Line, Tumor ; },
abstract = {The efficacy of chimeric antigen receptor (CAR) T cell therapy in solid tumours is limited by immunosuppression and antigen heterogeneity[1-3]. To overcome these barriers, 'armoured' CAR T cells, which secrete proinflammatory cytokines, have been developed[4]. However, their clinical application has been limited because of toxicity related to peripheral expression of the armouring transgene[5]. Here, we have developed a CRISPR knock-in strategy that leverages the regulatory mechanisms of endogenous genes to drive transgene expression in a tumour-localized manner. By screening endogenous genes with tumour-restricted expression, we have identified the NR4A2 and RGS16 promoters as promising candidates to support the delivery of cytokines such as IL-12 and IL-2 directly to the tumour site, leading to enhanced antitumour efficacy and long-term survival of mice in both syngeneic and xenogeneic models. This effect was concomitant with improved CAR T cell polyfunctionality, activation of endogenous antitumour immunity and a favourable safety profile, and was applicable in CAR T cells from patients.},
}
@article {pmid40602043,
year = {2025},
author = {Gong, Y and Li, G and Meng, Y and Wei, J and Tang, Q and Huang, L and Zhang, K and Liao, X},
title = {Development of a CRISPR/Cas13a-based electrochemiluminescence biosensing strategy for sensitive detection of α-synuclein oligomers in neurodegenerative diseases.},
journal = {Bioelectrochemistry (Amsterdam, Netherlands)},
volume = {166},
number = {},
pages = {109038},
doi = {10.1016/j.bioelechem.2025.109038},
pmid = {40602043},
issn = {1878-562X},
mesh = {*alpha-Synuclein/blood/analysis ; *Biosensing Techniques/methods ; Humans ; *CRISPR-Cas Systems ; *Neurodegenerative Diseases/blood/diagnosis ; *Luminescent Measurements/methods ; *Electrochemical Techniques/methods ; Limit of Detection ; },
abstract = {In this study, we report a highly sensitive CRISPR/Cas13a-based electrochemiluminescence (ECL) biosensor for detecting α-synuclein oligomers, early biomarkers for neurodegenerative diseases. The system integrates aptamer recognition, T7 transcription, CRISPR/Cas13a cleavage, and EXPAR amplification. α-Synuclein binding triggers the release of the T7 promoter, leading to RNA production that activates Cas13a, initiating collateral cleavage and EXPAR, generating double-stranded DNA that interacts with [Ru(phen)2dppz][2+] to produce a measurable ECL signal. The sensor achieved an ultralow detection limit of 1.025 aM with high specificity and stability. In serum samples, recovery ranged from 95.2 % to 99.8 %, demonstrating strong accuracy. No interference was observed from unrelated proteins. The biosensor showed excellent reproducibility (intra-day RSD = 0.78 %, inter-day RSD = 2.86 %) and stable performance over 14 days. Compared to other existing methods, this strategy offers superior sensitivity and comparable dynamic range, making it highly suitable for clinical use. Although the assay requires multiple steps and approximately two hours, the tradeoff is justified by its performance. This work highlights the potential of combining CRISPR/Cas13a with ECL for ultra-sensitive biomarker detection in complex samples, supporting early diagnosis and monitoring of Parkinson's and Alzheimer's disease.},
}
@article {pmid40581010,
year = {2025},
author = {Zhang, S and Bu, G and Jin, H and Zhang, B},
title = {Nucleic acid-guided genome editing of plant carbohydrate pathways for functional crops supporting glycogen replenishment in athletes.},
journal = {International journal of biological macromolecules},
volume = {319},
number = {Pt 4},
pages = {145574},
doi = {10.1016/j.ijbiomac.2025.145574},
pmid = {40581010},
issn = {1879-0003},
mesh = {*Gene Editing/methods ; *Glycogen/metabolism/biosynthesis ; *Ipomoea batatas/genetics/metabolism ; *Crops, Agricultural/genetics/metabolism ; *Carbohydrate Metabolism/genetics ; CRISPR-Cas Systems ; Starch/metabolism ; *Genome, Plant ; Humans ; },
abstract = {Nucleic acid-guided genome editing is transforming plant breeding by enabling precise modification of metabolic pathways to meet evolving food and nutritional needs. In this study, we applied multiplex CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats and CRISPR-associated protein 9) editing to improve Ipomoea batatas (sweet potato), a climate-resilient staple, for enhanced starch yield and customized digestibility suited for athletic performance nutrition. We targeted four key starch-related genes: AGPase (ADP-glucose pyrophosphorylase), GBSSI (granule-bound starch synthase I), SBEII (starch branching enzyme II), and GPT1 (glucose-6-phosphate translocator). Editing efficiency reached 85 to 92 % across multiple genome copies (homeologs), with no detectable off-target effects. The edited lines showed up to a 20 % increase in total starch and a fivefold reduction in amylose, which is the slowly digestible fraction of starch. This resulted in a high-amylopectin profile associated with rapid glucose release. Transcriptome analysis confirmed the metabolic shifts, including increased expression of GPT1 and sucrose synthase and reduced expression of invertase, which supports improved carbon allocation toward starch biosynthesis. In vitro digestibility and glycemic index (GI) assessments confirmed the functional impact of these traits for glycogen replenishment. Yield performance and drought resilience were retained, confirming agronomic viability. This study highlights the potential of CRISPR-based editing to develop climate-smart, nutritionally functional crops for both health-focused applications and global food security.},
}
@article {pmid40544550,
year = {2025},
author = {Jia, L and Kang, K and Yan, W and Jin, Y and Xu, K and Wang, J and Wang, Y and Jiang, M and Yuan, W and Liu, S and Niu, L},
title = {17β-Estradiol point-of-care test by commercial pregnancy test strips based on target-triggered CRISPR/Cas12a cleavage activity.},
journal = {Bioelectrochemistry (Amsterdam, Netherlands)},
volume = {166},
number = {},
pages = {109035},
doi = {10.1016/j.bioelechem.2025.109035},
pmid = {40544550},
issn = {1878-562X},
mesh = {*Estradiol/analysis/urine ; *CRISPR-Cas Systems ; Humans ; Female ; Pregnancy ; *Point-of-Care Testing ; *Pregnancy Tests/methods/instrumentation ; *Endodeoxyribonucleases/metabolism ; Chorionic Gonadotropin/chemistry ; Limit of Detection ; *Reagent Strips/analysis ; Biosensing Techniques/methods ; DNA, Single-Stranded/chemistry ; Bacterial Proteins ; CRISPR-Associated Proteins ; },
abstract = {17β-Estradiol (E2) serves as both a reproductive accelerator and growth promoter, yet its uncontrolled application induces severe endocrine dysfunction. Point-of-care testing (POCT) has emerged as a promising analytical platform for on-site E2 quantification, due to its inherent advantages in terms of sensitive, specific, cost-effective, and instrument-free. We developed a novel strategy combining the high collateral cleavage activity of CRISPR/Cas12a with the strong amplification power of dual-cycle reaction, translating target presence into a colorimetric signal on pregnancy test strip (PTS). In the presence of E2, the dual-cycle amplification was initiated to produce HP2-HP3 (Hairpin DNA 2-Hairpin DNA 3) duplexes. These duplexes then bound to the Cas12a-crRNA complex, activating its trans-cleavage activity. The activated Cas12a subsequently cleaved the ssDNA linkers in the MBs (magnetic beads)-ssDNA (single-stranded DNA)-hCG (human chorionic gonadotropin) conjugates, releasing hCG for quantitative E2 detection through strip color intensity measurement. By integrating CRISPR/Cas12a's high cleavage efficiency with dual-cycle amplification, a visible biosensor was developed, with a linear range of 1.0 × 10[-1]-200.0 pM and a detection limit of 0.0403 pM for E2. In conclusion, an E2 detection platform characterized by prominent sensitivity and convenience was established for monitoring spiked E2 in milk and urine, providing a significant reference for the POCT detection of other targets.},
}
@article {pmid40541695,
year = {2025},
author = {Ding, X and Lei, X and Yu, S},
title = {Split technology in sensors based on CRISPR/Cas12a system.},
journal = {Biotechnology advances},
volume = {83},
number = {},
pages = {108629},
doi = {10.1016/j.biotechadv.2025.108629},
pmid = {40541695},
issn = {1873-1899},
mesh = {*CRISPR-Cas Systems/genetics ; *Biosensing Techniques/methods ; Gene Editing ; DNA/genetics ; CRISPR-Associated Proteins/genetics ; Bacterial Proteins ; Endodeoxyribonucleases ; },
abstract = {CRISPR/Cas12a system has become a popular tool for nucleic acid analysis in recent years due to its high specificity, sensitivity and programmability. Recently, split technology has been applied to the CRISPR/Cas12a system for activators, crRNA, reporter and Cas12a. As a result, dsDNA without PAM, short ssDNA less than 15 nucleotides, and RNA can be directly detected, which are beyond the target scope of the canonical CRISPR/Cas12a system. Label-free reporter with lower cost can be incorporated into sensors based on the CRISPR/Cas12a system. Logic circuits with multiple inputs and outputs can be constructed in cells. Therefore, split technology can expand the target scope, enhance crRNA stability, increase strategy programmability, and reduce detection cost for the CRISPR/Cas12a system. In this review, we focus on the advancements of split technology in sensors based on the CRISPR/Cas12a system. We also summarize the advantages brought by split technology and discuss the challenges and perspectives of sensors based on the CRISPR/Cas12a system.},
}
@article {pmid40466204,
year = {2025},
author = {Zhou, Z and Li, Y and Wen, D and Zhang, X and Li, N and Shuai, J and Zhang, X and Zheng, X and Ban, Z and Farouk, A and Yu, X and Huang, J},
title = {A CRISPR-Cas13a powered electrochemical sensor based on reduced graphene oxide, polypyrrole and gold nanoparticles nanocomposites for PEDV detection.},
journal = {Bioelectrochemistry (Amsterdam, Netherlands)},
volume = {166},
number = {},
pages = {109019},
doi = {10.1016/j.bioelechem.2025.109019},
pmid = {40466204},
issn = {1878-562X},
mesh = {*Graphite/chemistry ; *Gold/chemistry ; *Pyrroles/chemistry ; *Porcine epidemic diarrhea virus/isolation &amp; purification/genetics ; *Polymers/chemistry ; *Metal Nanoparticles/chemistry ; *Nanocomposites/chemistry ; *Biosensing Techniques/methods ; *CRISPR-Cas Systems ; Animals ; *Electrochemical Techniques/methods ; Limit of Detection ; Swine ; Oxidation-Reduction ; },
abstract = {Porcine epidemic diarrhea virus (PEDV) is a highly infectious pathogen responsible for porcine epidemic diarrhea, which causes severe diarrhea and high mortality rates in neonatal piglets, leading to substantial economic losses in the swine industry. Therefore, there is an urgent need for rapid, sensitive, and accurate detection methods for PEDV. In this study, we develop a rapid, ultrasensitive electrochemical CRISPR-based biosensor for detecting PEDV with high sensitivity and specificity. The sensor integrates reduced graphene oxide-polypyrrole‑gold nanoparticle (rGO-PPy-AuNP) nanocomposites to enhance sensitivity and CRISPR-Cas13a for target-specific recognition. This dual-signal amplification strategy achieves an excellent analytical detection limit of 1.01 fg/mL and high linearity (R[2] = 0.9979) across a broad dynamic range (0.005-100,000 pg/mL) within 45 min, eliminating the need for nucleic acid amplification. By synergizing Cas13a-assisted signal enhancement with nanoparticle-mediated electron transfer, the biosensor outperforms conventional methods in sensitivity while maintaining excellent stability, and specificity, demonstrating strong potential for PEDV detection in clinical diagnostics.},
}
@article {pmid40450408,
year = {2025},
author = {Einhaus, A and Krieger, A and Köhne, L and Rautengarten, B and Jacobebbinghaus, N and Saudhof, M and Baier, T and Kruse, O},
title = {Genome editing of epigenetic transgene silencing in Chlamydomonas reinhardtii.},
journal = {Trends in biotechnology},
volume = {43},
number = {8},
pages = {1961-1981},
doi = {10.1016/j.tibtech.2025.04.019},
pmid = {40450408},
issn = {1879-3096},
mesh = {*Chlamydomonas reinhardtii/genetics ; *Gene Editing/methods ; *Transgenes/genetics ; *Gene Silencing ; *Epigenesis, Genetic/genetics ; CRISPR-Cas Systems/genetics ; },
abstract = {Eukaryotic microalgae are steadily advancing as promising green cell factories for sustainable modern biotechnology. However, one of the greatest hurdles to such use are the efficient transgene silencing mechanisms that drastically limit transgene expression levels and stability in engineered microalgal strains. Here, we used CRISPR/Cas9 to target multiple genes involved in epigenetic regulation in the model green microalga Chlamydomonas reinhardtii to identify key factors in epigenetic transgene silencing. Disruption of 11 candidate genes and subsequent systematic combination in double and triple knockout (KO) mutants, enabled a distinct reduction in transgene silencing and improved the stability of transgene expression compared with previously established strains. In addition, a split selectable marker system utilizing the Nostoc punctiforme DnaE split intein for dual-targeted genome editing was established. In summary, this work distinctly advances the biotechnological potential of C. reinhardtii and establishes a valuable mutant collection for further investigation of epigenetic regulation in green microalgae and potentially other eukaryotes.},
}
@article {pmid40391806,
year = {2025},
author = {Chen, Z and Xue, J and Wang, Z and Sun, J and Cui, Y and Zhu, T and Yang, H and Li, M and Wu, B},
title = {Small RNA Toxin-Assisted Evolution of GC-Preferred ErCas12a for Enhanced Genome Targeting Range.},
journal = {Advanced science (Weinheim, Baden-Wurttemberg, Germany)},
volume = {12},
number = {29},
pages = {e17105},
doi = {10.1002/advs.202417105},
pmid = {40391806},
issn = {2198-3844},
support = {XDB0810000//Strategic Priority Research Program of the Chinese Academy of Sciences/ ; 32225002//National Natural Science Foundation of China/ ; 32170033//National Natural Science Foundation of China/ ; 32422001//National Natural Science Foundation of China/ ; ZDBS-LY-SM014//Key Research Program of Frontier Sciences/ ; KFJ-BRP-009//Biological Resources Program/ ; KFJ-BRP-017-58//Biological Resources Program/ ; },
mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; Humans ; Animals ; *CRISPR-Associated Proteins/genetics ; },
abstract = {CRISPR/Cas12a, a promising gene editing technology, faces limitations due to its requirement for a thymine (T)-rich protospacer adjacent motif (PAM). Despite the development of Cas12a variants with expanded PAM profiles, many genomic loci, especially those with guanine-cytosine (GC)-rich PAMs, have remained inaccessible. This study develops a small RNA toxin-aided strategy to evolve ErCas12a for targeting GC-rich PAMs, resulting in the creation of enhanced ErCas12a (enErCas12a). EnErCas12a demonstrates the ability to recognize GC-rich PAMs and target five times more PAM sequences than the wild-type ErCas12a. Furthermore, enErCas12a achieves efficient gene editing in both bacterial and mammalian cells at various sites with non-canonical PAMs, including GC-rich PAMs such as GCCC, CGCC, and GGCC, which are inaccessible to previous Cas12a variants. Moreover, enErCas12a effectively targets PAM sequences with a GC content exceeding 75% in mammalian cells, providing a valuable alternative to the existing Cas12a toolkit. Importantly, enErCas12a maintains high specificity at targets with canonical PAMs, while also demonstrating enhanced specificity at targets with non-canonical PAMs. Collectively, this work establishes enErCas12a as a promising tool for gene editing in both eukaryotes and prokaryotes.},
}
@article {pmid40253584,
year = {2025},
author = {Jo, DH and Jang, H and Cho, CS and Lee, SJ and Heo, JH and Kim, JA and Kim, SJ and Ryu, W and Park, CW and Kang, BC and Gee, HY and Sung, YH and Kim, HH and Kim, JH},
title = {Intravitreal adenine base editing of RS1 improves vision in a preclinical mouse model of retinoschisis.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {33},
number = {8},
pages = {3955-3967},
doi = {10.1016/j.ymthe.2025.04.021},
pmid = {40253584},
issn = {1525-0024},
mesh = {Animals ; Mice ; *Gene Editing/methods ; Disease Models, Animal ; Humans ; *Retinoschisis/genetics/therapy ; Male ; *Adenine/metabolism ; Dependovirus/genetics ; *Eye Proteins/genetics ; Genetic Therapy/methods ; Genetic Vectors/genetics/administration &amp; dosage ; Retina/metabolism/pathology ; Intravitreal Injections ; RNA, Guide, CRISPR-Cas Systems/genetics ; *Vision, Ocular ; },
abstract = {Base editing offers high potential for treating genetic diseases, particularly those with limited treatment options. Retinoschisis, an X-linked retinal disease causing progressive vision loss, currently lacks effective therapies. We identified the c.422G>A (p.Arg141His) variant of the RS1 gene in six male patients with retinoschisis and generated a humanized mouse model harboring this variant, which mimicked the disease phenotype. By testing adenine base editors and single-guide RNAs, we identified an optimal combination of high editing efficiency and low bystander editing. Intravitreal injection of adeno-associated viral vectors encoding this adenine base editor achieved ∼40% editing efficiency in all retinal cells, restored retinal layer integrity, and preserved visual functions in 2-week-old male hemizygous mice. These mice exhibited retinal layer splitting at baseline, further validating the model. This study demonstrates a strategy for identifying effective base editing tools for clinical use through the preclinical evaluation of humanized mouse lines with patient-derived mutations and highlights their applicability in treating genetic diseases.},
}
@article {pmid40173114,
year = {2025},
author = {de Souza-Neto, RR and Cavalcante, LN and Carvalho, IGB and Curtolo, M and Benedetti, CE and Takita, MA and Wang, N and de Souza, AA},
title = {CRISPR/Cas9-Mediated Disruption of CsLIEXP1 Reveals Expansin as a Key Susceptibility Factor for Citrus Canker Disease.},
journal = {Molecular plant-microbe interactions : MPMI},
volume = {},
number = {},
pages = {MPMI12240151R},
doi = {10.1094/MPMI-12-24-0151-R},
pmid = {40173114},
issn = {0894-0282},
abstract = {The Citrus sinensis LATERAL ORGAN BOUNDERIES 1 (CsLOB1) gene, which is directly induced by the Xanthomonas citri subsp. citri effector PthA4, functions as a transcription factor and citrus canker susceptibility (S) gene. Genome editing of CsLOB1 has been shown to confer resistance to citrus canker disease. Previous studies revealed that the citrus CsLOB1-INDUCED EXPANSIN 1 gene (CsLIEXP1) is highly and directly upregulated by CsLOB1 in Xanthomonas citri subsp. citri-infected plants. Because expansins are associated with cell wall loosening, potentially facilitating bacterial colonization, the CsLOB1-dependent activation of CsLIEXP1 is thought to contribute to canker symptoms and disease progression. Thus, CsLIEXP1 likely represents a critical canker susceptibility gene. In this study, we employed CRISPR/Cas9 to disrupt the function of CsLIEXP1 by modifying its corresponding coding region in Citrus sinensis cultivar 'Hamlin' and evaluated the postinfection responses of edited plants. DNA sequencing confirmed the edition of the CsLIEXP1-edited plant, which exhibited 26.47% of CsLIEXP1 edited sequences. Furthermore, CsLIEXP1 protein accumulation was reduced in CsLIEXP1-edited plants compared with the wild type when infected with X. citri. Leaves of edited plants inoculated with X. citri showed significantly fewer canker symptoms, with lesions limited to the site of bacterial inoculation and less pronounced cellular hypertrophy compared with control plants. Our results show that CsLIEXP1 is a citrus canker S gene that acts downstream of CsLOB1, thus providing new insights into plant-pathogen interactions. [Formula: see text] Copyright © 2025 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.},
}
@article {pmid40119004,
year = {2025},
author = {Li, L and Bowling, S and Lin, H and Chen, D and Wang, SW and Camargo, FD},
title = {DARLIN mouse for in vivo lineage tracing at high efficiency and clonal diversity.},
journal = {Nature protocols},
volume = {20},
number = {8},
pages = {2319-2344},
pmid = {40119004},
issn = {1750-2799},
support = {32470700//National Science Foundation of China | National Natural Science Foundation of China-Yunnan Joint Fund (NSFC-Yunnan Joint Fund)/ ; },
mesh = {Animals ; Mice ; *Cell Lineage/genetics ; Single-Cell Analysis/methods ; *DNA Barcoding, Taxonomic/methods ; CRISPR-Cas Systems ; Gene Editing/methods ; },
abstract = {Lineage tracing is a powerful tool to study cell history and cell dynamics during tissue development and homeostasis. An increasingly popular approach for lineage tracing is to generate high-frequent mutations at given genomic loci, which can serve as genetic barcodes to label different cell lineages. However, current lineage tracing mouse models suffer from low barcode diversity and limited single-cell lineage coverage. We recently developed the DARLIN mouse model by incorporating three barcoding arrays within defined genomic loci and combining Cas9 and terminal deoxynucleotidyl transferase (TdT) to improve editing diversity in each barcode array. We estimated that DARLIN generates 10[18] distinct lineage barcodes in theory, and enables the recovery of lineage barcodes in over 70% of cells in single-cell assays. In addition, DARLIN can be induced with doxycycline to generate stable lineage barcodes across different tissues at a defined stage. Here we provide a step-by-step protocol on applying the DARLIN system for in vivo lineage tracing, including barcode induction, estimation of induction efficiency, barcode analysis with bulk and single-cell sequencing, and computational analysis. The execution time of this protocol is ~1 week for experimental data collection and ~1 d for running the computational analysis pipeline. To execute this protocol, one should be familiar with sequencing library generation and Linux operation. DARLIN opens the door to study the lineage relationships and the underlying molecular regulations across various tissues at physiological context.},
}
@article {pmid39948006,
year = {2025},
author = {Movahedi, A and Mu, Z and Yang, L},
title = {Advancing plant gene activation with CRISPR-Act3.0.},
journal = {Trends in biotechnology},
volume = {43},
number = {8},
pages = {1827-1830},
doi = {10.1016/j.tibtech.2025.01.008},
pmid = {39948006},
issn = {1879-3096},
mesh = {*CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; *Genome, Plant/genetics ; Plants, Genetically Modified/genetics ; *Plants/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {The pioneering plant genome-engineering tool CRISPR-Act3.0 is promising for precise gene activation, but faces challenges, such as unstable structures and inconsistent gene activation in polyploid genomes. Here, we propose strategic modifications to render CRISPR-Act3.0 more resilient and adaptable, especially in complex plant genomes, where conventional methods have struggled.},
}
@article {pmid39342431,
year = {2025},
author = {Sin, TN and Tng, N and Dragoli, J and Ramesh Kumar, S and Villafuerte-Trisolini, C and Chung, SH and Tu, L and Le, SM and Shim, JH and Pepple, KL and Ravindran, R and Khan, IH and Moshiri, A and Thomasy, SM and Yiu, G},
title = {Safety and efficacy of CRISPR-mediated genome ablation of VEGFA as a treatment for choroidal neovascularization in nonhuman primate eyes.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {33},
number = {8},
pages = {3939-3954},
doi = {10.1016/j.ymthe.2024.09.027},
pmid = {39342431},
issn = {1525-0024},
support = {R01 EY030431/EY/NEI NIH HHS/United States ; R01 EY032238/EY/NEI NIH HHS/United States ; R01 EY033733/EY/NEI NIH HHS/United States ; U24 EY029904/EY/NEI NIH HHS/United States ; },
mesh = {Animals ; *Choroidal Neovascularization/therapy/genetics/pathology/metabolism ; *Vascular Endothelial Growth Factor A/genetics ; *CRISPR-Cas Systems ; *Gene Editing/methods ; Dependovirus/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; Disease Models, Animal ; *Genetic Therapy/methods ; Genetic Vectors/genetics/administration &amp; dosage ; Macaca mulatta ; Humans ; Retina/metabolism/pathology ; Mice ; },
abstract = {CRISPR-based genome editing enables permanent suppression of angiogenic factors such as vascular endothelial growth factor (VEGF) as a potential treatment for choroidal neovascularization (CNV)-a major cause of blindness in age-related macular degeneration. We previously designed adeno-associated viral (AAV) vectors with S. pyogenes Cas 9 (SpCas9) and guide RNAs (gRNAs) to target conserved sequences in VEGFA across mouse, rhesus macaque, and human, with successful suppression of VEGF and laser-induced CNV in mice. Here, we advanced the platform to nonhuman primates and found that subretinal AAV8-SpCas9 with gRNAs targeting VEGFA may reduce VEGF and CNV severity as compared with SpCas9 without gRNAs. However, all eyes that received AAV8-SpCas9 regardless of gRNA presence developed subfoveal deposits, concentric macular rings, and outer retinal disruption that worsened at higher dose. Immunohistochemistry showed subfoveal accumulation of retinal pigment epithelial cells, collagen, and vimentin, disrupted photoreceptor structure, and retinal glial and microglial activation. Subretinal AAV8-SpCas9 triggered aqueous elevations in CCL2, but minimal systemic humoral or cellular responses against AAV8, SpCas9, or GFP reporter. Our findings suggest that CRISPR-mediated VEGFA ablation in nonhuman primate eyes may suppress VEGF and CNV, but can also lead to unexpected subretinal fibrosis, photoreceptor damage, and retinal inflammation despite minimal systemic immune responses.},
}
@article {pmid40770083,
year = {2025},
author = {Wang, S and Kou, Z and Jiang, Y and Tomberlin, JK and Zhang, W and Wang, S and Huang, Y},
title = {Truncated Higiant enhances the bioconversion ability of Hermetia illucens.},
journal = {Communications biology},
volume = {8},
number = {1},
pages = {1164},
pmid = {40770083},
issn = {2399-3642},
mesh = {Animals ; Gene Editing ; CRISPR-Cas Systems ; *Diptera/genetics/growth &amp; development/metabolism/physiology ; *Insect Proteins/genetics/metabolism ; },
abstract = {The black soldier fly (BSF), Hermetia illucens, has garnered attention for its proficiency in converting organic waste into valuable biomass. In the context of rapid population growth and urbanization, BSF with higher bioconversion efficiency is in urgent need. One promising approach to improve the efficiency is generating new lines by molecular breeding, which has succeeded in plants and various livestock. Here, we developed a BSF strain with an enhanced bioconversion efficiency using CRISPR/Cas9-mediated genome editing. By knocking out the gene giant (gt) in BSF, we obtained individuals with larger size and higher reproductive capacity. Notably, the mutant strain increased the bioconversion efficiency of food waste by 13.10%, and exhibited consistent performance across diverse organic waste substrates. In addition, our study elucidates multifaceted roles of gt in larval growth and developmental plasticity. Moreover, mutants showed reduced reproductive competitiveness against WT in mixed populations. In conclusion, our study provides a superior and safe insect chassis for biomanufacturing and underscores the potential of molecular breeding to enhance efficiency in BSF farming for sustainable organic waste recycling.},
}
@article {pmid40769560,
year = {2025},
author = {Yang, Y and Zhang, Z and Liu, J and Tang, L and Wei, Y and Nong, W and Yin, L and Li, S and Duan, P and Wang, Y and Rao, Y},
title = {[Identification of rice htd1 allelic mutant and its regulatory role in grain size].},
journal = {Sheng wu gong cheng xue bao = Chinese journal of biotechnology},
volume = {41},
number = {7},
pages = {2789-2802},
doi = {10.13345/j.cjb.250179},
pmid = {40769560},
issn = {1872-2075},
mesh = {*Oryza/genetics/growth &amp; development ; *Mutation ; *Edible Grain/genetics/growth &amp; development ; Alleles ; *Plant Proteins/genetics ; *Dioxygenases/genetics ; Lactones/metabolism ; Gene Expression Regulation, Plant ; Genes, Plant ; Gene Editing ; CRISPR-Cas Systems ; Phenotype ; },
abstract = {Rice is the world's largest food crop, and its yield and quality are directly related to food security and human health. Grain size, as one of the important factors determining the rice yield, has been widely concerned by breeders and researchers for a long time. To decipher the regulatory mechanism of rice grain size, we obtained a multi-tiller, dwarf, and small-grain mutant htd1 by ethyl methanesulfonate (EMS) mutation from the Japonica rice cultivar 'Zhonghua 11' ('ZH11'). Genetic analysis indicated that the phenotype of htd1 was controlled by a single recessive gene. Using the mutation site map (Mutmap) method, we identified the candidate gene OsHTD1, which encoded a carotenoid cleavage dioxygenase involved in the biosynthesis of strigolactone (SL). The SL content in htd1 was significantly lower than that in 'ZH11'. Cytological analysis showed that the grain size of the mutant decreased due to the reductions in the length and width of glume cells. The function of htd1 was further verified by the CRISPR/cas9 gene editing technology. The plants with the gene knockout exhibited similar grain size to the mutant. In addition, gene expression analysis showed that the expression levels of multiple grain size-related genes in the mutant changed significantly, suggesting that HTD1 may interact with other genes regulating grain size. This study provides a new theoretical basis for research on the regulatory mechanism of rice grain size and potential genetic resources for breeding the rice cultivars with high yields.},
}
@article {pmid40769554,
year = {2025},
author = {Lü, W and Yang, H and Xu, H and Zhang, Y},
title = {[Construction of a Sox17 activation vector based on the CRISPR/dCas9 system and its validation in sheep embryonic stem cells].},
journal = {Sheng wu gong cheng xue bao = Chinese journal of biotechnology},
volume = {41},
number = {7},
pages = {2707-2718},
doi = {10.13345/j.cjb.250136},
pmid = {40769554},
issn = {1872-2075},
mesh = {Animals ; *CRISPR-Cas Systems/genetics ; Sheep ; *SOXF Transcription Factors/genetics ; *Embryonic Stem Cells/metabolism/cytology ; *Genetic Vectors/genetics ; Cell Differentiation/genetics ; Transfection ; Gene Editing/methods ; },
abstract = {The CRISPR/dCas9 system is a gene editing tool that has proven to be highly efficient and precise. By utilizing transcriptional activators, such as VP64, p65, and Rta, the system can effectively and stably activate target genes. Sox17, a transcription factor belonging to the SOX family, plays a crucial role in the differentiation of the germ layers and the determination of cell fates during the early stages of embryonic development. Sheep embryonic stem cells (sESCs) are characterized by their capacity for self-renewal and multidirectional differentiation, serving as a significant in vitro model for studying the mechanisms of cell differentiation during early embryonic development. However, the importing of exogenous genes into sESCs is challenging due to their unique growth characteristics. The objective of this study was to investigate the conditions necessary for successfully activating Sox17 in sESCs. To this end, we employed the CRISPR/dCas9 system along with liposome transfection, lentivirus invasion, and electroporation to activate Sox17 in sESCs. The expression of Sox17 was then determined by fluorescence quantitative PCR, on the basis of which the performance of different transfection methods was compared. The results indicated that the electroporation group had the best transfection effect and the highest Sox17 expression among the three transfection methods. The efficient and stable gene activation protocol will provide a reference for embryonic stem cell research in other species, especially livestock animals, and lay the foundation for the subsequent study of gene function and realization of precise cell fate regulation by regulating gene expression in sheep embryonic stem cells.},
}
@article {pmid40769177,
year = {2025},
author = {Liu, Y and Yu, L and Gan, Z and Feng, Y and Tong, J and Xiao, Y},
title = {Lanthanide Metal-Organic Framework-Integrated CRISPR-Cas Technology for Amplification-free Gene Mutation Assay.},
journal = {Analytical chemistry},
volume = {},
number = {},
pages = {},
doi = {10.1021/acs.analchem.5c01949},
pmid = {40769177},
issn = {1520-6882},
abstract = {Nucleic acid amplification remains a major bottleneck in CRISPR-based molecular diagnostics, limiting assay speed, simplicity, and accuracy. Herein, we report a lanthanide metal-organic framework (Ln-MOF)-integrated CRISPR-Cas12a platform for amplification-free detection of gene mutations with high sensitivity and specificity. In this system, target recognition activates Cas12a trans-cleavage, degrading a single-stranded DNA linker and releasing alkaline phosphatase (ALP). The liberated ALP hydrolyzes p-nitrophenyl phosphate (pNPP) to generate phosphate ions, which interact with Eu-TPTC (TPTC: [1,1':4',1″] terphenyl-3,3″,5,5″-tetracarboxylic acid) MOF sensor, inducing a ratiometric fluorescence change by selectively quenching the Eu[3+] emission and enhancing the ligand emission. This mechanism enables quantitative, amplification-free detection of the oncogenic BRAF V600E mutation down to 0.1 pM, with excellent specificity and anti-interference ability. The assay demonstrates full agreement with conventional quantitative PCR when applied to clinical samples, accurately distinguishing mutant from wild-type genotypes. This strategy couples CRISPR-driven enzymatic signaling with Ln-MOF fluorescence modulation, offering an accurate and rapid readout. By eliminating amplification and streamlining workflow, this method holds significant potential for gene mutation analysis in clinical diagnostics.},
}
@article {pmid40768591,
year = {2025},
author = {Stundl, J and Desingu Rajan, AR and Urrutia, HA and Leyhr, J and Stundlova, J and Solovieva, T and Haitina, T and Sanchez, S and Musilova, Z and Martik, ML and Bronner, ME},
title = {Acquisition of neural crest promoted thyroid evolution from chordate endostyle.},
journal = {Science advances},
volume = {11},
number = {32},
pages = {eadv2657},
doi = {10.1126/sciadv.adv2657},
pmid = {40768591},
issn = {2375-2548},
mesh = {Animals ; *Neural Crest/embryology/metabolism ; *Thyroid Gland/embryology/metabolism ; *Biological Evolution ; *Chordata/embryology ; Gene Expression Regulation, Developmental ; CRISPR-Cas Systems ; },
abstract = {The endostyle is an endodermal organ unique to nonvertebrate chordates except for lamprey larvae, where it serves as forerunner to the adult thyroid. Here, we examine whether the acquisition of neural crest in the vertebrate lineage played a role in the elaboration of the endostyle. CM-DiI lineage tracing reveals a neural crest contribution to the endostyle, and CRISPR-Cas9 mutagenesis of key neural crest genes causes endostyle defects including formation of a single rather than bilobed structure. RNA sequencing reveals gene profiles characteristic of embryonic neural crest cells and Schwann cell precursors in the developing endostyle. Contrasting with the prevailing view that the endostyle is an endoderm-derived organ, we propose that the acquisition of the neural crest played a critical step in promoting thyroid evolution from chordate endostyle.},
}
@article {pmid40768573,
year = {2025},
author = {Bell, AG and Dunkley, ORS and Modi, NH and Huang, Y and Tseng, S and Reiss, R and Daivaa, N and Davis, JL and Vargas, DA and Banada, P and Xie, YL and Myhrvold, C},
title = {A streamlined CRISPR-based test for tuberculosis detection directly from sputum.},
journal = {Science advances},
volume = {11},
number = {32},
pages = {eadx2067},
doi = {10.1126/sciadv.adx2067},
pmid = {40768573},
issn = {2375-2548},
mesh = {*Sputum/microbiology ; Humans ; *Mycobacterium tuberculosis/genetics/isolation &amp; purification ; *Tuberculosis/diagnosis/microbiology ; *CRISPR-Cas Systems ; Sensitivity and Specificity ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Nucleic Acid Amplification Techniques ; },
abstract = {Mycobacterium tuberculosis (Mtb) is a major threat to global health, and there is an urgent need for affordable, simple tuberculosis (TB) diagnosis in underresourced areas. Here, we combine recombinase polymerase amplification with Cas13a and Cas12a detection to create two parallelized one-pot assays that detect two conserved elements of Mtb (IS6110 and IS1081) and a human DNA internal control. These assays are compatible with lateral flow and can be readily lyophilized. Our final assay showed a limit of detection of 69.0 CFU per milliliter for Mtb H37Rv and 80.5 CFU per milliliter for Mycobacterium bovis BCG in spiked sputum, with no cross-reactivity to diverse bacterial or fungal isolates. Clinical tests on 13 blinded sputum samples revealed 100% (six of six) sensitivity and 100% (seven of seven) specificity compared to culture. SHINE-TB streamlines TB diagnosis from sample to answer by combining amplification and detection while being compatible with lateral flow and lyophilization.},
}
@article {pmid40768535,
year = {2025},
author = {de Roest, RH and Buijze, M and Veth, M and de Lint, K and Pai, G and Rooimans, MA and Wolthuis, RMF and Brink, A and Poell, JB and Brakenhoff, RH},
title = {An unexpected role of CLASP1 in radiation response and S-phase regulation of head and neck cancer cells.},
journal = {PloS one},
volume = {20},
number = {8},
pages = {e0329731},
doi = {10.1371/journal.pone.0329731},
pmid = {40768535},
issn = {1932-6203},
mesh = {Humans ; *Head and Neck Neoplasms/genetics/radiotherapy/pathology/metabolism ; Cell Line, Tumor ; *Microtubule-Associated Proteins/genetics/metabolism ; Squamous Cell Carcinoma of Head and Neck/genetics ; *S Phase/radiation effects/genetics ; *Radiation Tolerance/genetics ; CRISPR-Cas Systems ; DNA Damage/radiation effects ; Gene Expression Regulation, Neoplastic/radiation effects ; Gene Knockout Techniques ; },
abstract = {Radiotherapy is a mainstay of treatment for head and neck squamous cell carcinoma (HNSCC), either definitive or adjuvant to surgery. Biological factors known to affect radiation response are hypoxia and DNA repair capacity, but several lines of evidence indicate that other genes and pathways in the tumor cells might be involved that have not been elucidated. Here, we report the results of a genome-wide CRISPR-Cas9 functional genomics screen in HNSCC cells to identify radiosensitizing genes. Remarkably, microtubule organizing genes were identified with CLASP1 as most unexpected radiosensitizing hit. Clonogenic assay confirmed the radiosensitizing effect of CLASP1 knockout. Functional analysis showed that CLASP1 knockout has major impact during S-phase, and resulted in mitotic cells with broken chromosomes and cell death. CLASP1 and possibly the microtubule machinery in broader sense seem involved in protection of HNSCC cells against radiation-induced DNA damage. This newly identified mechanism provides an outlook for novel treatment approaches in HNSCC.},
}
@article {pmid40768438,
year = {2025},
author = {Wang, Y and Zhao, W and He, Y and Li, J and Mao, R},
title = {Advanced Heterogeneous Network-Based Graph Neural Network Framework for Predicting Anti-CRISPR Protein Sequences.},
journal = {IEEE journal of biomedical and health informatics},
volume = {29},
number = {8},
pages = {6092-6104},
doi = {10.1109/JBHI.2025.3548463},
pmid = {40768438},
issn = {2168-2208},
mesh = {*Neural Networks, Computer ; *Computational Biology/methods ; *CRISPR-Cas Systems/genetics ; *Sequence Analysis, Protein/methods ; Algorithms ; Databases, Protein ; Bacteriophages/genetics ; Graph Neural Networks ; },
abstract = {Anti-CRISPR proteins play a crucial role in bacterial-phage interactions by inhibiting the CRISPR/Cas system and thus enhancing phage survival. Accurately predicting these proteins is essential for understanding phage-host immune interactions and progressing CRISPR/ Cas-based technologies. Current approaches primarily analyze proteins individually, which may overlook the intrinsic similarities and potential connections among protein sequences. This study introduces PACRGNN, a graph neural network framework that creates a heterogeneous protein network by integrating sequence and structural similarities, wherein nodes represent proteins and edges signify their relationships. By combining Graph Attention (GAT) and Graph Sample and Aggregation (GraphSAGE) layers, PACRGNN captures both local and global topological dependencies, while incorporating six protein feature categories to enrich node representations. PACRGNN achieves an accuracy of 0.9577, an F1-Score of 0.9572, and a PRAUC of 0.9876 on the validation set. The model demonstrated superior performance to existing methods on the independent test set derived from NCBI database (Jan.-Oct. 2024).},
}
@article {pmid40766843,
year = {2025},
author = {Zhang, Y and Chen, J and Feng, H and Yang, L and Hu, L and Gao, Y and Wang, Z and Feng, F and Zhao, J and Zhang, P and Zhang, R},
title = {Rapid generation of HCoV-229E and HCoV-OC43 reporter viruses and replicons for antiviral research.},
journal = {Frontiers in cellular and infection microbiology},
volume = {15},
number = {},
pages = {1614369},
pmid = {40766843},
issn = {2235-2988},
mesh = {*Antiviral Agents/pharmacology ; *Genes, Reporter ; *Replicon/genetics ; Humans ; *Coronavirus 229E, Human/genetics/drug effects ; *Coronavirus OC43, Human/genetics/drug effects ; *Reverse Genetics/methods ; CRISPR-Cas Systems ; Luciferases/genetics ; Recombination, Genetic ; Cloning, Molecular ; Saccharomyces cerevisiae/genetics ; Animals ; Adenosine Monophosphate/analogs &amp; derivatives ; Alanine/analogs &amp; derivatives ; },
abstract = {INTRODUCTION: The large size of coronavirus genome, along with the instability of certain genomic sequences, makes the construction of reverse genetics for coronaviruses particularly challenging. The rapid development and application of reverse genetics systems for coronaviruses require further exploration.<br><br>METHODS: Using transformation-associated recombination (TAR) cloning in yeast and the in vitro CRISPR-Cas9 system, reverse genetics systems of two mild coronaviruses HCoV-OC43 and HCoV-229E were rapidly established. Antiviral assays, high-content imaging, and NanoLuc luciferase assays were used to characterize reporter viruses and replicon systems.<br><br>RESULTS: We rapidly assembled infectious clones for two mild coronaviruses, HCoV-OC43 and HCoV-229E, using transformation-associated recombination (TAR) cloning in yeast. The infected clones could stably express the mGreenLantern reporter gene. We further generated T7 promoter-driven RNA replicon of HCoV-229E and CMV promoter-driven DNA replicon of HCoV-OC43, with the readout of NanoLuc luciferase activity. The effectiveness of these tools for antiviral study was evaluated using the broad-spectrum RNA-dependent RNA polymerase inhibitor remdesivir, exhibiting high sensitivity, efficiency, and convenience.<br><br>DISCUSSION: The application of yeast-based TAR cloning significantly facilitates the rapid assembly of large viral genome, and the establishment of HCoV-OC43 and HCoV-229E reverse genetics systems provides valuable platforms for studying the biology and developing antivirals against coronaviruses.},
}
@article {pmid40764301,
year = {2025},
author = {Aussel, C and Cathomen, T and Fuster-García, C},
title = {The hidden risks of CRISPR/Cas: structural variations and genome integrity.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {7208},
pmid = {40764301},
issn = {2041-1723},
mesh = {*CRISPR-Cas Systems/genetics ; Humans ; *Gene Editing/methods ; DNA Repair/genetics ; Translocation, Genetic ; Animals ; },
abstract = {CRISPR/Cas technology has revolutionized genome engineering, unlocking unprecedented therapeutic potential. However, beyond well-documented concerns of off-target (OT) mutagenesis, recent studies reveal a more pressing challenge: large structural variations (SVs), including chromosomal translocations and megabase-scale deletions, particularly in cells treated with DNA-PKcs inhibitors. These undervalued genomic alterations raise substantial safety concerns for clinical translation. As more CRISPR-based therapies progress toward the clinic, understanding and mitigating these risks is paramount. Here, we review emerging evidence on on-target aberrations and chromosomal translocations, identify key gaps in our understanding of the DNA repair pathways underlying these adverse effects, and discuss strategies to improve the safety of genome editing.},
}
@article {pmid40716589,
year = {2025},
author = {Du, H and Huang, X and Ren, R and Sun, Y and Wang, Y},
title = {TSPEAR S475TfsX79 mutation does not affect auditory function, tooth morphology or hair development in mice.},
journal = {Gene},
volume = {965},
number = {},
pages = {149687},
doi = {10.1016/j.gene.2025.149687},
pmid = {40716589},
issn = {1879-0038},
mesh = {Animals ; Mice ; *Tooth/growth &amp; development/metabolism ; Mutation ; Wnt Signaling Pathway/genetics ; Mice, Knockout ; *Hair/growth &amp; development/metabolism ; CRISPR-Cas Systems ; *Hearing/genetics ; },
abstract = {TSPEAR is a member of the EAR (epilepsy-associated repeat) protein family with poorly characterized function. Several lines of evidence suggest that mutations in the human TSPEAR gene are associated with hearing loss or ectodermal dysplasia. Although tooth abnormalities and a reduced capacity for caudal fin regeneration were observed in the Tspeara[-/-];Tspearb[-/-] knockout zebrafish model, there have been no reports of the Tspear knockout mouse model to date, which hampers further investigation of its physiological role. Here, we inactivated the Tspear gene in mice using CRISPR/Cas9-mediated genome editing. Intriguingly, stereociliary morphology and auditory function remain unaffected in TSPEAR S475TfsX79 mutant mice. Similarly, tooth morphology and hair development are unaltered in these mutants. Nevertheless, the S475TfsX79 mutation appears to perturb both Notch and Wnt signaling pathways. Specifically, Notch1 and several downstream target genes are downregulated, whereas Heyl expression is upregulated in the skin. Additionally, Wnt4 expression is elevated in both the skin and inner ear. In conclusion, our data demonstrate that TSPEAR S475TfsX79 mutation does not compromise auditory function, tooth morphology, or hair development in mice, but TSPEAR may modulate both Notch and Wnt signaling pathways in the mouse.},
}
@article {pmid40673518,
year = {2025},
author = {Wan, W and Cui, C and Zhou, Y and Wang, J and Zhao, X and Cui, X and Sun, J and Yu, P and Feng, J and Wang, T and Wang, L and Xu, J},
title = {Targeting Mettl14 Using an RNA-Targeting Clustered Regularly Interspaced Short Palindromic Repeat-High-Fidelity Cas13x System Attenuates Doxorubicin-Induced Cardiotoxicity.},
journal = {Journal of the American Heart Association},
volume = {14},
number = {15},
pages = {e040700},
doi = {10.1161/JAHA.124.040700},
pmid = {40673518},
issn = {2047-9980},
mesh = {Animals ; *Doxorubicin/toxicity/adverse effects ; *Methyltransferases/genetics/metabolism ; Male ; Cardiotoxicity ; *CRISPR-Cas Systems ; Mice ; Disease Models, Animal ; Mice, Inbred C57BL ; *Cardiomegaly/chemically induced/genetics/prevention &amp; control ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Physical Conditioning, Animal ; Myocytes, Cardiac/pathology/drug effects ; },
abstract = {BACKGROUND: Doxorubicin is an effective chemotherapy drug used to treat various types of cancer. However, doxorubicin treatment is associated with cardiotoxicity, which limits its clinical use. Exercise can benefit both cancer and cardiovascular disease. Clustered regularly interspaced short palindromic repeat (CRISPR)-Cas13 (CRISPR-associated protein 13) platforms have emerged as effective technologies for targeting the expression of RNA in transcript levels. To develop exercise mimetics that can mimic the beneficial effects of exercise training to attenuate doxorubicin-induced cardiotoxicity, we are using the CRISPR-hf (high-fidelity)Cas13x system.<br><br>METHODS: Adult male mice were swim-trained twice a day for 4&#x2009;weeks to induce exercise-induced physiological cardiac hypertrophy. Adeno-associated virus 9-mediated METTL14 (methyltransferase-like 14) overexpression under the cardiac-specific ctnt promoter was used to overexpression METTL14 in&#xa0;vivo. RNA N[6]-methyladenosine inhibitor STM2457 was used to modulate global total RNA m[6]A levels in&#xa0;vivo. CRISPR-cr3-4/hfCas13x system was generated by hfCas13x guided crRNA3 and crRNA4 targeting the Mettl14 expressed under ctnt promoter and packaged in an adeno-associated virus 9.<br><br>RESULTS: Swimming exercise alleviated doxorubicin-induced cardiotoxicity. METTL14 was increased in doxorubicin-treated hearts but decreased in exercised hearts. METTL14 overexpression inhibited exercise-induced physiological cardiac hypertrophy. Conversely, STM2457 treatment reversed the suppressive effects of METTL14 overexpression on the physiological cardiac hypertrophy induced by exercise. Treatment with CRISPR-cr3-4/hfCas13x effectively inhibiting the expression of METTL14 in the heart, alleviating doxorubicin treatment-induced cardiac dysfunction and cardiac fibrosis.<br><br>CONCLUSIONS: Our results suggest that the CRISPR-hfCas13x system has the potential for generating exercise mimetics. Mimicking exercise by RNA-targeting Mettl14 suppression could be a therapeutic strategy for doxorubicin-induced cardiotoxicity.},
}
@article {pmid40631538,
year = {2025},
author = {Jahani, R and Munusamy, S and Zheng, H and Kong, J and Chen, J and Zhou, S and Guan, X},
title = {Ultrasensitive Detection of Attomolar Neurofilament Light Chain Using Gold Nanoparticle-Assisted CRISPR-Cas12a Enhanced Fluorescent Assay.},
journal = {ACS chemical neuroscience},
volume = {16},
number = {15},
pages = {2844-2853},
doi = {10.1021/acschemneuro.5c00137},
pmid = {40631538},
issn = {1948-7193},
mesh = {*Gold/chemistry ; *Neurofilament Proteins/analysis/blood ; *Metal Nanoparticles/chemistry ; Humans ; *CRISPR-Cas Systems ; Biosensing Techniques/methods ; Limit of Detection ; },
abstract = {Neurofilament light chain (NfL) is a nonspecific biomarker that can indicate neuronal damage, and elevated NfL levels have been reported in several neurological disorders and traumatic brain injury and are correlated with disease severity and progression. However, quantitative detection of NfL in bodily fluids, especially in blood, is challenging due to its ultralow levels and potential interference from matrix components. Herein, we report a DNA-assisted CRISPR-Cas12a-enhanced fluorescent assay for ultrasensitive and specific detection of NfL. By utilizing antibody-functionalized magnetic beads for capturing the target NfL, dual-functionalized gold nanoparticles for DNA introduction and controlled release as well as signal amplification, and the CRISPR-Cas12a system for further enhancement in the sensor sensitivity, our method was able to detect NfL with a limit of detection reaching as low as 0.012 pg/mL. Furthermore, the high selectivity of the sensor was verified by examining its response to a number of nontarget proteins such as C-reactive protein, interleukin-6, interleukin-2β, bovine serum albumin, and human serum albumin. To validate its clinical utility, simulated serum samples were analyzed, with the percent recoveries determined by the use of our sensor ranging from 85% to 95%. Given the high sensitivity and selectivity as well its ability to accurately detect trace amounts of NfL in human biospecimens, the CRISPR-based florescence sensor constructed in this work may find useful application in neurological disease diagnosis.},
}
@article {pmid40550305,
year = {2025},
author = {León, E and Maya-Hoyos, M and Ramírez-Hernández, MH and Téllez, J and Contreras R, LE},
title = {Upregulation of nicotinamide/nicotinate mononucleotide adenylyl transferase increases resistance to oxidative stress and antimony in promastigotes of Leishmania braziliensis.},
journal = {Experimental parasitology},
volume = {275},
number = {},
pages = {108979},
doi = {10.1016/j.exppara.2025.108979},
pmid = {40550305},
issn = {1090-2449},
mesh = {*Oxidative Stress/drug effects ; Up-Regulation ; *Nicotinamide-Nucleotide Adenylyltransferase/genetics/metabolism ; *Leishmania braziliensis/drug effects/enzymology/genetics ; *Antimony/pharmacology ; Hydrogen Peroxide/pharmacology ; NAD/biosynthesis/metabolism ; CRISPR-Cas Systems ; Gene Editing ; *Antiprotozoal Agents/pharmacology ; Animals ; NADP/metabolism ; RNA, Messenger/metabolism ; Drug Resistance ; },
abstract = {Leishmaniasis is a prevalent parasitic neglected disease caused by protozoans of the genus Leishmania. Currently, no vaccines are available for humans, and existing treatments are ineffective because of their toxicity and the emergence of drug-resistant strains. Consequently, it is crucial to identify new potential therapeutic targets, thereby facilitating the development of effective therapies. This study indicates a correlation between the upregulated biosynthesis of nicotinamide adenine dinucleotide (NAD) and the resistance to hydrogen peroxide (H2O2) and trivalent antimony exposure (Sb[3+]) in L. braziliensis promastigotes. CRISPR/Cas9 gene-editing of the nicotinamide/nicotinate mononucleotide adenylyl transferase in L. braziliensis (Lbnmnat), the key gene in the NAD biosynthetic pathway, allowed upregulation at both the mRNA and protein levels, as well as elevated NAD and NADP contents in the Lbnmnat-edited parasites. A single guide RNA template, directed against the 5' end of the Lbnmnat and a donor DNA consisting of the mCherry reporter gene preceded by the 5' UTR from the Crithidia fasciculata phosphoglycerate kinase (CfPGKB) were electroporated into L. braziliensis promastigotes that constitutively express the Streptococcus pyogenes Cas9 and T7 RNA polymerase. PCR analysis and DNA sequencing demonstrated the successful editing of the Lbnmnat gene. Importantly, dose-dependent oxidative stress susceptibility assays to H2O2 and Sb[3+] revealed higher IC50 values in the Lbnmnat-edited parasites. These findings denote a correlation between the upregulated biosynthesis of NAD in L. braziliensis and its enhanced resistance to oxidative stress, indicating that the Lbnmnat gene may play a significant role in the survival and resistance mechanisms of the parasite against antimony compounds.},
}
@article {pmid40359132,
year = {2025},
author = {Park, SY and Feng, Z and Choi, SH and Zhang, X and Tang, Y and Gasser, GN and Richart, D and Yuan, F and Qiu, J and Engelhardt, JF and Yan, Z},
title = {Recombinant Adeno-Associated Virus Vector Mediated Gene Editing in Proliferating and Polarized Cultures of Human Airway Epithelial Cells.},
journal = {Human gene therapy},
volume = {36},
number = {15-16},
pages = {1067-1082},
doi = {10.1089/hum.2024.260},
pmid = {40359132},
issn = {1557-7422},
mesh = {Humans ; *Gene Editing/methods ; *Dependovirus/genetics ; *Cystic Fibrosis Transmembrane Conductance Regulator/genetics ; *Genetic Vectors/genetics ; *Epithelial Cells/metabolism/cytology ; *Cystic Fibrosis/genetics/therapy/pathology ; Cell Proliferation ; CRISPR-Cas Systems ; Cell Line ; Genetic Therapy ; },
abstract = {Cystic fibrosis (CF) is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. While CRISPR-based CFTR editing approaches have shown proof-of-concept for functional rescue in primary airway basal cells, induced pluripotent stem cells, and organoid cultures derived from patients with CF, their efficacy remains suboptimal. Here, we developed the CuFi[Cas9(Y66S)eGFP] reporter system by integrating spCas9 and a non-fluorescent Y66S eGFP mutant into CuFi-8 cells, an immortalized human airway epithelial cell line derived from a patient with CF with homozygous F508del mutations. These cells retain the basal cell phenotype in proliferating cultures and can differentiate into polarized airway epithelium at an air-liquid interface (ALI), enabling both visualized detection of gene editing and electrophysiological assessment of CFTR functional restoration. Using this system, recombinant adeno-associated virus (rAAV)-mediated homology-directed repair (HDR) was evaluated in proliferating cultures. A correction rate of 13.5 ± 0.8% was achieved in a population where 82.3 ± 5.6% of cells were productively transduced by AAV.eGFP630g2-CMVmCh, an rAAV editing vector with an mCherry reporter. Dual-editing of F508del CFTR and Y66S eGFP was explored using AAV.HR-eGFP630-F508(g03) to deliver two templates and single guide RNAs. eGFP[+] (Y66S-corrected) cells and eGFP[-] (non-corrected) cells were sorted via fluorescence-activated cell sorting and differentiated at an ALI to assess the recovery of CFTR function. Despite a low F508 correction rate of 2.8%, ALI cultures derived from the eGFP[-] population exhibited 25.2% of the CFTR-specific transepithelial Cl[-] transport observed in CuFi-ALI cultures treated with CFTR modulators. Next-generation sequencing revealed frequent co-editing at both genomic loci, with sixfold higher F508 correction rate in the eGFP[+] cells than eGFP[-] cells. In both populations, non-homology end joining predominated over HDR. This reporter system provides a valuable platform for optimizing editing efficiencies in proliferating airway basal cells, particularly for development of strategies to enhance HDR through modulation of DNA repair pathways.},
}
@article {pmid40763977,
year = {2025},
author = {Li, Q and Wu, Y and Yuan, J and Liu, F and Cheng, W},
title = {[Application of base editing techniques in the identification of functional sites of genes].},
journal = {Zhonghua yi xue yi chuan xue za zhi = Zhonghua yixue yichuanxue zazhi = Chinese journal of medical genetics},
volume = {42},
number = {6},
pages = {762-768},
doi = {10.3760/cma.j.cn511374-20241218-00666},
pmid = {40763977},
issn = {1003-9406},
mesh = {Humans ; *Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; Genome, Human ; Polymorphism, Single Nucleotide ; },
abstract = {The exploration of pathogenic single nucleotide polymorphisms in the genome plays a pivotal role in the study of human disease-associated genetic mutations. However, there remains a lack of suitable high-throughput screening platforms to investigate the impact of point mutations on genomic structure and function. CRISPR/Cas9-mediated base editors has enabled large-scale annotation of the human genome and phenotypic characterization of monogenic disorders. Base editors, a precise gene-editing technique capable of achieving targeted base substitutions, can be employed to induce mutations at specific functional sites, thereby observing their effects on gene expression, protein function, and cellular phenotypes. Furthermore, integrating base editors with high-throughput screening technologies allows for large-scale evaluation of multiple candidate sites, accelerating the identification of functional loci and providing a powerful tool for disease research and therapeutic target discovery. This article aims to introduce the working principles of various base editors, including cytosine base editors, adenine base editors, and prime editors, and summarize recent advances in high-throughput screening of functional genomic sites using base-editing techniques.},
}
@article {pmid40763823,
year = {2025},
author = {Xu, L and Wu, X},
title = {Viral detection using Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated protein and Argonaute nucleases.},
journal = {Clinica chimica acta; international journal of clinical chemistry},
volume = {},
number = {},
pages = {120526},
doi = {10.1016/j.cca.2025.120526},
pmid = {40763823},
issn = {1873-3492},
abstract = {Viral pandemics pose severe threats to human health and societal stability, exemplified by the COVID-19 outbreak in 2019. Conventional viral detection methods such as Polymerase chain reaction (PCR) typically require trained personnel, expensive equipment, and 2-4 h for processing. Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated protein (Cas) and Argonaute (Ago) system-based detection methods achieve attomolar sensitivity or single-copy detection limits with single-base specificity within 1 h, without requiring complex or costly instruments. This review firstly introduces the mechanisms and functions of CRISPR/Cas systems (Cas9, Cas12, Cas13) and Ago systems. It also introduces viruses with significant social impact, and continued with reviewing applications of these systems in single and multiplex virus detection. Single viral detection includes recently developed DNA/RNA-activated Cas9 detection (DACD/RACD) using Cas9 trans-cleavage activity, Cas12-based DNA Endonuclease-targeted CRISPR Trans Reporter (DETECTR) with attomolar sensitivity, CRISPR/Cas13a-based Fluorescent Nanoparticle SARS-CoV-2 (CFNS) achieving 1 copy/mL sensitivity with quantum dot reporters, and amplification-free mobile phone detection detecting 31 copies/μL without amplification. Multiplex viral detection includes Microfluidic Device Integrated with CRISPR/Cas12a and Multiplex Recombinase Polymerase Amplification (MiCaR) enabling 30-plex detection through microfluidic chips with spatial discrimination, PfAgo-mediated Nucleic acid Detection (PAND) utilizing Ago-produced guide sequences for 5-plex detection, Specific High-Sensitivity Enzymatic Reporter UnLOCKing v2 (SHERLOCKv2) achieving 4-plex detection with multi-enzyme single-reaction systems, and Multiplexed Evaluation of Nucleic acids (CARMEN) supporting over 100 target assays. Finally, this review discusses challenges in CRISPR/Cas and Ago-based detection methods, including Protospacer Adjacent Motif (PAM) sequence requirements for Cas9/12, prolonged reaction times due to nucleic acid extraction/amplification, and instability of core components like nucleases and crRNAs. Detection specificity and multiplex capabilities could be further improved. Future directions are outlined for improving detection specificity, developing multiplex capabilities and advancing POCT. Developing diagnostic tools using CRISPR/Cas and Ago systems could transform molecular diagnostics, such tools promise to be easily accessible worldwide. They are essential for precise identification and strategic containment of infectious disease transmission.},
}
@article {pmid40763014,
year = {2025},
author = {Chen, L and Zhou, X and Huang, C and Zhang, Y and Xin, C and Hong, J and Wang, Y},
title = {Engineered Un1Cas12f1 with boosted gene-editing activity and expanded genomic coverage.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {122},
number = {32},
pages = {e2501292122},
doi = {10.1073/pnas.2501292122},
pmid = {40763014},
issn = {1091-6490},
support = {2023YFA0915000//Nation Key Research and Development Program of China/ ; 82273967//National Natural Science Foundation of China/ ; 2021QN020576//Department of Science and Technology of Guangdong Province/ ; 82425015//MOST | NSFC | National Science Fund for Distinguished Young Scholars (NSF for Distinguished Young Scholars)/ ; 82171102//National Natural Science Foundation of China/ ; 82271044//National Natural Science Fundation of China/ ; QWF158001//National Youth talent support program/ ; 22Y21900900//Shanghai Medical Innovation Research Program/ ; 24SG11//(the "Dawn" Program of) Shanghai Municipal Education Commission/ ; 24J22800500//Shanghai Science and Technology Innovation Action Plan for Cell and Gene Therapy/ ; 24CL2900802//Shanghai Science and Technology Innovation Action Plan for Advanced Materials/ ; 20254Z0019//Shanghai Municipal Commission of Health/ ; },
mesh = {Animals ; *Gene Editing/methods ; Mice ; Dependovirus/genetics ; Humans ; CRISPR-Cas Systems/genetics ; Genetic Vectors/genetics ; PTEN Phosphohydrolase/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; Genetic Therapy/methods ; HEK293 Cells ; },
abstract = {Compact programmable nucleases provide versatile genome editing tools with therapeutic potential, particularly when delivered via adeno-associated virus (AAV) vectors. However, their limited editing efficacy and stringent protospaceradjacent motif (PAM) requirements impose significant limitations in practical application. Here, we engineered MiniCasUltra, an optimized Un1Cas12f1 variant, through rational mutagenesis. MiniCasUltra exhibits sixfold higher editing activity than Un1Cas12f1, minimal off-target effects (on/off-target ratio > 10), and an expanded PAM preference (5'-WBTR). Using a single AAV vector encoding MiniCasUltra and two single-guide RNAs, we achieved simultaneous editing of two disease-causing genes (Pten and Fah) in mouse liver, with indel rates of 15.82% and 29.39%, respectively- significantly surpassing CasMINI V3.1 (3.45% and 10.98%). Furthermore, AAV delivery of MiniCasUltra targeting a noncanonical 5'-TCTG PAM site in human vascularendothelial growth factor A reduced choroidal neovascularization (CNV) lesions in a laser-induced CNV mouse model of neovascular age-related macular degeneration, a leading global cause of blindness. The broad and effective targeting capabilities of MiniCasUltra, coupled with its compact size, highlight its potential for in vivo genome editing and therapeutic interventions.},
}
@article {pmid40761563,
year = {2025},
author = {Saraswat, P and Ranjan, R},
title = {Unlocking the potential of CRISPR tools and databases for precision genome editing.},
journal = {Frontiers in plant science},
volume = {16},
number = {},
pages = {1563711},
pmid = {40761563},
issn = {1664-462X},
abstract = {Recent breakthroughs in CRISPR/Cas genome editing have transformed molecular biology research and offer significant potential across biotechnology and medicine. This has created a broad spectrum of computational tools and databases that aim to optimize each phase of the genome-edited workflow, from guide RNA design and off-target prediction through screening analysis and biological validation. Here, we survey major CRISPR tools and analyse their features in the context of precision genome editing. CRISPOR and CHOPCHOP versatile platform that provides robust guide RNA design for several species, integrated off-target scoring, and intuitive genomic locus visualization. This review gives an overview of these new resources that have been developed, grouped based on their functionalities like design of guide RNA, off-target predictions, genome-wide screens, and visualizations of the data. Furthermore, we discuss new trends in database development like their integration with genome browsers and implementation of machine learning. This review thus gives a useful overview of the dynamic field of CRISPR/Cas genome editing tools. It also serves as a helpful guide for researchers looking to utilize these tools in their research.},
}
@article {pmid40760280,
year = {2025},
author = {Nguyen, PDT and Nguyen, NH},
title = {Revolutionizing molecular plant breeding through genetic engineering of in vivo haploid induction genes.},
journal = {Molecular biology reports},
volume = {52},
number = {1},
pages = {791},
pmid = {40760280},
issn = {1573-4978},
mesh = {*Haploidy ; *Plant Breeding/methods ; Gene Editing/methods ; *Genetic Engineering/methods ; CRISPR-Cas Systems/genetics ; Plants, Genetically Modified/genetics ; Genes, Plant/genetics ; Genome, Plant ; Oryza/genetics ; },
abstract = {Haploid and doubled haploid plants serve critical functions in plant genetics and breeding studies, as they enable the production of genetically uniform lines (pure lines) and thereby accelerate the development of new cultivars. Nowadays, various key genes regulating haploid induction have been found in maize and other plant species (e.g. Arabidopsis, rice, and wheat). These genes offer insights into the mechanisms underlying in vivo haploid induction and provide targets for genetic manipulation. Recent advancements in gene editing and genetic engineering technologies, such as CRISPR/Cas9, have revolutionized our ability to understand gene functions and precisely modify genomes. By leveraging these technologies, scientists can introduce targeted genetic changes, optimize the haploid induction process, and broaden the range of species amenable to haploid production. The application of gene editing and genetic engineering in haploid production holds immense promise for various fields, including agriculture and biotechnology. This focused review aims to advance our understanding of haploid induction genes as well as the potential applications of gene editing/genetic engineering technologies in haploid production, paving the way for innovative solutions to pressing challenges in future plant breeding and agriculture.},
}
@article {pmid40760060,
year = {2025},
author = {Liang, S and Jia, S and Lu, W and Wang, J and Huang, M and Chen, C and Huang, C and Zhou, D and Guo, T and Liu, H},
title = {Generation and characterization of rice OsCENH3 mutants for haploid induction.},
journal = {Plant cell reports},
volume = {44},
number = {8},
pages = {190},
pmid = {40760060},
issn = {1432-203X},
support = {No. 2022YFD1200703//National Key Research and Development Project/ ; No. 2022B0202060006//Guangdong Province Key Research and Development/ ; No.&#x202f;2024NBZ00003//Guangdong Province Revitalization of Seed Industry/ ; CARS-018//National Rice Industry Technology System of China/ ; },
mesh = {*Oryza/genetics ; *Haploidy ; *Mutation/genetics ; CRISPR-Cas Systems/genetics ; Gene Editing ; *Plant Proteins/genetics/metabolism ; Aneuploidy ; Chromosome Segregation/genetics ; Chromosomes, Plant/genetics ; Histones/genetics/metabolism ; Plants, Genetically Modified ; Pollen/genetics ; },
abstract = {CRISPR/Cas9-mediated modification of OsCENH3 induces aneuploidy but fails to trigger haploid formation in rice, underscoring limited efficiency and the need for strategy refinement. The centromeric histone H3 variant (CENH3) is essential for kinetochore assembly and accurate chromosome segregation during cell division. Alterations in CENH3 have been shown to trigger haploid induction in various plant species; however, its utility in rice remains largely unexplored. In this study, six OsCENH3 mutant lines were generated through CRISPR/Cas9-mediated genome editing, complemented by a GFP-tagged construct. To assess their haploid induction potential, both self pollination and outcrossing with the cultivar Nipponbare (NIP) were performed. Flow cytometry analysis revealed that none of the mutants produced true haploids. However, two aneuploid individuals (Het-C1-1-1 and Het-C1-5-1) were identified among the hybrid progeny derived from tail domain mutants C1-1 and C1-5, suggesting partial chromosome missegregation. These aneuploid plants exhibited reduced pollen viability, abnormal morphology, and compromised agronomic performance. In addition, significantly elevated rates of embryo/endosperm abortion were observed across different crosses, which far exceeded the frequency of aneuploid production, implying that early-stage chromosomal instability may result in embryo lethality. Collectively, while specific OsCENH3 mutations can induce limited chromosomal instability, their haploid induction efficiency remains low in rice. Further refinement of editing strategies and exploration of favorable genetic backgrounds will be essential for developing effective centromere-based haploid induction systems. This study provides a theoretical framework and technical reference for engineering haploid inducers in rice through centromere manipulation.},
}
@article {pmid40719616,
year = {2025},
author = {Zhou, X and Zhang, R and Cai, L and Fu, H and Wang, D and Yuan, R and Yuan, Y},
title = {Efficient Entropy-Driven Cross-Double-Loop Reaction Cascade Asymmetric CRISPR/Cas12a Cleavage for Ultrasensitive Electrochemical Biosensing.},
journal = {Analytical chemistry},
volume = {97},
number = {30},
pages = {16355-16363},
doi = {10.1021/acs.analchem.5c02014},
pmid = {40719616},
issn = {1520-6882},
mesh = {*Biosensing Techniques/methods ; *Entropy ; *Electrochemical Techniques/methods ; *CRISPR-Cas Systems ; *CRISPR-Associated Proteins/metabolism ; *Endodeoxyribonucleases/metabolism ; DNA, Single-Stranded/chemistry ; Limit of Detection ; Electrodes ; DNA/analysis ; Bacterial Proteins ; },
abstract = {The limited signal amplification efficiency of the conventional CRISPR/Cas12a-cleavage system was primarily due to the structural constraints of crRNA and single-trigger activation. Herein, an efficient target-induced entropy-driven cross-double-loop strand displacement reaction (SDR) cascade asymmetric CRISPR/Cas12a cleavage was developed to construct an ultrasensitive and reliable signal-off electrochemical biosensor. The desirable entropy-driven modulation could spontaneously undergo a cross-double-loop reaction that possessed self-accelerating ability, effectively improving the rate of chain replacement and avoiding the usage of extra fuel chains with generation of two abundant distinct DNA outputs, significantly improving target conversion efficiency. More importantly, all the targets and two distinct DNA outputs could simultaneously act as activators in the asymmetric CRISPR/Cas12a system, which cooperatively bound to both split and full-sized crRNAs to accomplish the highly efficient discharge of ferrocene-labeled single-stranded DNA (Fc-reporter) on the electrode, thereby markedly improving the detection sensitivity and reliably compared to that of traditional ones. The experimental results suggested that the proposed biosensor had a wide linear range spanning from 1 fM to1 nM with a detection limit as low as 0.23 fM. By integrating entropy-driven amplification with CRISPR-enhanced signal transduction, this work established a versatile and robust analytical tool for early cancer diagnosis and precision biomolecular detection.},
}
@article {pmid40698640,
year = {2025},
author = {Dai, X and Meng, C and Huang, S and Wang, Y and He, J and Chen, Z and Ji, Y and Tai, J and Zhang, J and Ni, H and Zhuang, Z and Chen, J and Zhang, H and Qu, J and Shao, Y},
title = {Attomolar Nucleic Acid Detection Using CRISPR Enhanced Phase-Sensitive Surface Plasmon Resonance Imaging.},
journal = {Analytical chemistry},
volume = {97},
number = {30},
pages = {16296-16303},
doi = {10.1021/acs.analchem.5c01772},
pmid = {40698640},
issn = {1520-6882},
mesh = {*Surface Plasmon Resonance/methods ; *SARS-CoV-2/genetics/isolation &amp; purification ; Gold/chemistry ; Metal Nanoparticles/chemistry ; *CRISPR-Cas Systems ; *DNA, Viral/analysis/genetics ; Humans ; COVID-19/diagnosis/virology ; *Clustered Regularly Interspaced Short Palindromic Repeats ; DNA, Single-Stranded/chemistry ; Limit of Detection ; },
abstract = {Driven by the growing need for real-time, amplification-free, and label-free nucleic acid detection in clinical diagnostics and pathogen surveillance, traditional methods often fall short due to limited sensitivity, a narrow dynamic range, and difficulties in detecting low-concentration nucleic acids and single-nucleotide mutations. To address these challenges, we developed a clustered regularly interspaced short palindromic repeats (CRISPR) enhanced Phase-interrogation Surface Plasmon Resonance imaging (CRISPR-PSPRi) sensor that employs phase delay modulation for highly sensitive extraction of SPR phase signals and a wavelength scanning strategy to extend its dynamic range. By harnessing CRISPR-Cas12a for target DNA recognition and activating trans-cleavage to cleave ssDNA-linked gold nanoparticle probes, our platform converts extremely weak signals from low-concentration DNA into readily detectable cleavage signals. Achieving a sensitivity of 1.436 × 10[-6] RIU and a dynamic range of 0.0111 RIU, this system successfully detects specific DNA from the SARS-CoV-2 Omicron BA.2 variant and monkeypox virus, and it can detect single-nucleotide mutations down to 1 aM. This breakthrough offers a real-time, high-throughput, and ultrasensitive nucleic acid detection approach, promising significant advancements in clinical diagnostics and pathogen monitoring.},
}
@article {pmid40689881,
year = {2025},
author = {Xu, X and Tang, K and Yang, L and Wu, Y and Gu, Y and Li, Y},
title = {Split crRNA-Mediated Regulation of CRISPR-Cas12a Enables Construction of Biochemical Circuits for Sensitive Molecular Diagnostics.},
journal = {Analytical chemistry},
volume = {97},
number = {30},
pages = {16645-16651},
doi = {10.1021/acs.analchem.5c03196},
pmid = {40689881},
issn = {1520-6882},
mesh = {*CRISPR-Cas Systems/genetics ; *CRISPR-Associated Proteins/genetics/metabolism ; *Endodeoxyribonucleases/genetics/metabolism ; *RNA/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats ; DNA/analysis ; Humans ; *Bacterial Proteins/genetics/metabolism ; *Molecular Diagnostic Techniques/methods ; },
abstract = {The clustered regularly interspaced short palindromic repeat (CRISPR) system represents a fundamental tool for biotechnological applications. However, the ability to engineer intrinsic components of the CRISPR system to regulate its activity remains limited. Here, we present a strategy for the regulation of CRISPR-Cas12a by engineering an enhanced split CRISPR RNA (split en-crRNA) variant. Reassembly of this variant autonomously restores the full activity of Cas12a nuclease without the need for chemical modifications or exogenous activation stimuli. Leveraging this strategy, we developed CREST (CRISPR Reactions Elicited by Split en-crRNA Transcription) for molecular diagnostics. CREST integrates biochemical circuits that ingeniously harness isothermal amplification to trigger the on-demand synthesis and reassembly of a universal split en-crRNA, addressing longstanding challenges in CRISPR diagnostics, including the low efficiency of one-pot assays, protospacer adjacent motif restriction, the need for crRNA reoptimization, and limited reagent stability. The CREST assay enables robust detection of attomolar DNAs or RNAs within 15 to 20 min and demonstrates capability for identifying bacterial infections in patients. This study provides new insights into Cas12a regulation, which is expected to advance the development of controllable CRISPR technologies for diagnostics as well as broader applications such as genome editing and programmable gene expression.},
}
@article {pmid40505899,
year = {2025},
author = {Bai, Y and Gao, BJ and Shi, XY and Hu, TH and Shi, LD and Duan, JX and Zhang, ZH and Shang, GH and Xu, J and Li, DL and He, ZQ and Li, K},
title = {The tyrosine hydroxylase regulates organs development via affecting hormones level in Gryllus bimaculatus.},
journal = {International journal of biological macromolecules},
volume = {319},
number = {Pt 2},
pages = {145159},
doi = {10.1016/j.ijbiomac.2025.145159},
pmid = {40505899},
issn = {1879-0003},
mesh = {Animals ; *Tyrosine 3-Monooxygenase/genetics/metabolism ; *Juvenile Hormones/metabolism ; Mutation ; Gene Expression Regulation, Developmental ; CRISPR-Cas Systems ; Phenotype ; Pigmentation ; },
abstract = {Tyrosine hydroxylase (TH) is the initial enzyme in the melanin biosynthesis pathway and catalyzes the conversion of tyrosine to dihydroxyphenylalanine (DOPA). Extensive research has demonstrated that TH exhibits evolutionarily conserved function in insects, regulating multiple physiological pathways including integumentary repair mechanisms, cuticular sclerotization processes, and melanization in response to pathogenic challenges. However, few studies have addressed the role of TH in Gryllus bimaculatus (G. bimaculatus), a hemimetabolous model insect. In this study, we investigated the in vivo role of GbTH gene by using CRISPR/Cas9 gene-editing tool to generate mosaic mutants (GbTH F0) and homozygous mutants (GbTH[-/-]). The GbTH F0 mutants displayed less pigmented phenotypes, meanwhile the pronotum became thinner, leading to defects in chirping and motor behaviors. The GbTH[-/-] mutants exhibited pleiotropic developmental abnormalities, characterized by complete lethality at the nymphal stage. Phenotypic analysis revealed compromised cuticular integrity with impaired chitin matrix deposition in pre-nymphal exoskeletons, accompanied by significantly enhanced desiccation susceptibility and progressive body mass reduction in mutant nymphs. RNA-sequencing transcriptome analysis indicated that GbTH was involved in regulation the synthesis and coordination of juvenile hormone (JH) and ecdysone (20E). Furthermore, GbTH knockout significantly reduced the expression of the lipid transport protein NPC1b. This suppression led to impaired cholesterol uptake, which consequently disrupted steroid hormone biosynthesis and ultimately compromised key developmental transitions, including molting cycles and metamorphic progression. Collectively, these findings establish that GbTH serves as a master regulatory enzyme for the fine-tuning of developmental processes in hemimetabolic insects, extending beyond its canonical function in cuticular pigmentation regulation.},
}
@article {pmid40503836,
year = {2025},
author = {Liu, W and Wang, Y and Zhao, J and Zhang, X and Li, Z and Jia, H and Zhu, C and Zhang, L and Pan, L and Zhang, Z},
title = {Identification of Mycobacterium tuberculosis intracellular survival-related virulence factors via CRISPR-based eukaryotic-like secretory protein mutant library screen.},
journal = {Microbiology spectrum},
volume = {13},
number = {8},
pages = {e0076725},
doi = {10.1128/spectrum.00767-25},
pmid = {40503836},
issn = {2165-0497},
support = {32394013//National Natural Science Foundation of China/ ; 82172279//National Natural Science Foundation of China/ ; 82402641//National Natural Science Foundation of China/ ; 82070012//National Natural Science Foundation of China/ ; G2024-2-007//Beijing High-level Public Health Project/ ; },
mesh = {*Mycobacterium tuberculosis/genetics/pathogenicity/metabolism ; Macrophages/microbiology/immunology ; *Virulence Factors/genetics/metabolism ; *Bacterial Proteins/genetics/metabolism ; Animals ; Clustered Regularly Interspaced Short Palindromic Repeats ; Mice ; CRISPR-Cas Systems ; Mutation ; Tuberculosis/microbiology ; Humans ; Gene Library ; RAW 264.7 Cells ; Gene Editing ; },
abstract = {UNLABELLED: Tuberculosis (TB), caused by Mycobacterium tuberculosis (M.tb), remains a serious infectious disease posing significant global health challenges. A critical evolutionary feature of M.tb is its genome encoding a set of eukaryotic-like secretory proteins, which facilitate intracellular survival by manipulating host immune responses. However, the specific eukaryotic-like secretory proteins that facilitate M.tb intracellular survival and their regulatory mechanisms on host immunity remain uncharacterized. In this study, a mutant library comprising 137 potential eukaryotic-like secretory proteins was constructed using clustered regularly interspaced short palindromic repeats (CRISPR)-non-homologous end joining genome editing technology. Subsequently, macrophages were infected with the mutant library, and CRISPR sequencing enabled preliminary identification of virulence factors associated with bacterial intracellular persistence. To validate the screen, two genes (Rv0066c and Rv3139) exhibiting the most pronounced reduction in intracellular survival rates when mutated were selected for the construction of large-fragment knockout strains (ΔRv0066c and ΔRv3139). Subsequent macrophage infection assays reconfirmed the impaired intracellular survival of these two mutants. RNA-seq analysis was conducted to characterize host gene expression profiles during ΔRv0066c-infected macrophage interactions. RNA-seq analysis of macrophages infected with wild-type and ΔRv0066c strains identified 138 differentially expressed genes, with 75 upregulated and 63 downregulated in ΔRv0066c. Gene ontology clustering of these differentially expressed genes highlighted molecular functions related to chemokine binding, chemokine-mediated signaling pathways, Ras protein signal transduction, and calcineurin-mediated signaling. Collectively, this work established a potential eukaryotic-like secretory protein mutant library and identified two novel M.tb effectors governing intracellular survival, providing potential new targets for anti-TB drug development.<br><br>IMPORTANCE: Eukaryotic-like secretory proteins that subvert host immunity to enable intracellular persistence are a key evolutionary adaptation of Mycobacterium tuberculosis (M.tb). In this study, we established a mutant library targeting 137 potential eukaryotic-like secretory proteins through clustered regularly interspaced short palindromic repeats (CRISPR)-non-homologous end joining genome editing technology. The library was subjected to macrophage infection assays, and CRISPR sequencing enabled identification of M.tb persistence-associated virulence determinants. Validation screens highlighted two genes (Rv0066c and Rv3139) that displayed the most significant intracellular survival defects to generate large-fragment knockout strains (&#x394;Rv0066c and &#x394;Rv3139). Macrophage infection experiments reconfirmed the compromised intracellular viability of both mutants. RNA-seq profiling of &#x394;Rv0066c-infected macrophages identified 138 differentially expressed genes, with functional enrichment in chemokine signaling, Ras protein signal transduction, and calcineurin-mediated signaling. To conclude, this study identified two novel M.tb effectors contributing to intracellular survival as potential new targets for anti-TB drug development.},
}
@article {pmid40399576,
year = {2025},
author = {Gu, P and Yuan, Y and Qi, L and Lan, F},
title = {RPA-CRISPR Cas13a-Based Point-of-Care Testing established for rapid detection of Methicillin-Resistant Staphylococcus aureus (MRSA) resistance genes.},
journal = {European journal of clinical microbiology &amp; infectious diseases : official publication of the European Society of Clinical Microbiology},
volume = {44},
number = {8},
pages = {1959-1966},
pmid = {40399576},
issn = {1435-4373},
mesh = {*Methicillin-Resistant Staphylococcus aureus/genetics/isolation &amp; purification/drug effects ; Humans ; *Bacterial Proteins/genetics ; Penicillin-Binding Proteins/genetics ; Staphylococcal Infections/microbiology/diagnosis ; *CRISPR-Cas Systems ; *Point-of-Care Testing ; Polymorphism, Single Nucleotide ; *Nucleic Acid Amplification Techniques/methods ; Recombinases/metabolism ; Sensitivity and Specificity ; Mutation ; },
abstract = {PURPOSE: This study aimed to develop a streamlined and efficient method for detecting single nucleotide polymorphism (SNP), specifically the S643N mutation in the mecA gene of Staphylococcus aureus (S. aureus). Detecting this mutation is crucial for guiding appropriate antibiotic therapy, avoiding antibiotic misuse, and enhancing infection control measures, especially in the cases of methicillin-resistant S. aureus (MRSA) infections.<br><br>METHODS: We employed the RPA-CRISPR Cas13a method to detect the S643N mutation. This approach integrated recombinase polymerase amplification (RPA) with CRISPR Cas13a-mediated detection into a single-step procedure, significantly reducing detection time compared to the conventional two-step process.<br><br>RESULTS: The established RPA-CRISPR Cas13a-Based Point-of-Care Testing method achieved high sensitivity, detecting as few as 10 copies per reaction and successfully differentiating between wild-type and mutant mecA genes. The one-step procedure streamlined the workflow and reduced detection time to less than 30&#xa0;min, while delivering results that were consistent with the conventional two-step method.<br><br>CONCLUSION: The one-step RPA-CRISPR Cas13a method significantly facilitates the rapid and accurate detection of single nucleotide mutations, such as the S643N mutation in the mecA gene. This advancement holds substantial clinical value by guiding precise antibiotic therapy and improving the management of S. aureus infections.},
}
@article {pmid40393858,
year = {2025},
author = {Tenjo-Castaño, F and Rout, SS and Dey, S and Montoya, G},
title = {Unlocking the potential of CRISPR-associated transposons: from structural to functional insights.},
journal = {Trends in genetics : TIG},
volume = {41},
number = {8},
pages = {660-677},
doi = {10.1016/j.tig.2025.04.005},
pmid = {40393858},
issn = {0168-9525},
mesh = {*DNA Transposable Elements/genetics ; Humans ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Animals ; Transposases/genetics ; },
abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR)-associated transposons (CASTs) are emerging genome-editing tools that enable RNA-guided DNA integration without inducing double-strand breaks (DSBs). Unlike CRISPR-associated (Cas) nucleases, CASTs use transposon machinery to insert large DNA segments with high precision, potentially reducing off-target effects and bypassing DNA damage responses. CASTs are categorized into classes 1 and 2, each employing distinct mechanisms for DNA targeting and integration. Recent structural insights have elucidated how CASTs recognize target sites, recruit transposases, and mediate insertion. These advances position CASTs as promising tools for genome engineering in bacteria and possibly in mammalian cells. Key challenges remain in enhancing efficiency and specificity, particularly for therapeutic use. Ongoing research aims to evolve CAST systems for precise, large-scale genome editing in human cells.},
}
@article {pmid40335327,
year = {2025},
author = {Chen, W and Choi, J},
title = {Molecular circuits for genomic recording of cellular events.},
journal = {Trends in genetics : TIG},
volume = {41},
number = {8},
pages = {647-659},
doi = {10.1016/j.tig.2025.04.004},
pmid = {40335327},
issn = {0168-9525},
mesh = {Humans ; *Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; *Genomics/methods ; *Synthetic Biology/methods ; Animals ; *Gene Regulatory Networks ; Cell Lineage/genetics ; Genome ; },
abstract = {Advances in precise genome editing are enabling genomic recordings of cellular events. Since the initial demonstration of CRISPR-based genome editing, the field of genomic recording has witnessed key strides in lineage recording, where clonal lineage relationships among cells are indirectly recorded as synthetic mutations. However, methods for directly recording and reconstructing past cellular events are still limited, and their potential for revealing new insights into cell fate decisions has yet to be realized. The field needs new sensing modules and genetic circuit architectures that faithfully encode past cellular states into genomic DNA recordings to achieve such goals. Here we review recently developed strategies to construct diverse sensors and explore how emerging synthetic biology tools may help to build molecular circuits for genomic recording of diverse cellular events.},
}
@article {pmid39865841,
year = {2025},
author = {Choi, BJ and Kim, BR and Choi, HJ and Kim, OH and Kim, SJ},
title = {Enhanced membrane protein production in HEK293T cells via ATF4 gene knockout: A CRISPR-Cas9 mediated approach.},
journal = {Biomolecules &amp; biomedicine},
volume = {25},
number = {9},
pages = {1961-1971},
doi = {10.17305/bb.2024.11519},
pmid = {39865841},
issn = {2831-090X},
mesh = {Humans ; HEK293 Cells ; *CRISPR-Cas Systems/genetics ; *Gene Knockout Techniques ; *Activating Transcription Factor 4/genetics/metabolism ; *Membrane Proteins/genetics/biosynthesis/metabolism ; Gene Editing ; Cadherins/genetics/metabolism/biosynthesis ; RNA, Guide, CRISPR-Cas Systems/genetics ; },
abstract = {HEK293T cells are extensively utilized for therapeutic protein production due to their human origin, which enables accurate post-translational modifications. This study aimed to enhance membrane protein production in HEK293T cells by knocking out the ATF4 gene using CRISPR-Cas9 technology. The ATF4 gene was edited by infecting HEK293T cells with a lentivirus carrying optimized single-guide RNA (ATF4-KO-3) and Cas9 genes. Comparative evaluations were conducted using all-in-one and two-vector systems. Genome sequencing and membrane protein productivity of ATF4-knockout (KO) cells were compared to wild-type (WT) cells using next-generation sequencing (NGS) and a membrane protein isolation kit, respectively. Single-cell analysis confirmed gene editing patterns, with NGS verifying the intended deletions. Membrane protein production was also assessed indirectly via flow cytometry, analyzing cells expressing Membrane-GFP. Compared to WT cells, ATF4-KO cells exhibited a significant increase in membrane protein production, with a 52.2 ± 19.0% improvement. Gene editing efficiency was compared between the two delivery systems, with the two-vector system demonstrating higher efficiency based on T7 endonuclease I assays. Western blot analysis confirmed ATF4 suppression and increased expression of membrane proteins, including E-cadherin and CD63. Quantitative analysis via PAGE revealed a 77.2 ± 30.6% increase in purified membrane protein yields, consistent with the observed enhancements. Flow cytometry using Membrane-GFP further demonstrated a 22.9 ± 9.7% increase in productivity. In summary, ATF4 knockout significantly enhances membrane protein production in HEK293T cells, offering potential improvements in biopharmaceutical manufacturing by enabling more efficient protein synthesis.},
}
@article {pmid40760018,
year = {2025},
author = {Haider, D and Bauer, R and Grempels, A and Roscher, R and Aslan, CC and Mauerer, S and Spellerberg, B},
title = {The stress of carrying CRISPR-Cas.},
journal = {Virulence},
volume = {16},
number = {1},
pages = {2541701},
doi = {10.1080/21505594.2025.2541701},
pmid = {40760018},
issn = {2150-5608},
mesh = {*CRISPR-Cas Systems ; *Stress, Physiological ; *Streptococcus anginosus/genetics/radiation effects/physiology/growth &amp; development ; Ultraviolet Rays ; Hydrogen Peroxide ; Microbial Viability/radiation effects ; Hot Temperature ; Plasmids ; CRISPR-Associated Protein 9/genetics ; Bacterial Proteins/genetics ; },
abstract = {Streptococcus anginosus (S. anginosus) is a commensal that can cause severe invasive bacterial infections. A considerable percentage of S. anginosus strains harbour CRISPR-Cas systems, which apart from being a bacterial immunity system, can play an important role in adaptation to environmental stress. The functionality of S. anginosus CRISPR-Cas systems has not been investigated previously. To address this, we created a set of deletion mutants in the CRISPR-Cas type II-A system of the S. anginosus SK52 type strain, targeting the nuclease Cas9 and CRISPR array. We were able to confirm CRISPR-Cas activity by testing these strains in a plasmid clearance assay. Furthermore, the impact of the CRISPR-Cas system on the viability of S. anginosus was investigated under various stress conditions, such as UV light, hydrogen peroxide exposure, and high temperatures in wild-type S. anginosus and CRISPR-Cas mutant strains. Under these conditions, survival was significantly lower in the strains carrying cas9. Bacterial growth and metabolic activity in Alamar Blue assays were also negatively affected by the presence of cas9 in S. anginosus. In summary, we found that the presence of a functional CRISPR-Cas system in S. anginosus leads to measurable metabolic and fitness costs in the wild-type strain. Carrying cas9 was associated with an impaired stress response in our experiments and may explain why many strains of this species lack CRISPR-Cas.},
}
@article {pmid40759662,
year = {2025},
author = {Leprince, A and Lefrançois, J and Millen, AM and Magill, D and Horvath, P and Romero, DA and Rousseau, GM and Moineau, S},
title = {Strengthening phage resistance of Streptococcus thermophilus by leveraging complementary defense systems.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {7142},
doi = {10.1038/s41467-025-62408-3},
pmid = {40759662},
issn = {2041-1723},
mesh = {*Streptococcus thermophilus/virology/genetics/immunology ; *CRISPR-Cas Systems/genetics ; *Bacteriophages/genetics/physiology ; *Streptococcus Phages/genetics/physiology ; },
abstract = {CRISPR-Cas and restriction-modification systems represent the core defense arsenal in Streptococcus thermophilus, but their effectiveness is compromised by phages encoding anti-CRISPR proteins (ACRs) and other counter-defense strategies. Here, we explore the defensome of 263 S. thermophilus strains to uncover other anti-phage systems. The defense landscape of S. thermophilus is enriched by 21 accessory defense systems, 13 of which have never been investigated in this species. Experimental validation of 17 systems with 14 phages reveals a range of anti-phage activities, highlighting both broad and narrow specificities across the five viral genera infecting S. thermophilus. Synergies are observed when combining CRISPR immunity with accessory systems. We also assess the fitness cost associated with the chromosomal integration of these systems in their native context and find no impact under laboratory or industrial conditions. These findings underscore the potential of these accessory defense systems to enhance the resistance of S. thermophilus, particularly against ACR-encoding phages.},
}
@article {pmid40758882,
year = {2025},
author = {Vera, AO and Truex, NL and Sreekanth, V and Pentelute, BL and Choudhary, A and Raines, RT},
title = {Protective antigen-mediated delivery of an anti-CRISPR protein for precision genome editing.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {122},
number = {32},
pages = {e2426960122},
doi = {10.1073/pnas.2426960122},
pmid = {40758882},
issn = {1091-6490},
support = {R01 GM137606/GM/NIGMS NIH HHS/United States ; R35 GM148220/GM/NIGMS NIH HHS/United States ; },
mesh = {*Gene Editing/methods ; Humans ; *CRISPR-Cas Systems ; *Bacterial Toxins/genetics/metabolism ; *Antigens, Bacterial/genetics/metabolism ; HEK293 Cells ; CRISPR-Associated Protein 9 ; },
abstract = {Precise control over the dosage of Cas9-based technologies is essential because off-target effects, mosaicism, chromosomal aberrations, immunogenicity, and genotoxicity can arise with prolonged Cas9 activity. Type II anti-CRISPR proteins (Acrs) inhibit and control Cas9 but are generally impermeable to the cell membrane due to their size and anionic charge. Moreover, existing Acr delivery methods are long-lived and operate within hours (e.g., viral and nonviral vectors) or require external devices (e.g., electroporation), limiting therapeutic applications. To address these problems, we developed a protein-based anti-CRISPR delivery platform, LFN-Acr/PA, which delivers Acrs into cells within minutes. LFN-Acr/PA is a nontoxic, two-component protein system derived from anthrax toxin, where protective antigen (PA) proteins bind receptors widespread in human cells, forming a pH-triggered endosomal pore that an engineered Acr (LFN-Acr) binds and uses to enter the cell. In the presence of PA, LFN-Acr enters human cells (e.g., immortalized cell lines, embryonic stem cells, and 3D cell cultures) at concentrations as low as 2.5 pM to inhibit up to 95% of Cas9-mediated knockout, knock-in, transcriptional activation, and base editing. Timing LFN-Acr delivery reduces off-target base editing and increases Cas9 specificity by 41%. LFN-Acr/PA is the most potent known cell-permeable CRISPR-Cas inhibition system, significantly improving the utility of CRISPR for genome editing.},
}
@article {pmid40758756,
year = {2025},
author = {Angst, P and Thivolle, A and Haden, Z and Wale, N and Ebert, D},
title = {Genomic analysis of the zooplankton-associated pathogenic bacterium Spirobacillus cienkowskii reveals its functional and metabolic capacities.},
journal = {Microbial genomics},
volume = {11},
number = {8},
pages = {},
doi = {10.1099/mgen.0.001463},
pmid = {40758756},
issn = {2057-5858},
mesh = {Animals ; *Genome, Bacterial ; *Zooplankton/microbiology ; Genomics ; Daphnia/microbiology ; Whole Genome Sequencing ; CRISPR-Cas Systems ; Carotenoids/metabolism ; },
abstract = {Genomic information can yield new insights into the molecular and physiological mechanisms that underpin pathogen virulence and transmission. We decode the genome of Spirobacillus cienkowskii Metchnikoff 1889, a gram-negative bacterium and one of the first described parasites of Daphnia. We use long-read sequencing and extensive annotation to assemble the complete circular genome of 2.81 Mbp with 2,486 protein-coding genes. In addition to antiviral systems, including CRISPR-Cas and restriction-modification systems, we describe the likely molecular basis of the unusual red phenotype of S. cienkowskii, which results from carotenoid production. We further describe genetic modules that may mediate this bacterium's interactions with its host and environment. Our study provides insight into the metabolic and functional capacities of a parasite through the assessment of its genome. More generally, it demonstrates what can be learnt by applying recent advances in high-throughput sequencing to the study of parasites.},
}
@article {pmid40758696,
year = {2025},
author = {Thavorasak, T and Santajit, S and Tunyong, W and Kong-Ngoen, T and Reamtong, O and Ampawong, S and Saelim, N and Srisai, T and Aiumurai, P and Pumirat, P and Chaicumpa, W and Indrawattana, N},
title = {CRISPR/Cas9-targeted smpB mutation revealing roles in biofilm formation, motility, and antibiotic susceptibility in Acinetobacter baumannii.},
journal = {PloS one},
volume = {20},
number = {8},
pages = {e0329638},
doi = {10.1371/journal.pone.0329638},
pmid = {40758696},
issn = {1932-6203},
mesh = {*Biofilms/drug effects/growth &amp; development ; *Acinetobacter baumannii/genetics/drug effects/pathogenicity/physiology ; *CRISPR-Cas Systems/genetics ; *Bacterial Proteins/genetics/metabolism ; *Anti-Bacterial Agents/pharmacology ; Mutation ; Animals ; Microbial Sensitivity Tests ; Virulence/genetics ; Gene Editing ; Acinetobacter Infections/microbiology/drug therapy ; },
abstract = {BACKGROUND: Acinetobacter baumannii is a multidrug-resistant pathogen and a major cause of hospital-acquired infections worldwide. Its ability to survive in harsh environments and evade antibiotic treatments underscores the urgent need for new therapeutic targets. Emerging evidence suggests that the small protein B (SmpB) may also play broader roles in bacterial virulence, including regulation of biofilm formation, motility, and stress adaptation. However, the specific contributions of SmpB to these pathogenic traits in A. baumannii remain poorly defined. Addressing this knowledge gap is essential for evaluating SmpB as a potential antimicrobial target and developing new strategies to combat multidrug-resistant infections.<br><br>METHODS: CRISPR/Cas9-mediated gene editing was used to generate a targeted smpB mutant in A. baumannii. The smpB mutant was assessed for growth, biofilm formation, motility, antibiotic susceptibility, and virulence. Biofilm was quantified via crystal violet staining and microscopy, while motility was examined using swimming, swarming, and twitching assays. Antibiotic susceptibility was evaluated using disk diffusion. Virulence was tested in the Galleria mellonella infection model. Proteomic analysis was performed to identify changes in protein expression associated with smpB disruption.<br><br>RESULTS: CRISPR/Cas9-mediated editing successfully introduced a C212T nucleotide substitution in the smpB gene, resulting in an A89G amino acid change. Growth curve analysis showed no significant difference between the wild-type and smpB mutant strains under nutrient-rich conditions. However, the mutant exhibited a significant reduction in biofilm formation (p = 0.0079) and impaired twitching motility, while swimming and swarming motility remained unaffected. Antibiotic susceptibility testing revealed increased sensitivity to ceftizoxime, piperacillin/tazobactam, and gentamicin, alongside decreased susceptibility to cefepime, tetracycline, and spectinomycin. In the G. mellonella infection model, the smpB mutant showed reduced virulence, with 84% larval survival compared to 72% in the wild type (p = 0.4183). Proteomic analysis revealed downregulation of key stress response and virulence-associated proteins, including GroEL, DnaK, RecA, and PirA, while proteins involved in ribosome maturation and transcription, such as RimP and RpoA, were upregulated. STRING network analysis supported the broad regulatory role of SmpB in biofilm formation, motility, stress adaptation, and pathogenesis.<br><br>CONCLUSION: This study demonstrates that SmpB is a key regulator of biofilm formation, twitching motility, antibiotic response, and virulence in A. baumannii. While not essential for growth under optimal conditions, smpB disruption impairs multiple pathogenic traits and alters stress-related proteomic pathways. These findings highlight the potential of SmpB as a novel antimicrobial target, offering a promising strategy to weaken bacterial virulence without promoting resistance. Targeting the trans-translation system may pave the way for innovative therapies against multidrug-resistant A. baumannii.},
}
@article {pmid40758649,
year = {2025},
author = {Qu, L and Meng, L and Sun, X and Cui, W and Shi, J and Wang, Y and Hu, Q and Xu, S and Sun, B and Liang, C},
title = {CRISPR/Cas-Based Electrochemical Biosensor for Human Immunodeficiency Virus-1 Nucleic Acid Amplification-Free Detection to the Attomolar Level.},
journal = {ACS sensors},
volume = {},
number = {},
pages = {},
doi = {10.1021/acssensors.5c00363},
pmid = {40758649},
issn = {2379-3694},
abstract = {Aiming at realizing field detection during HIV-1 patient screening in developing countries and point of care testing (POCT) of virus levels in HIV-1 patients receiving medication, an urgent demand for portable nucleic acid detection technology with low cost and sensitivity is raised. To solve this, a CRISPR/Cas13a-based electrochemical detection platform by a multiple combined crRNAs strategy is developed. This sensing platform is based on the Ion Current Rectification regulation through a Porous Anodic Alumina membrane decorated with ssRNA chains, which are trans-cleavage substrates for activated Cas13a and become shorter when the target gene exists, resulting in an altered motion of transmembrane ions. This platform was proven to be feasible and ultrasensitive for tracing the HIV-1 virus by multiple applicable crRNAs combination, and its clinical sample detection limit arrives at 60 copies/μL for HIV-1 patients' serum samples. Importantly, this method is amplification-free, and its sensitivity is beyond the nonamplification fluorescence-based nucleic acid strategy by one order of magnitude. This sensor is facile to use and cost-effective, and can also be extended to other RNA-based pathogenic microorganism detections. This POCT method facilitates self-monitoring of drug resistance in HIV-1 patients, regardless of medication status.},
}
@article {pmid40758593,
year = {2025},
author = {Carreira de Paula, J and Solano Parada, J and Rosel Miñarro, JF and García Olmedo, P and Orantes, FJ and Osuna, A and de Pablos Torró, LM},
title = {On-site DNA Detection of Trypanosomatid Parasites and Nosema ceranae Through Alkaline Lysis Coupled to RPA/CRISPR/Cas12a System.},
journal = {Journal of visualized experiments : JoVE},
volume = {},
number = {221},
pages = {},
doi = {10.3791/68874},
pmid = {40758593},
issn = {1940-087X},
mesh = {*Nosema/genetics/isolation &amp; purification ; Animals ; Bees/parasitology/microbiology ; *CRISPR-Cas Systems ; *Nucleic Acid Amplification Techniques/methods ; *DNA, Protozoan/analysis/genetics ; Recombinases/chemistry/metabolism/genetics ; *DNA, Fungal/analysis/genetics ; *Trypanosomatina/genetics/isolation &amp; purification ; },
abstract = {Early on-site detection of microscopic pathogens is essential for the effective management and health of honeybee colonies. Currently, the gold standard for molecular detection of bee pathogens is qPCR or RT-qPCR. Here, we present a rapid, sensitive, and cost-effective alternative for field-deployable DNA pathogen detection. This method combines direct, amplification-ready cell lysis of worker bee abdomens with recombinase polymerase amplification (RPA), CRISPR/Cas12a-mediated trans-cleavage of reporter probes, and detection via lateral flow assays. We successfully validated this protocol in honeybees infected with Nosema ceranae and Lotmaria passim. The described protocol can be performed using a simple heat block or at room temperature and is potentially applicable to any DNA-based pathogen or gut microbiota of the honeybee. The entire process takes approximately 180 min and achieves a sensitivity comparable to qPCR, detecting as few as 96 parasite copies/µL. In conclusion, this approach offers a promising tool for reliable and rapid field diagnosis of honeybee infections without the need for complex laboratory equipment, making it ideal for on-site colony surveillance.},
}
@article {pmid40756515,
year = {2025},
author = {Hendi, NN and Nemer, G},
title = {SDR42E1 modulates vitamin D absorption and cancer pathogenesis: insights from an in vitro model.},
journal = {Frontiers in endocrinology},
volume = {16},
number = {},
pages = {1585859},
pmid = {40756515},
issn = {1664-2392},
mesh = {Humans ; *Vitamin D/metabolism ; HCT116 Cells ; *Neoplasms/metabolism/pathology/genetics ; *Vitamin D Deficiency/genetics/metabolism ; Proteomics ; Cell Proliferation ; CRISPR-Cas Systems ; *Colorectal Neoplasms/metabolism/genetics/pathology ; Signal Transduction ; },
abstract = {INTRODUCTION: Vitamin D is a pleiotropic hormone essential for bone health and overall physiological function. Despite its significance, vitamin D deficiency remains widespread and is often influenced by genetic factors.<br><br>METHODS: This study investigates the role of SDR42E1, a gene encoding a short-chain dehydrogenase/reductase enzyme, in vitamin D regulation and sterol metabolism. Using CRISPR/Cas9 gene-editing, we generated an SDR42E1 knock-in model in HCT116 colorectal cells, which exhibit high endogenous SDR42E1 expression, harboring a nonsense variant associated with vitamin D deficiency.<br><br>RESULTS: Integrated transcriptomic and proteomic analyses revealed significant dysregulation of sterol absorption and metabolism (fold change (FC) = 1.8, P = 0.007) and cancer-related signaling pathways (FC = -1.7, P = 0.02). Notably, key differentially expressed genes included upregulated LRP1B and ABCC2, alongside downregulated WNT16 and SLC7A5. Proteomic profiling confirmed alterations in cell proliferation-related proteins, including reduced ALDOA expression (FC = -0.37, P = 0.0005). Functionally, SDR42E1 deficiency reduced cell viability by 53% (P = 0.0001), an effect reversed by transient SDR42E1 overexpression with restoring ABCC2 expression.<br><br>CONCLUSION: These findings establish SDR42E1 as a key modulator of vitamin D-related pathways and highlight its potential as a therapeutic target for addressing vitamin D deficiency and associated pathologies, including cancer.},
}
@article {pmid40755799,
year = {2025},
author = {Kong, H and Yin, Y and Zeng, N and Zhu, Y and Cui, Y},
title = {Rnf32 is not essential for spermatogenesis and male fertility in mice.},
journal = {PeerJ},
volume = {13},
number = {},
pages = {e19794},
pmid = {40755799},
issn = {2167-8359},
mesh = {Animals ; *Spermatogenesis/genetics ; Male ; Mice ; *Fertility/genetics ; Mice, Knockout ; *Ubiquitin-Protein Ligases/genetics/metabolism ; Sperm Motility/genetics ; Testis/metabolism ; CRISPR-Cas Systems ; },
abstract = {BACKGROUND: Ring finger motifs are found in a variety of proteins with diverse functions, often involved in protein-DNA or protein-protein interactions. The Rnf32-encoded protein contains two such motifs and is predominantly expressed in the testes and ovaries, suggesting that its expression may be regulated by elements within the Rnf32 promoter region. Rnf32 is active during spermatogenesis, mainly in spermatocytes and spermatids, indicating a potential role in sperm development.<br><br>METHODS: We established an Rnf32 knockout (Rnf32 [-/-]) mouse model using CRISPR/Cas9 technology. Gene expression was analyzed via reverse transcription quantitative polymerase chain reaction (RT-qPCR). Testicular and epididymal phenotypes were assessed through histological and immunofluorescence staining, and fertility and sperm motility were evaluated.<br><br>RESULTS: Here, we successfully established an Rnf32 knockout mouse model using CRISPR/Cas9 technology. Surprisingly, male Rnf32 [-/-] mice exhibited normal fertility, with no significant differences in testicular and epididymal histology, spermatogenesis, sperm count, or motility compared to Rnf32 [+/+] mice. These findings suggest that Rnf32 may not be essential for male fertility in mice, and its potential functions warrant further investigation.},
}
@article {pmid40754411,
year = {2025},
author = {Kiga, K},
title = {[The Need for Phage Therapy in Combating Antimicrobial Resistance].},
journal = {Yakugaku zasshi : Journal of the Pharmaceutical Society of Japan},
volume = {145},
number = {8},
pages = {679-688},
doi = {10.1248/yakushi.24-00190-4},
pmid = {40754411},
issn = {1347-5231},
mesh = {*Phage Therapy/trends/methods ; Humans ; *Bacteriophages/genetics ; *Drug Resistance, Bacterial ; Genetic Engineering ; CRISPR-Cas Systems ; RNA, Transfer ; *Bacterial Infections/therapy ; *Drug Resistance, Microbial ; Bacteria/virology ; },
abstract = {The escalating crisis of antimicrobial resistance poses a grave threat to global health and medicine in the 21st century. Phage therapy has emerged as a promising alternative to conventional antibiotics in addressing this urgent issue. Phages, unlike traditional antibiotics, leave the healthy microbiome largely undisturbed by selectively targeting and infecting their bacterial host. Additionally, phages can be readily genetically engineered to enhance their efficacy against specific bacterial strains. While some countries are slowly developing new regulations and implementing phage therapy in the clinic, widespread societal adoption remains limited. Phage therapy has the potential to revolutionize infection treatment; however, the unique biological properties of phages necessitate a multifaceted approach for the societal implementation of phage therapy. Recent research has focused on genetically engineering phages to enhance their capabilities or confer novel functions. Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas systems have facilitated the development of phages that target specific genes. Furthermore, the emergence of tRNA-carrying phages and phages that inhibit bacterial defense systems represents new classes of genetically engineered phages with enhanced bactericidal properties.},
}
@article {pmid40745005,
year = {2025},
author = {Ruis, BL and Bielinsky, AK and Hendrickson, EA},
title = {Gene editing and CRISPR-dependent homology-mediated end joining.},
journal = {Experimental &amp; molecular medicine},
volume = {57},
number = {7},
pages = {1409-1418},
doi = {10.1038/s12276-025-01442-z},
pmid = {40745005},
issn = {2092-6413},
support = {CA266254//U.S. Department of Health &amp; Human Services | NIH | National Cancer Institute (NCI)/ ; R35GM148181//U.S. Department of Health &amp; Human Services | National Institutes of Health (NIH)/ ; },
mesh = {*Gene Editing/methods ; Humans ; *DNA End-Joining Repair ; *CRISPR-Cas Systems ; Animals ; DNA Breaks, Double-Stranded ; },
abstract = {Gene editing is the intentional modification of a genetic locus in a living cell and is used for two general applications of great importance and wide interest. One is the inactivation of genes ('knockouts'), a process utilized to delineate the loss-of-function phenotype(s) of a particular gene. The second application ('knock-ins') is essentially the process of gene therapy, which predominately involves correcting a pre-existing mutated allele(s) of a gene back to wild-type to ameliorate some pathological phenotype associated with the mutation. Importantly, although these applications are conceptually exact reciprocal opposites of one another, they are achieved via mechanistically different pathways. In the case of knockouts, breakage (usually in the form of double-stranded breaks) of the chromosomal DNA at the site of targeting is used to engage a repair process (nonhomologous end joining) that is error prone. The ensuing repair frequently results in insertions/deletions at the cleavage site, which, in turn, results in out-of-frame mutations and, hence, a knockout of the gene in question. In the case of knock-ins, breakage (again, usually in the form of double-stranded breaks) of the DNA is used to engage a repair process (homology-dependent repair/recombination) in which homologous sequences between an incoming donor DNA (containing new genetic information) and the chromosomal DNA are exchanged. Although homology-directed repair was known to predominate in bacteria and lower eukaryotes, the competing process of nonhomologous end joining predominates in higher eukaryotes and was presumed to prevent the use of knock-in gene editing in human somatic cells in culture. A series of molecular and technical advances disproved this notion but still resulted in a process that was cumbersome, labor intensive, highly inefficient and slow. In 2013, however, a new RNA-programmable nuclease, CRISPR-Cas9 was described that has revolutionized the field and made gene editing accessible to anyone with even a rudimentary knowledge of molecular biology. Thus, gene editing in a wide variety of model organisms, as well as human somatic cells in culture, has become not only extremely feasible but also extremely facile, and it harbingers a golden age for directed mutagenesis, directed evolution and improvements in gene therapy.},
}
@article {pmid40745003,
year = {2025},
author = {Kwon, J and Park, Y and Ha, T},
title = {Unveiling the invisible genomic dynamics.},
journal = {Experimental &amp; molecular medicine},
volume = {57},
number = {7},
pages = {1400-1408},
doi = {10.1038/s12276-025-01434-z},
pmid = {40745003},
issn = {2092-6413},
mesh = {Humans ; *CRISPR-Cas Systems ; *Genomics/methods ; Chromatin/genetics/metabolism ; Animals ; Gene Editing/methods ; RNA, Guide, CRISPR-Cas Systems/genetics ; },
abstract = {CRISPR-based imaging technologies have emerged as powerful tools for visualizing specific genomic loci, providing groundbreaking insights into chromatin structure and dynamics. Here, in this Review, we discuss the development and recent advances in these techniques, highlighting key strategies such as signal amplification, background reduction, multiplexing and enhanced genomic resolution. By engineering Cas proteins and guide RNAs, and incorporating peptide and aptamer tags, researchers have remarkably improved the sensitivity, specificity and resolution of CRISPR-based imaging, enabling the detection of nonrepetitive genomic regions and single-nucleotide polymorphisms. Recent studies have further pushed the boundaries of CRISPR-based imaging with the introduction of degron-mediated fluorogenic labeling and light-controllable background reduction. Despite remaining challenges, such as the bulkiness of signal amplification systems, limitations in guide RNA design and the effects of fixation on chromatin-protein interactions, CRISPR-based imaging holds great promise for advancing our understanding of chromatin dynamics, genomic interactions and their roles in various biological processes.},
}
@article {pmid40744998,
year = {2025},
author = {Lee, J and Kweon, J and Kim, Y},
title = {Emerging trends in prime editing for precision genome editing.},
journal = {Experimental &amp; molecular medicine},
volume = {57},
number = {7},
pages = {1381-1391},
doi = {10.1038/s12276-025-01463-8},
pmid = {40744998},
issn = {2092-6413},
mesh = {*Gene Editing/methods/trends ; Humans ; CRISPR-Cas Systems ; Animals ; Genome ; },
abstract = {Prime editing is an advanced genome editing technology that enables precise genetic modifications without inducing double-strand breaks or requiring donor DNA templates. Prime editing has rapidly become a versatile tool, supporting a wide range of genetic modifications, including point mutations, insertions and deletions. Here we examine the evolution of prime editing technologies, detailing advancements from the initial prime editing systems to recent innovations that enhance editing efficiency. Through structural modifications and improved delivery methods, prime editing has expanded its applicability across eukaryotic systems. By enabling access to previously challenging mutations, prime editing opens new avenues for therapeutic development and precision genetic research, with efficiency, specificity and accessibility expected to shape its future impact in genome engineering.},
}
@article {pmid40720544,
year = {2025},
author = {Walsh, B and Summers, RL and Rangel, GW and Hagenah, LM and Mok, S and Llinás, M and Fidock, DA and Wirth, DF and Bopp, S},
title = {The plasmepsin-piperaquine paradox persists in Plasmodium falciparum.},
journal = {PLoS pathogens},
volume = {21},
number = {7},
pages = {e1012779},
doi = {10.1371/journal.ppat.1012779},
pmid = {40720544},
issn = {1553-7374},
mesh = {*Plasmodium falciparum/drug effects/genetics/enzymology ; *Aspartic Acid Endopeptidases/genetics/metabolism ; *Antimalarials/pharmacology ; *Quinolines/pharmacology ; *Drug Resistance/genetics ; *Protozoan Proteins/genetics/metabolism ; Humans ; *Malaria, Falciparum/drug therapy/parasitology ; Artemisinins/pharmacology ; CRISPR-Cas Systems ; Piperazines ; },
abstract = {Malaria remains a pressing global health challenge, with rising drug resistance threatening current treatment strategies. Partial resistance to dihydroartemisinin-piperaquine (DHA-PPQ) has emerged in Southeast Asia, particularly in Plasmodium falciparum strains from Cambodia. While artemisinin partial resistance is associated with mutations in kelch13, reduced PPQ sensitivity has been linked to increased copy numbers of the aspartic protease genes plasmepsin II and III and mutations in the chloroquine resistance transporter. In this study, we demonstrate the effective use of CRISPR-Cas9 technology to generate single knockouts (KO) of plasmepsin II and plasmepsin III, as well as a double KO of both genes, in two isogenic Cambodian parasites with varying numbers of plasmepsin gene copies. The deletion of plasmepsin II and/or III increased parasite sensitivity to PPQ. We explored several hypotheses to understand how an increased plasmepsin gene copy number might influence parasite survival under high PPQ pressure. Our findings indicate that protease inhibitors have a minimal impact on parasite susceptibility to PPQ. Additionally, parasites with higher plasmepsin gene copy numbers did not exhibit significantly increased hemoglobin digestion, differences in peptide composition, nor did they produce different amounts of free heme following PPQ treatment compared to wildtype (single copy) parasites. Interestingly, hemoglobin digestion was slowed in parasites with plasmepsin II deletions. We also found that culturing parasites with different plasmepsin II and III copies in amino acid-limited media had little impact on parasite sensitivity to high-dose PPQ. By treating parasites with modulators of digestive vacuole (DV) homeostasis, we found that changes in DV pH potentially affect their response to PPQ. Our research highlights the crucial role of increased plasmepsin II and III gene copy numbers in modulating response to PPQ and begins to uncover the molecular and physiological mechanisms underlying the contribution of plasmepsin II and III amplification to PPQ resistance in Cambodian parasites.},
}
@article {pmid40640612,
year = {2025},
author = {Yu, H and Qian, W and Song, Y and Welch, JD},
title = {PerturbNet predicts single-cell responses to unseen chemical and genetic perturbations.},
journal = {Molecular systems biology},
volume = {21},
number = {8},
pages = {960-982},
pmid = {40640612},
issn = {1744-4292},
support = {R01HG010883//HHS | NIH | National Human Genome Research Institute (NHGRI)/ ; U01HG011952//HHS | NIH | National Human Genome Research Institute (NHGRI)/ ; },
mesh = {*Single-Cell Analysis/methods ; Humans ; GATA1 Transcription Factor/genetics/chemistry/metabolism ; CRISPR-Cas Systems ; *Computational Biology/methods ; Gene Expression Regulation/drug effects ; Mutation, Missense ; },
abstract = {Chemical and genetic perturbations, such as those induced by small molecules and CRISPR, effect complex changes in the molecular states of cells. Despite advances in high-throughput single-cell perturbation screening technology, the space of possible perturbations is far too large to measure exhaustively. Here, we introduce PerturbNet, a flexible deep generative model designed to predict the distribution of cell states induced by unseen chemical or genetic perturbations. PerturbNet accurately predicts gene expression changes in response to unseen small molecules based on their chemical structures while also accounting for key covariates such as dosage and cell type. Moreover, PerturbNet accurately predicts the distribution of single-cell gene expression states following CRISPR activation or CRISPR interference by leveraging gene functional annotations. Our approach significantly outperforms previous methods, particularly for predicting the effects of perturbing completely unseen genes. Finally, we demonstrate for the first time that amino acid sequence embeddings can be used to predict gene expression changes induced by missense mutations. We use PerturbNet to predict the effects of all point mutations in GATA1 and nominate variants that significantly impact the cell state distribution of human hematopoietic stem cells. Using a crystal structure of GATA1 bound to DNA, we validate that these large-effect variants occur in the core DNA-contact region of GATA1 and tend to involve large changes in amino acid side-chain volume.},
}
@article {pmid40633258,
year = {2025},
author = {Liberty, JT and Lin, H and Sipos, Y and Ihedioha, OC and Kwaji, MJ},
title = {Synergizing gene editing and cellular agriculture for a sustainable and healthy food future.},
journal = {Current opinion in biotechnology},
volume = {94},
number = {},
pages = {103334},
doi = {10.1016/j.copbio.2025.103334},
pmid = {40633258},
issn = {1879-0429},
mesh = {*Gene Editing/methods ; Humans ; *Agriculture/methods ; CRISPR-Cas Systems ; Food Supply ; Food Security ; Animals ; },
abstract = {The intersection of gene editing and cellular agriculture is transforming food production by offering sustainable, ethical alternatives to conventional agriculture. Cellular agriculture uses tissue engineering and fermentation technologies to produce animal-free food, whereas gene editing tools like CRISPR-Cas9 optimize cellular efficiency, nutritional value, and sustainability. While some researchers emphasize the environmental and food security benefits, others raise concerns about high costs, regulatory challenges, and consumer acceptance. This paper critically examines existing literature, compares breakthroughs and controversies, and provides an expert perspective on the challenges and opportunities in gene-edited cellular agriculture. By tackling key scientific, economic, regulatory, and ethical issues, this article presents a roadmap for responsibly advancing these technologies and integrating them into global food systems. To our knowledge, this is the first work to explore how gene editing and cellular agriculture can be synergized to advance sustainability, food security, and global health.},
}
@article {pmid40616517,
year = {2025},
author = {Spataru, I and Mahoro, F and Badea, I},
title = {Development of Inorganic and Hybrid Nanosystems for Delivery of CRISPR-Based Gene Editors.},
journal = {Molecular pharmaceutics},
volume = {22},
number = {8},
pages = {4373-4393},
doi = {10.1021/acs.molpharmaceut.5c00251},
pmid = {40616517},
issn = {1543-8392},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; *Nanoparticles/chemistry ; Animals ; *Drug Delivery Systems/methods ; Liposomes/chemistry ; Lipids/chemistry ; Gene Transfer Techniques ; Transfection/methods ; },
abstract = {The CRISPR-Cas9 system is a gene editing tool, replacing specific target sequences of eukaryotic DNA via a distinct molecular pathway, with minimal off-target effects. In this manner, the effects are long-lasting and potentially require only one dose of medication to be effective. The package of bacterial nucleases is susceptible to the same degradative pathways as other nucleic acid therapeutics. Similarly, the CRISPR-Cas9 system is incapable of traversing biological membranes on its own. Therefore, a delivery mechanism is needed for effective transfection. In recent literature reviews, the focus has been on viral and lipid-based drug delivery systems, with little attention paid to solid-core nanoparticles, such as gold or silica nanoparticles, which present unique physicochemical properties and delivery opportunities. While viral delivery systems are efficient carriers, they can be highly immunogenic and unstable, and the production of high-viral titers is limited. Lipid-based drug delivery systems, such as liposomes, possess good shelf life, encapsulation, and transfection efficiency, but their biological stability and biodistribution profile limit their in vivo use. Thus, the arsenal of delivery agents could be complemented by solid-core nanoparticles. Their unique structural properties could lead to improved delivery strategies to mitigate disease outcomes and promote organ-specific delivery.},
}
@article {pmid40613419,
year = {2025},
author = {Rochussen, AM and Ma, CY and Griffiths, GM},
title = {Transcriptional adaptation after deletion of Cdc42 in primary T cells.},
journal = {Journal of cell science},
volume = {138},
number = {15},
pages = {},
doi = {10.1242/jcs.263826},
pmid = {40613419},
issn = {1477-9137},
support = {217100/WT_/Wellcome Trust/United Kingdom ; 220543/WT_/Wellcome Trust/United Kingdom ; 102163/WT_/Wellcome Trust/United Kingdom ; //University of Cambridge/ ; },
mesh = {*cdc42 GTP-Binding Protein/genetics/metabolism ; Animals ; *Transcription, Genetic ; *T-Lymphocytes, Cytotoxic/metabolism/immunology ; Mice ; *Gene Deletion ; CRISPR-Cas Systems/genetics ; *T-Lymphocytes/metabolism ; Mice, Inbred C57BL ; },
abstract = {Cdc42 is a Rho family GTPase known for its central role in cell polarity and cytoskeletal regulation. To understand the role of Cdc42 in polarised secretion from cytotoxic T lymphocytes (CTLs) we used CRISPR/Cas9 gene deletion. Although Cdc42-deleted CTLs initially showed reduced cytotoxicity for up to 2 days after CRISPR-mediated deletion, full secretion and cytotoxicity was rapidly restored and even enhanced while CDC42 protein remained absent. In contrast, chemical inhibition of CDC42 using CASIN consistently decreased secretion in wild-type cells, but had no impact on Cdc42-deleted CTLs, confirming the specificity of this inhibitor. Comparative proteomics and transcriptomics of CTLs after Cdc42 deletion revealed transcriptional changes that could support improved T cell function, including compensation via other Rho GTPases. Targeting the promoter region of Cdc42 did not trigger transcriptional adaptation, consistent with a nonsense-mediated decay mechanism of genetic compensation. Our work highlights the importance of taking orthogonal approaches to study protein function and reveals the remarkable robustness of primary T cells to adapt to loss of an essential gene.},
}
@article {pmid40371725,
year = {2025},
author = {Kalniņa, Z and Liekniņa, I and Koteloviča, S and Petrovska, R and Žvinys, G and Petrosiute, A and Zubrienė, A and Laugalis, MT and Skeltona, V and Jansons, J and Kreishmane, M and Čapkauskaitė, E and Matulis, D and Tārs, K},
title = {Development of 4T1 breast cancer mouse model system for preclinical carbonic anhydrase IX studies.},
journal = {FEBS open bio},
volume = {15},
number = {8},
pages = {1285-1302},
doi = {10.1002/2211-5463.70052},
pmid = {40371725},
issn = {2211-5463},
support = {1.1.1.2/VIAA/4/20/728//European Regional Development Fund (ERDF)/ ; },
mesh = {Animals ; *Carbonic Anhydrase IX/metabolism/genetics ; Mice ; Female ; Cell Line, Tumor ; Mice, Inbred BALB C ; Disease Models, Animal ; *Triple Negative Breast Neoplasms/pathology/metabolism/genetics ; Humans ; CRISPR-Cas Systems ; },
abstract = {Triple-negative breast cancer (TNBC) is the most aggressive type of breast cancer, for which targeted treatment is currently lacking. Carbonic anhydrase IX (CAIX) is a known cancer target due to its selective overexpression in hypoxia, a hallmark of many solid cancers including TNBC. This study aimed to develop a robust murine TNBC cell line 4T1-based model system that could be used in the comprehensive preclinical evaluation of targeting CAIX. The model is based on the original 4T1 breast cancer cell line and two genetically edited versions of it-one with biallelic CRISPR/Cas9-mediated Car9 inactivation and another with constitutively expressed Car9, thus ensuring negative and positive controls for CAIX production in the model system, respectively. The generated cell lines were validated for CAIX production and characterised functionally in vitro and in vivo after orthotopic implantation in syngeneic BALB/c mice. Results demonstrated significantly reduced primary tumour growth and metastatic progression rates in animals with CAIX-deficient tumours, while the CAIX-expressing tumours had vascularised phenotypes with prominent central areas of coagulative necrosis. The differential CAIX expression levels in the model were preserved during tumour growth in syngeneic mice, as verified by in vivo imaging using a novel high-affinity CAIX-specific near-infrared (NIR) fluorescent imaging probe, GZ22-4. Constitutive overexpression of autologous CAIX did not elicit specific autoantibody responses in vivo, demonstrating the suitability of this model for evaluating the efficacy of anti-CAIX vaccination as a therapeutic strategy. The in vivo study was repeated as an independent experiment and demonstrated good robustness of the developed model.},
}
@article {pmid40367935,
year = {2025},
author = {Egashira, S and Maehara, K and Tanaka, K and Nakamura, M and Takemoto, T and Ohkawa, Y and Harada, A},
title = {Histone H2B isoform H2bc27 is expressed in the developing brain of mouse embryos.},
journal = {Journal of biochemistry},
volume = {178},
number = {2},
pages = {109-119},
doi = {10.1093/jb/mvaf026},
pmid = {40367935},
issn = {1756-2651},
support = {//Autonomous Medical Research Center in Kyushu University/ ; //CURE:JPMXP1323015486/ ; //MRCI for High Depth Omics/ ; //MEXT Promotion of Development of a Joint Usage/Research System Project:Pan-Omics DDRIC/ ; JP18H05528, JP24H02323, JP23H00372, JP22H04676 and JP22K19275//JSPS KAKENHI/ ; JP22H04696 and JP23H04288//JSPS KAKENHI/ ; JP21H05292, JP23K27087 and JP23H02394//JSPS KAKENHI/ ; JP23gm1810008//AMED-CREST/ ; JP22ama121017j0001//AMED BINDS/ ; JPMJPR2026//JST PRESTO/ ; JPMJCR23N3//JST CREST/ ; },
mesh = {Animals ; Mice ; *Histones/genetics/metabolism ; *Brain/metabolism/embryology ; Protein Isoforms/genetics/metabolism ; Mice, Knockout ; *Gene Expression Regulation, Developmental ; CRISPR-Cas Systems ; *Embryo, Mammalian/metabolism ; Mice, Inbred C57BL ; },
abstract = {Histones bind directly to DNA and play a role in regulating gene expression in part by influencing chromatin structure. The DNA sequences of these histone genes are quite similar, which has hindered individual analyses. The exact function of the 13 different isoforms of histone H2B remains unclear. In this study, we performed a comprehensive gene expression analysis of the H2B isoforms, focusing on tissue specificity. Our results revealed that the H2bc27 gene exhibited brain-specific expression in mice at E14.5. We generated mice lacking the H2bc27 gene using the CRISPR/Cas9 system. While the phenotype of H2bc27 knockout mouse brains was not different from that of wild-type mouse brains, transcriptome analysis indicated that H2bc27 is associated with regulating the expression of several functional genes involved in mouse brain development. The methods used in this study may serve to facilitate comprehensive H2B isoform analysis.},
}
@article {pmid40756867,
year = {2025},
author = {Samson, C and du Rand, A and Hunt, J and Whitford, W and Jacobsen, J and Sheppard, H},
title = {A bioinformatic analysis of gene editing off-target loci altered by common polymorphisms, using 'PopOff'.},
journal = {Journal of the Royal Society of New Zealand},
volume = {55},
number = {6},
pages = {2440-2463},
pmid = {40756867},
issn = {1175-8899},
abstract = {Gene editing therapies are designed to minimise off-target editing. However, it is not widespread practice for common polymorphisms to be considered when identifying potential off-target sites in silico. Nevertheless, genetic variants should be included as they have the potential to alter existing, or to generate new, off-target sites. To facilitate the consideration of common polymorphisms when designing targeted gene therapies we developed PopOff, a web-based tool that integrates minor allele frequencies from the gnomAD variant database into an off-target analysis. We used PopOff to analyse predicted off-target loci from guide RNAs used in four clinical trials and thirty-four research publications. From an analysis of sixty guides, we identified that approximately 20% of off-target loci overlap with a common polymorphism. Of these sites, 6.93% contained variants that reduce the level of mismatch between the off-target locus and guide, and therefore may increase off-target cleavage. In addition, we identified that 0.34% of common polymorphisms generated novel PAM sites, resulting in off-target loci that standard workflows would miss. Our findings demonstrate that common polymorphisms should be considered when designing guides to maximise the safety of CRISPR-based gene therapies. However, this may be problematic in populations where the breadth of genetic diversity remains uncharacterised.},
}
@article {pmid40753163,
year = {2025},
author = {Wolkoff, M and Yoshida, M and Lowmiller, T and Sim, C and Meuti, ME},
title = {The cycle gene is essential for both daily responses and seasonal reproduction in the Northern house mosquito, Culex pipiens.},
journal = {Scientific reports},
volume = {15},
number = {1},
pages = {28279},
doi = {10.1038/s41598-025-06637-y},
pmid = {40753163},
issn = {2045-2322},
support = {IOS-1944323//National Science Foundation/ ; 2023-67013-39915//U.S. Department of Agriculture/ ; R01AI188567/NH/NIH HHS/United States ; },
mesh = {Animals ; *Culex/genetics/physiology ; Reproduction/genetics ; Seasons ; Female ; Photoperiod ; *Circadian Rhythm/genetics ; Circadian Clocks/genetics ; Male ; *Insect Proteins/genetics/metabolism ; Diapause, Insect/genetics ; },
abstract = {The insect circadian clock synchronizes daily activities, such as feeding, eclosion, and mating to diurnal light:dark cycles, while the photoperiodic clock synchronizes development and reproduction to the appropriate season. Despite mounting evidence that the circadian and photoperiodic timekeeping systems are linked, it is unclear whether the circadian clock as a whole or individual genes within the circadian clock are responsible for measuring daylength, releasing hormones and/or inducing diapause phenotypes. To interrogate the role of a core circadian transcription factor, cycle, in mediating both seasonal and daily behaviors we used CRISPR/Cas-9 genome editing to introduce a deletion mutation into cycle in the Northern house mosquito, Culex pipiens. Females homozygous for this deletion exhibited high rates of reproductive arrest characteristic of an overwintering diapause even when reared in summer-like conditions, while a minority remained reproductive even when reared in winter-like conditions. Nearly all (79%) homozygotes lacked robust behavioral rhythms in constant darkness, while 52% of heterozygotes carrying one wild-type copy of the cycle gene lacked robust rhythmicity in constant darkness. Heterozygotes and homozygotes were more active in constant darkness than WT mosquitoes, but this difference was abolished under light:dark conditions. Lastly, the daily transcription profiles of various circadian genes, especially timeless and vrille, were disrupted in homozygous females even when they were reared under long and short days with light:dark cycles. These results demonstrate that cycle is essential for coordinating both daily activity and transcript abundance and seasonal diapause in Cx. pipiens.},
}
@article {pmid40752531,
year = {2025},
author = {Liu, Q and Zhuo, R and He, W and Li, C},
title = {The new SCCmec type methicillin-resistant Staphylococcus aureus carried CRISPR-Cas system isolated from a pig in China.},
journal = {Microbial pathogenesis},
volume = {},
number = {},
pages = {107943},
doi = {10.1016/j.micpath.2025.107943},
pmid = {40752531},
issn = {1096-1208},
abstract = {Methicillin-resistant Staphylococcus aureus (MRSA) lineages circulate globally in healthcare, community, and livestock-associated (LA) settings. Nine MRSA isolates were recovered from swine in China, all exhibiting resistance to ampicillin and multidrug resistance phenotypes. Among eight ST9-t899 isolates, SCCmec type XII(9C2) predominated. However, we identified a novel staphylococcal cassette chromosome mec (SCCmec) type, designated XIII (9A), in an LA-MRSA strain (LS45). Structural analysis revealed SCCmec XIII(9A) comprises a CRISPR-Cas system (cas10-csm2-csm3-csm4-csm5-csm6). Functional analysis demonstrated this CRISPR-Cas system provided partial protection against phage infection at low multiplicities of infection (MOIs ≤10[-7]), but conferred no detectable immunity against spacer-matched plasmids, with no significant change in cas10 expression during plasmid challenge. The co-location of this novel SCCmec element and a functional CRISPR-Cas system within an LA-MRSA strain demonstrates that S. aureus can maintain a defense system active against phages while accommodating SCCmec-mediated horizontal gene transfer. These findings provide new insights into the genomic adaptations of MRSA across different hosts.},
}
@article {pmid40752020,
year = {2025},
author = {Camborde, L and Jaturapaktrarak, C and Gouzy, J and Lopez-Roques, C and Krajaejun, T and Gaulin, E and Badis, Y},
title = {Adenine Phosphoribosyltransferase Is a Universal Counter-Selectable Marker for DNA-Free Genome Editing in Oomycetes.},
journal = {Molecular plant-microbe interactions : MPMI},
volume = {},
number = {},
pages = {},
doi = {10.1094/MPMI-05-25-0063-TA},
pmid = {40752020},
issn = {0894-0282},
abstract = {CRISPR-Cas genome editing is a powerful tool for understanding the pathogenicity of oomycetes, a group that includes several destructive plant parasites. While few Phytophthora species have benefited from plasmid-based transformation methods for gene overexpression and RNAi silencing, these techniques remain inefficient for other oomycete genera such as Pythium and Aphanomyces. Here, we explored the applicability of DNA-free endogenous counter-selection in filamentous oomycetes, using CRISPR-Cas9 ribonucleoproteins (RNPs). We used biolistics to deliver RNPs targeting the Adenine phosphoribosyltransferase (APT) gene, and generated selectable 2-fluoroadenine-resistant mutants in Aphanomyces, Pythium, and Phytophthora species. Targeted mutagenesis resulted in various deletions at the expected cut-sites, confirming efficient genome editing. Knockout mutants exhibited no alterations in growth or virulence, making APT a suitable selectable marker gene for oomycete research. Whole genome comparative analyses on CRISPR-edited mutants revealed no or very few additional mutations in A. euteiches and P. oligandrum, and substantial off-target effects in P. capsici. This one-step approach circumvents the need for protoplast generation and can be broadly applied to oomycetes producing zoospores or oospores.},
}
@article {pmid40750796,
year = {2025},
author = {Li, Z and Ee, A and Amaya, L and Hamad, JL and Yadav, PK and Wang, SK and Chang, HY and Wender, PA},
title = {Discrete Immolative Guanidinium Transporters deliver mRNA to specific organs and red blood cells.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {7055},
pmid = {40750796},
issn = {2041-1723},
support = {R01CA245533//U.S. Department of Health &amp; Human Services | NIH | National Cancer Institute (NCI)/ ; },
mesh = {Animals ; *RNA, Messenger/metabolism/administration &amp; dosage/chemistry/genetics ; *Erythrocytes/metabolism ; Mice ; Female ; *Guanidine/chemistry/metabolism ; Humans ; Lung/metabolism ; *Drug Delivery Systems/methods ; Spleen/metabolism ; Cell Survival ; },
abstract = {RNA medicine is an emerging groundbreaking technology for the prevention and treatment of disease. However, tools to deliver messenger RNA (mRNA) and other polyanions (circRNA, saRNA, pDNA, CRISPR-Cas, reprogramming factors) are required to advance current RNA therapies and address next generation challenges. Existing delivery systems often suffer from laborious syntheses, limited organ selectivity, formulation complexity, and undesired inflammatory responses. Here, we report novel mRNA delivery systems termed Discrete Immolative Guanidinium Transporters (DIGITs), which are synthesized convergently in as few as 4 steps. Unlike most cationic (ammonium) delivery systems, DIGITs are based on cationic guanidinium moieties, which complex mRNA at acidic pH and undergo irreversible neutralization at physiological pH to enable efficient RNA release. Systematic evaluation of structural variations and formulations have led to DIGIT/mRNA complexes that selectively target lung, spleen, and immature red blood cells in peripheral blood in female mice model. DIGIT/mRNA delivery systems show minimal toxicity based on cell viability and biochemical assays, supporting their future utility in biomedical applications.},
}
@article {pmid40460689,
year = {2025},
author = {Liu, T and Wang, Y and Liao, Z and Li, X and Tang, S and Zhang, Z and Chu, M and Wei, L},
title = {RPA-CRISPR/Cas12a detection based on HCMV-UL123 gene: A way with higher detection rate than clinical detection methods.},
journal = {Journal of infection and public health},
volume = {18},
number = {9},
pages = {102845},
doi = {10.1016/j.jiph.2025.102845},
pmid = {40460689},
issn = {1876-035X},
mesh = {Humans ; *Cytomegalovirus/genetics/isolation &amp; purification ; Sensitivity and Specificity ; *Cytomegalovirus Infections/diagnosis/virology ; *CRISPR-Cas Systems ; DNA Primers/genetics ; *Molecular Diagnostic Techniques/methods ; *Viral Proteins/genetics ; DNA, Viral/genetics ; },
abstract = {OBJECTIVE: Human cytomegalovirus (HCMV), a prevalent double-stranded DNA enveloped virus, poses a threat to immunocompromised individuals. The current clinical detection methods are insufficient in sensitivity, highlighting the need for more effective approaches.<br><br>METHODS: We designed and screened RPA primers and crRNA based on the UL123 gene of HCMV. Evaluate the HCMV-RPA-CRISPR detection method using cloned plasmids and the whole-genome samples of HCMV-infected cells. Conduct RPA-CRISPR/Cas12a reactions with 48 clinical samples and compare the results with those of PCR-Fluorescent Probe Method in clinical applications and the qPCR method for detecting the UL123 gene.<br><br>RESULTS: The optimized RPA-CRISPR system exhibited high sensitivity and specificity for HCMV detection. The positive rate of clinical sample detection was approximately 20.5&#x202f;% (6/48) higher than that of the clinical detection method.<br><br>CONCLUSION: Currently, the sensitivity and early detection of HCMV in clinical settings are still limited. The UL123 gene of HCMV is characterized by high transcription in the early stage and high conservation. The RPA-CRISPR/Cas12a technology exhibits high sensitivity in detecting the HCMV UL123 gene, and it is expected to provide a more effective method for the early specific detection of HCMV infection.},
}
@article {pmid40750194,
year = {2025},
author = {Li, T and Chen, S and Chen, X and Yang, S and Huang, H and Xu, J and Liu, Y and Zhang, J and Cao, L and Kang, Z and Li, M and Wang, H},
title = {CRISPR-based sensing platform for the Group B streptococcus screening in pregnant women.},
journal = {Analytica chimica acta},
volume = {1370},
number = {},
pages = {344390},
doi = {10.1016/j.aca.2025.344390},
pmid = {40750194},
issn = {1873-4324},
mesh = {Female ; *Streptococcus agalactiae/isolation &amp; purification/genetics ; Pregnancy ; Humans ; *Streptococcal Infections/diagnosis ; *CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Nucleic Acid Amplification Techniques ; *Prenatal Diagnosis ; },
abstract = {BACKGROUND: Group B Streptococcus (GBS) is a major cause of perinatal infections. Prenatal screening is critical to prevent maternal and neonatal GBS infections, reduce adverse outcomes, and guide clinical interventions. While bacterial culture is the gold standard, it is time-consuming and delays decision-making. Rapid molecular tests like PCR are sensitive and specific but require expensive equipment and skilled personnel. Point-of-care tests (e.g., Xpert GBS LB) offer speed and sensitivity but remain costly and underutilized. This study aims to develop a portable CRISPR/Cas13a-based device for rapid, on-site GBS detection.<br><br>RESULTS: A palm-sized CRISPR detection platform, PalmCS, was developed. PalmCS includes two key components: an integrated tube for nucleic acid extraction, gene amplification, and CRISPR-based reaction, and a multifunctional device for thermal regulation, fluorescence detection, and automatic result interpretation. The sealed plug needle valve controls fluid flow for sample input and result output. By optimizing crRNA selection, Cas13a/crRNA ribonucleoprotein (RNP) complex concentrations, and isothermal recombinase polymerase amplification primers, we established a one-pot CRISPR/Cas13a-RPA method for GBS detection. Results showed that 3&#xa0;M guanidinium salt combined with 5&#xa0;% Tween 20 achieved the highest extraction efficiency. The system extracted nucleic acids from samples in 5&#xa0;min at room temperature, demonstrating its potential for rapid diagnostics. PalmCS was validated using 40 clinical samples, achieving a limit of detection (LOD) of 20 copies/reaction, 97.5&#xa0;% sensitivity, and 100&#xa0;% specificity.<br><br>SIGNIFICANCE AND NOVELTY: This study used a one-step nucleic acid extraction combined with a closed one-pot RPA-CRISPR reaction for rapid, sensitive, and specific GBS detection. This provides a new platform for prenatal screening in pregnant women. In resource-limited settings with emergency deliveries, PalmCS enables on-site GBS testing, allowing antibiotic administration during delivery for positive cases to significantly reduce neonatal infection risk.},
}
@article {pmid40750193,
year = {2025},
author = {Zhu, C and Li, J and Yu, H and Xu, K and He, X and Liu, Z and Chen, J},
title = {CRISPR/Cas13a combined with reverse transcription-multienzyme isothermal rapid amplification for hepatitis B virus RNA detection.},
journal = {Analytica chimica acta},
volume = {1370},
number = {},
pages = {344389},
doi = {10.1016/j.aca.2025.344389},
pmid = {40750193},
issn = {1873-4324},
mesh = {*Hepatitis B virus/genetics/isolation &amp; purification ; *Nucleic Acid Amplification Techniques/methods ; *RNA, Viral/genetics/analysis/blood ; Humans ; *CRISPR-Cas Systems/genetics ; Reverse Transcription ; Limit of Detection ; Molecular Diagnostic Techniques ; },
abstract = {BACKGROUND: Hepatitis B virus (HBV) infection represents a major global public health challenge. The covalently closed circular DNA (cccDNA), which serves as the key reservoir for viral persistence, currently requires invasive liver biopsy for clinical monitoring. Recent studies have identified serum HBV pregenomic RNA (pgRNA) and its splicing variants emerging as potential noninvasive and reliable biomarkers for tracking disease progression and forecasting prognosis in chronic HBV infection. Precise detection of pgRNA is therefore essential for informed clinical decision-making and optimized patient management.<br><br>RESULTS: In this study, we developed a rapid, accurate, and clinically applicable method for HBV RNA detection by integrating CRISPR/Cas13a with reverse transcription-multienzyme isothermal rapid amplification (RT-MIRA). This innovative platform enabled simultaneous amplification of two pgRNA targets within a single-tube RT-MIRA reaction, allowing dual-target detection via one-step amplification. The optimized system achieved efficient isothermal amplification with a detection sensitivity of 10[3] copies/mL for both total and spliced pgRNA and exhibited no cross-reactivity with other common clinical viruses. Validation using both RT-qPCR and the RT-MIRA-Cas13a assay on clinical samples from 48 HBV-infected patients demonstrated a positive prediction value of 97.2&#xa0;% and a negative predictive value of 100&#xa0;%. These results conclusively validate the assay's reliability for clinical application in detecting and quantifying both total pgRNA and its splicing variants.<br><br>SIGNIFICANCE: This study presents a rapid and user-friendly HBV RNA detection platform capable of accurately identifying both total and spliced pgRNA. The RT-MIRA-Cas13a assay demonstrates excellent clinical potential for prognosis assessment and disease monitoring in HBV infection. Its robust performance and operational simplicity suggest strong suitability for point-of-care applications, potentially transforming chronic HBV monitoring through accessible nucleic acid testing.},
}
@article {pmid40748632,
year = {2025},
author = {Zhao, Z and Li, Y and Xiu, L and Wang, F and Peng, J},
title = {Development of a CRISPR/Cas-Based Detection Platform for Tracking Decreased Susceptibility to Cephalosporins in Neisseria gonorrheae.},
journal = {Analytical chemistry},
volume = {},
number = {},
pages = {},
doi = {10.1021/acs.analchem.5c01400},
pmid = {40748632},
issn = {1520-6882},
abstract = {Gonorrhea has become an escalating public health issue due to the rapid emergence of antimicrobial resistance (AMR). Developing efficient and accurate detection of resistant strains is urgently needed for their management and treatment. We have developed the Multiplex Integrated RPA-CRISPR/Cas12a detection Assay (MIRCA) for simultaneous detection of Neisseria gonorrheae (Ng) and mutations with decreased susceptibility to cephalosporins. MIRCA enables multiplex detection of Ng and single-nucleotide polymorphisms in resistance-associated genes within 40 min, with high specificity and sensitivity (10-20 copies/reaction). Clinical evaluation showed 100% concordance with qPCR for Ng identification and Sanger sequencing for FC428 strain tracking. For predicting decreased-susceptibility strains with A501 mutations, MIRCA achieved 98.33% agreement with Sanger sequencing. Simulated tests demonstrated 100% consistency between MIRCA results in centrifuge tubes and microfluidic chips. This robust and cost-effective approach addresses current challenges in AMR surveillance. Its integration with microfluidic chip offers an affordable and user-friendly diagnostic solution, making it highly valuable for timely infectious disease diagnosis and resistance monitoring. It also holds significant potential for point-of-care testing in resource-limited areas.},
}
@article {pmid40516634,
year = {2025},
author = {Wu, Y and Wang, Y and Wang, Y and Qiu, H and Yuan, X and Xiong, H and Zou, Y},
title = {Exploring synthetic lethality in cancer therapy: CRISPR-Cas9 technology offers new hope.},
journal = {Biochimica et biophysica acta. Reviews on cancer},
volume = {1880},
number = {4},
pages = {189370},
doi = {10.1016/j.bbcan.2025.189370},
pmid = {40516634},
issn = {1879-2561},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Neoplasms/genetics/therapy/drug therapy ; *Synthetic Lethal Mutations ; *Gene Editing/methods ; Drug Resistance, Neoplasm/genetics ; *Genetic Therapy/methods ; Animals ; },
abstract = {Synthetic lethality (SL) is a breakthrough concept in cancer therapy that describes a scenario in which the simultaneous inactivation of two genes leads to cell death, whereas inactivation of either gene alone does not. The rise of clustered regularly interspaced short palindromic repeat (CRISPR)-CRISPR-associated nuclease 9 (Cas9) technology has provided a new tool for exploring this phenomenon, enabling genome editing and screening. This review evaluates the advancements achieved by CRISPR technology in identifying novel therapeutic targets and comprehending the processes of drug resistance using the concept of SL in cancer cells. This review explores the fundamental concept of SL and its application in cancer therapy, highlighting how the CRISPR-Cas9 system functions and how CRISPR-based screening can be leveraged to identify synthetic lethal genes and investigate the mechanisms of drug resistance. We summarize important research in related fields from recent years, demonstrating the role of CRISPR screening in revealing cancer cellular pathways and identifying new drug targets. We also summarize the clinical trials of related drugs currently underway, and anticipate that with the continuous development of CRISPR technology, its integration with cancer genetics and immuno-oncology will bring new hope to patients with drug-resistant cancers.},
}
@article {pmid40425316,
year = {2025},
author = {Rao, Z and Zhang, M and Huang, S and Wu, C and Zhou, Y and Zhang, W and Lin, X and Zhao, D},
title = {Cancer driver topologically associated domains identify oncogenic and tumor-suppressive lncRNAs.},
journal = {Genome research},
volume = {35},
number = {8},
pages = {1842-1858},
pmid = {40425316},
issn = {1549-5469},
mesh = {*RNA, Long Noncoding/genetics/metabolism ; Humans ; *Neoplasms/genetics ; Gene Expression Regulation, Neoplastic ; *Oncogenes ; *Genes, Tumor Suppressor ; Computational Biology/methods ; CRISPR-Cas Systems ; },
abstract = {Cancer long noncoding RNAs (lncRNAs) have been identified by experimental and in silico methods. However, current approaches for identifying cancer lncRNAs are not sufficient and effective. To uncover them, we focus on the core cancer driver lncRNAs, which directly interact with cancer driver protein-coding genes (PCGs). We investigate various aspects of cancer lncRNAs, including their expression patterns, genomic locations, and direct interactions with cancer driver PCGs, and developed a pipeline to identify candidate cancer driver lncRNAs. Finally, we validate the reliability of potential cancer driver lncRNAs through functional analysis of bioinformatics data and CRISPR-Cas9 knockout experiments. We find that cancer lncRNAs are more concentrated in cancer driver topologically associated domains (CDTs), and CDT is an important feature in identifying cancer lncRNAs. Moreover, cancer lncRNAs show a high tendency to be coexpressed with and bind to cancer driver PCGs. Utilizing these distinctive characteristics, we develop a pipeline CAncer Driver Topologically Associated Domains (CADTAD) to identify candidate cancer driver lncRNAs in pan-cancer, including 256 oncogenic lncRNAs, 177 tumor-suppressive lncRNAs, and 75 dual-function lncRNAs, as well as in three individual cancer types, and validate their cancer-related functions. More importantly, the function of 10 putative cancer driver lncRNAs in prostate cancer is subsequently validated to influence cancer phenotype through cell studies. In light of these findings, our study offers a new perspective from the 3D genome to study the roles of lncRNAs in cancer. Furthermore, we provide a valuable set of potential lncRNAs that could deepen our understanding of the oncogenic mechanism of cancer driver lncRNAs.},
}
@article {pmid40745870,
year = {2025},
author = {Sui, Z and Chen, B and Zhao, J and Yang, H and Guo, L and Xu, J},
title = {Self-Sustaining miRNA Amplification Diagnostics via Catalytic Hairpin Assembly and Split Activator-Initiated CRISPR/Cas12a Hybrid Feedback Circuit with Target Regeneration and Recycling.},
journal = {Analytical chemistry},
volume = {},
number = {},
pages = {},
doi = {10.1021/acs.analchem.5c02845},
pmid = {40745870},
issn = {1520-6882},
abstract = {To meet the growing demand for ultrasensitive diagnostics, representative hybrid platforms integrating nonenzymatic isothermal nucleic acid amplification such as catalytic hairpin assembly (CHA) with CRISPR/Cas systems have been developed. However, two major challenges remain: background leakage from spontaneous hairpin hybridization and inherent fluorescence from conventional ssDNA reporters. Here, we present a self-sustaining isothermal biosensing platform that addresses these limitations by combining CHA with a split activator-initiated CRISPR/Cas12a feedback circuit for the ultrasensitive detection of miRNA-155, a key biomarker of breast cancer. In our design, miRNA-155 initiates CHA to form a DNA duplex, which, along with the miRNA, acts as split activators to trigger CRISPR/Cas12a. Cas12a cleaves a ds-loop DNA reporter, releasing fluorescence and regenerating the target. This dual-recognition mechanism ensures strict target dependence, reduces background noise, and, with the reporter design, minimizes leakage. The released miRNA reactivates CHA, enabling continuous signal amplification through a self-sustaining feedback loop involving successive CHA and Cas12a trans-cleavage cycles, enhancing detection sensitivity. Via these features, the platform achieves attomolar sensitivity and excellent specificity, even distinguishing single-base miRNA variants. Direct detection of endogenous miRNA-155 in serum samples from breast cancer patients demonstrated clear differentiation from healthy controls. This strategy provides a robust molecular detection platform for the accurate and ultrasensitive detection of low-abundance miRNAs in biomedical studies.},
}
@article {pmid40745619,
year = {2025},
author = {Bezerra, AM and Leite, AB and de Souza Reis, CR and de Lemos Padilha Pitta, JL and Maran, SR and Moretti, NS and Moura, DMN and Rezende, AM},
title = {Impact on the Leishmania mexicana transcriptome due to knockout of genes encoding orthologs of methyltransferases involved in m1A and m5C mRNA modifications.},
journal = {Parasites &amp; vectors},
volume = {18},
number = {1},
pages = {315},
pmid = {40745619},
issn = {1756-3305},
support = {IBPG-1257-2.1319//Funda&#xe7;&#xe3;o de Amparo &#xe0; Ci&#xea;ncia e Tecnologia do Estado de Pernambuco/ ; Financial Code 001//Coordena&#xe7;&#xe3;o de Aperfei&#xe7;oamento de Pessoal de N&#xed;vel Superior/ ; 23/16672-0//Funda&#xe7;&#xe3;o de Amparo &#xe0; Pesquisa do Estado de S&#xe3;o Paulo/ ; 2021/13477-6//Funda&#xe7;&#xe3;o de Amparo &#xe0; Pesquisa do Estado de S&#xe3;o Paulo/ ; 2018/09948-0//Funda&#xe7;&#xe3;o de Amparo &#xe0; Pesquisa do Estado de S&#xe3;o Paulo/ ; 314103/2021-0//Conselho Nacional de Desenvolvimento Cient&#xed;fico e Tecnol&#xf3;gico/ ; 400739/2019-4//Instituto Aggeu Magalh&#xe3;es/FIOCRUZ - CNPq/ ; },
mesh = {*Leishmania mexicana/genetics/enzymology/growth &amp; development ; *Methyltransferases/genetics/metabolism ; *Transcriptome ; *RNA, Messenger/metabolism/genetics ; Protozoan Proteins/genetics/metabolism ; Gene Knockout Techniques ; CRISPR-Cas Systems ; *RNA Processing, Post-Transcriptional ; },
abstract = {BACKGROUND: Chemical modifications of mRNAs constitute an alternative mechanism for gene expression regulation, which involves proteins responsible for adding, recognizing and removing these modifications. While orthologs of enzymes involved in adding m1A (TRMT6/TRMT61A) and m5C (NSUN2) modifications are present in trypanosomatid species, a clear understanding of their biological role in these parasites is necessary.<br><br>METHODS: To shed light on this, we genetically manipulated the TRMT61A and NSUN2 protein-encoding genes in the Leishmania mexicana species using the CRISPR-Cas9 editing technique and analyzed the impact on cell growth and differentiation as well as the global gene expression profile.<br><br>RESULTS: Deletion of the genes investigated here caused changes in the normal pattern of L. mexicana differentiation, and functional analyses of differentially expressed genes in the mutants unveiled significant biological effects. For the TRMT61A gene, transcripts related to nucleotide metabolism, translation, protein folding and refolding were affected. For the NSUN2 genes, enrichment analysis indicated impacts on biological processes mostly related to nucleotide metabolism and DNA binding.<br><br>CONCLUSIONS: Our findings provide insights into the role of these methyltransferases orthologs in the regulation of trypanosomatid transcriptome, contributing to our understanding of gene expression control in this parasite.},
}
@article {pmid40745004,
year = {2025},
author = {Cheng, H and Jeong, E and Cho, SW},
title = {Applications of multiplexed CRISPR-Cas for genome engineering.},
journal = {Experimental &amp; molecular medicine},
volume = {},
number = {},
pages = {},
pmid = {40745004},
issn = {2092-6413},
abstract = {The CRISPR-Cas system has become a worldwide genome editing tool for various organisms. Its precision and efficiency have facilitated basic research, drug discovery and therapeutic interventions. In contrast to other genome editing agents, CRISPR-Cas is modulated by a short guide RNA. Due to its simplicity, CRISPR-Cas is recognized as the best candidate for multiplexed genome editing. With simultaneous targeting, efficient knockout of genes with large deletions is possible. In addition, CRISPR-Cas can induce complex structural variations, such as inversions, translocations and duplications. Moreover, by utilizing engineered CRISPR-Cas proteins specialized for direct repression or activation of gene expression, one can perform multiplexed epigenetic editing. Lastly, multiplexed targeting enables killing of specific types of cells by accumulating stress mediated by simultaneous DNA damages. Here we discuss how CRISPR-based editing technologies for multiple targets are applied in recent studies.},
}
@article {pmid40568870,
year = {2025},
author = {Narendradev, ND and Marathe, S and Baboo, S and McClatchy, DB and Diedrich, JK and Jain, P and Purwar, R and Yates, JR and Srinivasula, SM},
title = {Quantitative Proteomic Analysis Reveals JMJD6 and DNAJB11 as Endogenous Substrates of E3 Ligase RFFL.},
journal = {Journal of proteome research},
volume = {24},
number = {8},
pages = {3913-3930},
doi = {10.1021/acs.jproteome.5c00086},
pmid = {40568870},
issn = {1535-3907},
mesh = {Humans ; *Proteomics/methods ; *Ubiquitin-Protein Ligases/metabolism/genetics ; *HSP40 Heat-Shock Proteins/metabolism/genetics ; *Jumonji Domain-Containing Histone Demethylases/metabolism/genetics ; Ubiquitination ; Proteolysis ; HEK293 Cells ; Proteasome Endopeptidase Complex/metabolism ; Gene Knockout Techniques ; CRISPR-Cas Systems ; },
abstract = {The ubiquitin-proteasome system contributes to protein quality control, involving E3 ligases that ubiquitinate proteins and leading to their degradation. The dysregulation of protein degradation results in the abnormal accumulation of proteins and is implicated in the pathology of diverse diseases, making targeted protein degradation a promising therapeutic strategy. Here, we focus on RFFL, an endosome-associated RING E3 ligase involved in mitochondrial homeostasis and the clearance of misfolded cystic fibrosis transmembrane conductance regulator proteins. Using label-free quantitative mass spectrometry based proteomics for interactome and differential expression analyses, we systematically investigated and identified putative substrates of RFFL. For more confident identification, we performed these analyses on three cell lines that we generated: an RFFL knockout cell line generated using CRISPR/Cas9, another cell line rescuing RFFL expression when complemented with KO cells with stably expressing RFFL cDNA, and wild-type cells. We validated JMJD6 and DNAJB11 as substrates of endogenous RFFL, providing orthogonal validation and confidence in our screening approach. We demonstrated that RFFL ubiquitinates and degrades JMJD6 and DNAJB11 via the proteasomal pathway using in vivo assays. Interestingly, we also discovered a hitherto unknown role of RFFL in lipid metabolism. Collectively, this study provides the first comprehensive and unbiased analysis of RFFL substrates employing multiple complementary approaches.},
}
@article {pmid40462261,
year = {2025},
author = {Liu, Z and Liu, L and Liu, S and Ba, Y and Zuo, A and Xu, H and Zhang, Y and Wang, S and Wang, L and Han, X},
title = {Genome-Scale CRISPR-Cas9 Analysis Reveals Tumor Heterogeneity and Identifies NDC80 as Novel Biomarker in HCC.},
journal = {Journal of gastroenterology and hepatology},
volume = {40},
number = {8},
pages = {2078-2090},
doi = {10.1111/jgh.17016},
pmid = {40462261},
issn = {1440-1746},
support = {221100310100//Major Science and Technology Projects of Henan Province/ ; xzy022024018//Basic Scientific Research Business of Central Universities/ ; },
mesh = {Humans ; *Carcinoma, Hepatocellular/genetics/pathology/mortality ; *Liver Neoplasms/genetics/pathology/mortality ; *Biomarkers, Tumor/genetics ; *CRISPR-Cas Systems/genetics ; Prognosis ; Cell Proliferation/genetics ; Female ; Male ; Cell Line, Tumor ; Cell Movement/genetics ; *Nuclear Proteins/genetics ; Gene Expression Regulation, Neoplastic ; },
abstract = {BACKGROUND AND AIMS: Hepatocellular carcinoma (HCC) is a malignant tumor with a poor prognosis and is characterized by severe intratumoral heterogeneity. Identifying key genomic features and more reliable classifications is helpful for clinical management.<br><br>METHODS: Cancer essential genes (CEGs) were identified using genome-scale CRISPR-Cas9 and univariate Cox regression analyses. Based on gene expression, nonnegative matrix factorization (NMF) was used to generate distinct molecular subtypes. The nearest template prediction (NTP) algorithm was used to validate the accuracy and robust classifications among three independent cohorts, including GSE14520, GSE54236, and ICGC-LIRI. Specifically, potential biomarkers were screened for clinical transformation based on their prognostic characteristics and biological function features. EdU, colony formation, and Transwell assays were utilized to confirm the effect of biomarkers in vitro.<br><br>RESULTS: The C1 subtype had the worst prognosis and was characterized by advanced AJCC stages and high genomic instability. The NTP approach confirmed that the molecular subtypes were practical, robust, and reproducible. We further identified NDC80 as a gene specifically expressed in C1 subtype, indicative of prognosis solely for this subtype. Based on overrepresentation analysis (ORA), it was found that the biological function of NDC80 was mainly enriched in proliferation. In vitro cellular assays verified that promoted tumor growth and migration.<br><br>CONCLUSIONS: Our study identified three robust molecular subtypes and revealed tumor heterogeneity. Meanwhile, the potential biomarker NDC80 served as a characteristic gene of the C1 subtype, correlating with poor prognosis and promoting tumor growth and migration, providing new insights for prognostic treatment strategies in HCC.},
}
@article {pmid40327605,
year = {2025},
author = {Lee, S and Kim, K and Jeong, HJ and Choi, S and Cheng, H and Kim, D and Heo, S and Mun, J and Kim, M and Lee, E and Choi, YJ and Lee, SG and Lee, EA and Jang, Y and Lim, K and Kim, HS and Jeong, E and Myung, SJ and Jung, DB and Yu, CS and Song, IH and Corces, MR and Kang, JH and Myung, K and Kwon, T and Park, TE and Joo, J and Cho, SW},
title = {Combining Multiplexed CRISPR/Cas9-Nickase and PARP Inhibitors Efficiently and Precisely Targets Cancer Cells.},
journal = {Cancer research},
volume = {85},
number = {15},
pages = {2890-2904},
doi = {10.1158/0008-5472.CAN-24-2938},
pmid = {40327605},
issn = {1538-7445},
support = {IBS-R022-D1//Institute for Basic Science (IBS)/ ; RS-2024-00399800//National Research Foundation of Korea (NRF)/ ; RS-2023-00213043//National Research Foundation of Korea (NRF)/ ; RS-2024-00509412//National Research Foundation of Korea (NRF)/ ; RS-2023-00209822//National Research Foundation of Korea (NRF)/ ; 2021R1A6A1A03040260//National Research Foundation of Korea (NRF)/ ; HU20C0094//Ministry of Science and ICT, South Korea (MSIT)/ ; HI19C1095//Ministry of Health and Welfare (MOHW)/ ; //POSCO TJ Park Foundation (&#xd3ec;&#xc2a4;&#xcf54;&#xccad;&#xc554;&#xc7ac;&#xb2e8;)/ ; 1.240016.01//Ulsan National Institute of Science and Technology (UNIST)/ ; },
mesh = {Humans ; *Poly(ADP-ribose) Polymerase Inhibitors/pharmacology ; *CRISPR-Cas Systems ; Animals ; Mice ; *Neoplasms/genetics/drug therapy/pathology/therapy ; Xenograft Model Antitumor Assays ; RNA, Guide, CRISPR-Cas Systems/genetics ; Cell Line, Tumor ; DNA Repair/drug effects ; DNA Breaks, Double-Stranded/drug effects ; Female ; },
abstract = {UNLABELLED: Triggering cancer cell death by inducing DNA damage is the primary aim of radiotherapy; however, normal cells are also damaged. In this study, we showed that delivery of only four synthetic guide RNAs with Cas9 endonuclease efficiently induced simultaneous DNA double-strand breaks, resulting in efficient cell death in a cell type-specific manner. Off-target effects of Cas9 endonuclease were prevented by using Cas9-nickase to induce DNA single-strand breaks and blocking their repair with PARP inhibitors (PARPi). When recombinant Cas9-nickase protein and multiple synthetic guide RNAs were delivered with PARPis into cultured cells, in vivo xenografts, and patient-derived cancer organoids via lipid nanoparticles, cancer cells were unable to tolerate the induced DNA damage even in the presence of a functional BRCA2 gene. This approach has the potential to expand the use of PARPis with verified safety and thus is a potentially powerful tool for personalized genome-based anticancer therapy.<br><br>SIGNIFICANCE: Targeting cancer-specific variants with CRISPR/Cas9-nickase induces cancer-specific cell death in combination with DNA repair pathway inhibitors, demonstrating the potential of CRISPR cancer therapy for treating a broad range of cancers.},
}
@article {pmid40192983,
year = {2025},
author = {Bao, W and Fan, W and Zhang, Y and Lan, F and Ma, S},
title = {Circular CRISPR Edits Human Pluripotent Stem Cells for Disease Modeling.},
journal = {Stem cell reviews and reports},
volume = {21},
number = {5},
pages = {1512-1522},
pmid = {40192983},
issn = {2629-3277},
support = {82400587//National Natural Science Foundation of China/ ; 2022M720498//China Postdoctoral Science Foundation/ ; B2302048//Shenzhen Medical Research Fund/ ; },
mesh = {Humans ; *Gene Editing/methods ; *Pluripotent Stem Cells/metabolism/cytology ; *CRISPR-Cas Systems/genetics ; *RNA, Circular/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; },
abstract = {The CRISPR system has been widely used for human pluripotent stem cell (hPSC) disease modeling. Circular RNA can effectively reduce RNA immunogenicity and improve RNA stability, thus contributing to in vivo DNA editing. In this study, we briefly describe the process of circularizing guide RNA and CRISPR base editing elements and using them to establish stem cell disease models. Our work provides step-by-step guidance for constructing gene point editing cell lines, offering a reliable, low-immunogenic alternative for disease modeling and therapeutic research.},
}
@article {pmid40745002,
year = {2025},
author = {Park, EJ and Kim, H},
title = {Live genome imaging by CRISPR engineering: progress and problems.},
journal = {Experimental &amp; molecular medicine},
volume = {},
number = {},
pages = {},
pmid = {40745002},
issn = {2092-6413},
abstract = {CRISPR-Cas-based genome imaging opened a new era of genome visualization in living cells. While genomic loci with repetitive sequences, such as centromeres and telomeres, can be reliably imaged, applying the technique to nonrepetitive genomic loci has remained challenging. Recent advancements in the design of CRISPR RNAs and Cas proteins, the development of novel fluorophores and the combination of CRISPR-Cas with other molecular machinery amplified target-specific signals and suppressed background signals, revolutionizing this unique genome imaging technique and enabling the tracking of genomic loci with a small number of CRISPR-Cas complexes, down to a single complex. Here we review the latest advancements in CRISPR-Cas-based genome imaging techniques and their application to imaging nonrepetitive genomic loci. The challenges that these techniques are currently facing are the cellular toxicity and genomic instability induced by the expression of CRISPR-Cas and its interference with DNA metabolism, which impacts DNA replication and genome maintenance. Recently reported adverse effects of CRISPR-Cas-based genome labeling are discussed here, along with perspectives on how to overcome the problem.},
}
@article {pmid40745001,
year = {2025},
author = {Varshney, GK and Burgess, SM},
title = {CRISPR-based functional genomics tools in vertebrate models.},
journal = {Experimental &amp; molecular medicine},
volume = {},
number = {},
pages = {},
pmid = {40745001},
issn = {2092-6413},
support = {ZIAHG000183-24//U.S. Department of Health &amp; Human Services | NIH | National Human Genome Research Institute (NHGRI)/ ; R24OD034438//U.S. Department of Health &amp; Human Services | National Institutes of Health (NIH)/ ; },
abstract = {The advent of CRISPR-Cas technologies has revolutionized functional genomics by enabling precise genetic manipulations in various model organisms. In popular vertebrate models, including mice and zebrafish, CRISPR has been adapted to high-throughput mutagenesis workflows, knock-in alleles and large-scale screens, bringing us closer to understanding gene functions in development, physiology and pathology. The development of innovative technologies, such as base editors, capable of single-nucleotide modifications, and prime editors, offering precision edits without double-strand breaks, exemplifies the expanding toolkit. In addition to gene editing, transcriptional modulation, that is, CRISPR interference and CRISPR activation systems, can elucidate the mechanisms of gene regulation. Newer methods, such as MIC-Drop and Perturb-seq, which increase screening throughput in vivo, hold significant promise to improve our ability to dissect complex biological processes and mechanisms. Furthermore, CRISPR-based gene therapies for treating sickle cell disease and other monogenic diseases have already demonstrated their potential for clinical translation. Here this Review covers the transformative impact of CRISPR-based tools in vertebrate models, highlighting their utility in functional genomics research and disease modeling.},
}
@article {pmid40745000,
year = {2025},
author = {Kim, MG and Go, MJ and Kang, SH and Jeong, SH and Lim, K},
title = {Revolutionizing CRISPR technology with artificial intelligence.},
journal = {Experimental &amp; molecular medicine},
volume = {},
number = {},
pages = {},
pmid = {40745000},
issn = {2092-6413},
support = {RS-2023-00210965 and RS-2024-00339116//National Research Foundation of Korea (NRF)/ ; 2V10573 and 2E33791//Korea Institute of Science and Technology (KIST)/ ; },
abstract = {Genome engineering has made remarkable strides, evolving from DNA-binding proteins such as zinc fingers and transcription activator-like effectors to CRISPR-Cas systems. CRISPR technology has revolutionized the field through its simplicity and ability to target specific genome regions via guide RNA and Cas proteins. Progress in CRISPR tools-CRISPR nucleases, base editors and prime editors-has expanded the toolkit to induce targeted insertions or deletions, nucleotide conversions and a wider array of genetic alterations. Nevertheless, variations in editing outcomes across cell types and unintended off-target effects still present substantial hurdles. Artificial intelligence (AI), which has seen rapid advances, provides high-level solutions to these problems. By leveraging large datasets from diverse experiments, AI enhances guide RNA design, predicts off-target activities and improves editing efficiency. In addition, AI aids in discovering and designing novel CRISPR systems beyond natural limitations. These developments provide new modalities essential for the innovation of personalized therapies and help to ensure efficiency, precision and safety. Here we discuss the transformative role of AI in advancing CRISPR technology. We highlight how AI contributes to refining nuclease-based editing, base editing and prime editing. Integrating AI with CRISPR technology enhances existing tools and opens doors to next-generation medicine for gene therapy.},
}
@article {pmid40744946,
year = {2025},
author = {Lysne, DP and Stewart, MH and Susumu, K and Leski, TA and Stenger, DA and Medintz, IL and Díaz, SA and Green, CM},
title = {Quantum dot molecular beacons achieve sub-10 pM CRISPR-Cas detection in field-ready assays.},
journal = {Scientific reports},
volume = {15},
number = {1},
pages = {27950},
pmid = {40744946},
issn = {2045-2322},
mesh = {*Quantum Dots/chemistry ; *CRISPR-Cas Systems/genetics ; Fluorescence Resonance Energy Transfer/methods ; *Biosensing Techniques/methods ; Limit of Detection ; Humans ; },
abstract = {CRISPR-Cas systems have revolutionized molecular diagnostics through their specificity and programmability, yet their broad adoption is hindered by the reliance on expensive and complex instrumentation. Here, we present an optimized quantum dot (QD) molecular beacon (QD-MB) platform that integrates Förster resonance energy transfer (FRET)-based detection with CRISPR-Cas functionality, achieving sub-picomolar sensitivity without the need for target amplification. By systematically tuning components, including His-tag modifications for improved QD conjugation, nucleic acid hairpin structures for enhanced enzyme interaction, and QD surface passivation strategies, we demonstrate a two-order-of-magnitude improvement in detection sensitivity. Using LwaCas13a and RNA targets, the limit of detection (LOD) decreased to under 1 pM with plate-reader-based fluorescence measurements and below 10 pM with a lamp-and-smartphone setup, establishing the feasibility of portable, field-ready applications. This work highlights the transformative potential of QD-MBs in biosensing and sets a foundation for further advances in CRISPR-based diagnostics and nanotechnology-enabled sensing platforms.},
}
@article {pmid40744920,
year = {2025},
author = {Dib, C and Queenan, JA and Swartzrock, L and Willner, H and Denis, M and Ahmed, N and Moulana Zada, F and Borges, B and Charlesworth, CT and Lum, T and Yates, BP and Kwon, CY and Scorzo, AV and Davis, SC and Davis, JR and He, R and Xie, J and Gao, G and MacKenzie, TC and Liu, DR and Newby, GA and Czechowicz, AD},
title = {GFP-on mouse model for interrogation of in vivo gene editing.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {7017},
doi = {10.1038/s41467-025-61449-y},
pmid = {40744920},
issn = {2041-1723},
mesh = {Animals ; *Gene Editing/methods ; *Green Fluorescent Proteins/genetics/metabolism ; Dependovirus/genetics ; Mice ; CRISPR-Cas Systems ; Genetic Vectors/genetics ; Genes, Reporter ; Female ; RNA, Guide, CRISPR-Cas Systems/genetics ; Humans ; Codon, Nonsense ; },
abstract = {Gene editing technologies have revolutionized therapies for numerous genetic diseases. However, in vivo gene editing hinges on identifying efficient delivery vehicles for editing in targeted cell types, a significant hurdle in fully realizing its therapeutic potential. A model system to rapidly evaluate systemic gene editing would advance the field. Here, we develop the GFP-on reporter mouse, which harbors a nonsense mutation in a genomic EGFP sequence correctable by adenine base editor (ABE) among other genome editors. The GFP-on system was validated using single and dual adeno-associated virus (AAV9) encoding ABE8e and sgRNA. Intravenous administration of AAV9-ABE8e-sgRNA into adult GFP-on mice results in EGFP expression consistent with the tropism of AAV9. Intrahepatic delivery of AAV9-ABE8e-sgRNA into GFP-on fetal mice restores EGFP expression in AAV9-targeted organs lasting at least six months post-treatment. The GFP-on model provides an ideal platform for high-throughput evaluation of emerging gene editing tools and delivery modalities.},
}
@article {pmid40744728,
year = {2025},
author = {Sabin, CE and Lauderdale, JD and Menke, DB},
title = {Anolis Lizards as a Model System for Studies of Gene Function in Reptile Development and Evolution.},
journal = {Cold Spring Harbor protocols},
volume = {},
number = {},
pages = {},
doi = {10.1101/pdb.top108535},
pmid = {40744728},
issn = {1559-6095},
abstract = {Anolis lizards are an ecologically diverse group that includes more than 400 described species. These reptiles have been the subject of wide-ranging studies, from speciation and convergent evolution to climate adaptation and tail regeneration. While CRISPR-based gene editing has tremendous potential to reveal new insights into these and other aspects of Anolis biology, the reproductive biology of these reptiles has presented significant barriers to gene editing. Here, we briefly summarize gene editing approaches in vertebrates and discuss some of the major challenges associated with the performance of gene editing in anoles. We then introduce a recently established surgical procedure that enables the injection of CRISPR-Cas into the developing oocytes of female lizards. This approach circumvents the need to manipulate early-stage embryos and permits the production of gene-edited anoles. This method has recently been successfully adapted for use in other reptiles, suggesting that it may be effective in a wide range of species and will broadly enable studies of gene function in reptiles.},
}
@article {pmid40744574,
year = {2025},
author = {Zhang, Z and Wu, S and Long, Y and Huang, W and Bramlett, M and Yang, Y and Wu, Y},
title = {Identification of SfABCC2 as the critical receptor for Cry1Fa and Cry1Ab in Spodoptera frugiperda via CRISPR-mediated gene knockouts.},
journal = {Pesticide biochemistry and physiology},
volume = {213},
number = {},
pages = {106526},
doi = {10.1016/j.pestbp.2025.106526},
pmid = {40744574},
issn = {1095-9939},
mesh = {Animals ; *Spodoptera/genetics/metabolism/drug effects ; *Bacillus thuringiensis Toxins ; *Endotoxins/metabolism/pharmacology ; *Hemolysin Proteins/metabolism/pharmacology ; *Bacterial Proteins/metabolism/pharmacology ; Gene Knockout Techniques ; CRISPR-Cas Systems ; *Insect Proteins/genetics/metabolism ; Insecticide Resistance/genetics ; Insecticides/pharmacology ; Bacillus thuringiensis ; },
abstract = {Insecticidal proteins from Bacillus thuringiensis (Bt) have been widely used to control major agricultural pests through genetically modified (GM) Bt crops. However, the development of resistance in target pests could undermine the effectiveness of Bt crops. Understanding the mechanisms of action of Bt insecticidal proteins and the resistance mechanisms in pests is crucial for developing effective resistance management strategies to sustain the use of Bt crops. In this study, CRISPR/Cas9 gene editing was used to investigate the functional roles of four genes (SfABCC2, SfABCC3, SfCad1, and SfAPN1) that encode putative receptors for Cry1 proteins in the fall armyworm, Spodoptera frugiperda, a globally significant pest. We created five homozygous knockouts, each with a substantial fragment deletion: SfCad1-KO, SfAPN1-KO, SfABCC2-KO, SfABCC3-KO, and SfC2/C3-KO (a double knockout of SfABCC2 and SfABCC3). Bioassay results revealed that SfCad1-KO, SfAPN1-KO, and SfABCC3-KO strains exhibited no resistance to Cry1Fa or Cry1Ab. In contrast, SfABCC2-KO and SfC2/C3-KO strains demonstrated high levels of resistance to Cry1Fa (>3300-fold) and Cry1Ab (>450-fold), demonstrating that SfABCC2 is pivotal to the insecticidal action of these two Bt proteins. As anticipated, all five knockouts generated in this study did not significantly impact susceptibility to Vip3Aa compared with the control strain. Our findings underscore the critical role of SfABCC2 in mediating Cry1Ab and Cry1Fa toxicity in S. frugiperda. Therefore, resistance monitoring program and resistance management tactics should focus on SfABCC2 mutations in field populations of S. frugiperda.},
}
@article {pmid40744495,
year = {2025},
author = {Sokirniy, I and Inam, H and Tomaszkiewicz, M and Reynolds, J and McCandlish, D and Pritchard, J},
title = {A side-by-side comparison of variant function measurements using deep mutational scanning and base editing.},
journal = {Nucleic acids research},
volume = {53},
number = {14},
pages = {},
doi = {10.1093/nar/gkaf738},
pmid = {40744495},
issn = {1362-4962},
support = {2141650//NSF/ ; T32GM108563/GF/NIH HHS/United States ; R35 GM133613/GF/NIH HHS/United States ; U01CA265709/BC/NCI NIH HHS/United States ; 2033673//NSF RECODE/ ; //Simons Center for Quantitative Biology at Cold Spring Harbor Laboratory/ ; },
mesh = {Humans ; *Gene Editing/methods ; RNA, Guide, CRISPR-Cas Systems/genetics ; High-Throughput Nucleotide Sequencing/methods ; *Mutation ; DNA, Complementary/genetics ; CRISPR-Cas Systems ; Molecular Sequence Annotation/methods ; Genetic Variation ; Genomics/methods ; Gene Library ; },
abstract = {Variant annotation is a crucial objective in mammalian functional genomics. Deep mutational scanning (DMS) using saturation libraries of complementary DNAs (cDNAs) is a well-established method for annotating human gene variants, but CRISPR base editing (BE) is emerging as an alternative. However, questions remain about how well high-throughput BE measurements can annotate variant function and the extent of downstream experimental validation required. This study is the first direct comparison of cDNA DMS and BE in the same lab and cell line. We focus on how well short guide RNA (sgRNA) depletion or enrichment is explained by the predicted edits within the editing "window" defined by the sgRNA. The most likely predicted edits enhance the agreement between a "gold standard" DMS dataset and a BE screen. A simple filter for sgRNAs making single edits in their window could sufficiently annotate a large proportion of variants directly from sgRNA sequencing of large pools. When multi-edit guides are unavoidable, directly measuring edits in medium-sized validation pools can recover high-quality variant annotation data. Our data show a surprisingly high degree of correlation between base editor data and gold standard DMS. We suggest that the main variable measured in base editor screens is the desired base edits.},
}
@article {pmid40744490,
year = {2025},
author = {Cattle, MA and Aguado, LC and Sze, S and Venkittu, S and Wang, Y and Papagiannakopoulos, T and Smith, S and Rice, CM and Schneider, WM and Poirier, JT},
title = {An enhanced Eco1 retron editor enables precision genome engineering in human cells without double-strand breaks.},
journal = {Nucleic acids research},
volume = {53},
number = {14},
pages = {},
doi = {10.1093/nar/gkaf716},
pmid = {40744490},
issn = {1362-4962},
support = {P30CA016087//NYU Laura &amp; Isaac Perlmutter Cancer Center/ ; P30CA016087//NYU Laura &amp; Isaac Perlmutter Cancer Center/ ; //Robertson Foundation/ ; R35GM149355/GM/NIGMS NIH HHS/United States ; },
mesh = {Humans ; *Gene Editing/methods ; CRISPR-Cas Systems/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics/metabolism ; DNA Breaks, Double-Stranded ; Zika Virus/genetics ; *Retroelements/genetics ; 3' Untranslated Regions/genetics ; HEK293 Cells ; RNA, Untranslated/genetics/metabolism ; CRISPR-Associated Protein 9/metabolism/genetics ; Genome, Human ; },
abstract = {Retrons are a retroelement class found in diverse prokaryotes that can be adapted to augment CRISPR-Cas9 genome engineering technology to efficiently rewrite short stretches of genetic information in bacteria and yeast. However, efficiency in human cells has been limited by unknown factors. We identified non-coding RNA (ncRNA) instability and impaired Cas9 activity due to 5' sgRNA extension as key contributors to low retron editor efficiency in human cells. We re-engineered the Eco1 ncRNA to incorporate an exoribonuclease-resistant RNA pseudoknot from the Zika virus 3' UTR and devised an RNA processing strategy using Csy4 ribonuclease to minimize 5' sgRNA extension. This strategy increased steady-state ncRNA levels and rescued sgRNA activity, leading to increased templated repair. This work reveals a previously unappreciated role for ncRNA stability in retron editor efficiency in human cells and presents an enhanced Eco1 retron editor capable of precise genome editing in human cells from a single integrated lentivirus and, in the context of the nCas9 H840A nickase, without creating double-strand breaks.},
}
@article {pmid40743876,
year = {2025},
author = {Gardoul, M and Rached, B and Mbarki, A and Ajdig, M and Belouad, EM and Chouati, T and Chauiyakh, O and Lahcen Ouchari, and Harlé, O and Al-Nakeeb, K and Melloul, M and El Fahime, E},
title = {Comprehensive whole-genome analysis of Streptococcus infantarius strains from Moroccan farmhouse dairy products: Genomic insights into dairy adaptation, safety, and biotechnological potential.},
journal = {International journal of food microbiology},
volume = {442},
number = {},
pages = {111358},
doi = {10.1016/j.ijfoodmicro.2025.111358},
pmid = {40743876},
issn = {1879-3460},
abstract = {Streptococcus infantarius, a lactic acid bacterium prevalent in Moroccan dairy products, holds significant probiotic and biotechnological potential, yet its genomic features remain poorly characterized. This study aims to provide a comprehensive genomic characterization of six S. infantarius isolates (B50-6, B50-7, B51-6, O53-2, T41-8, K85-8) from Moroccan farmhouse dairy products. Whole-genome sequencing and comparative analysis were conducted using advanced bioinformatics tools to assess their genetic diversity, functional attributes, adaptive mechanisms, probiotic properties, safety profile, and potential applications in food fermentation. All strains were confirmed as S. infantarius with average nucleotide identity (ANI) >98.2 % and digital DNA-DNA hybridization (dDDH) >85.9 %. Genomes sizes ranging from 1.81 (K85-8) to 1.91 (T41-8) Mb, with GC content ∼37.4 %. Functional profiling identified 1343, 1343, 1345, 1342, 1359 and 1345 proteins with functional assignments; 519, 521, 536, 520, 542 and 502 hypothetical proteins; and 1752, 1754, 1778, 1752, 1781 and 1740 proteins with COG (Clusters of Orthologous Groups) assignments, in B50-6, B50-7, B51-6, O53-2, T41-8 and K85-8, respectively. Gene enrichment analysis highlighted key metabolic pathways and functional categories relevant to dairy adaptation and biotechnological potential. The total number of genes encoding carbohydrate-active enzymes was 45 (B50-6), 45 (B50-7), 48 (B51-6), 45 (O53-2), 48 (T41-8) and 48 (K85-8). Safety assessments identified 13 (K85-8) to 16 (B51-6) virulence-associated genes per strain. However, no antimicrobial resistance genes or plasmids were detected, while the presence of CRISPR-Cas systems was detected in most strains. A total of 88 mobile genetic elements were identified ranging from 9 (B51-6) to 18 (O53-2) per strain. In addition, prophages were detected in all strains with the exception of T41-8 and K85-8. All Strains exhibited diverse biosynthetic gene clusters (BGCs), including RiPP-like, T3PKS and terpene precursor pathways. These findings position S. infantarius as a relatively stable core genome, metabolically versatile candidate for dairy fermentation, probiotic and biotechnological applications.},
}
@article {pmid40742466,
year = {2025},
author = {Zhang, K and Wang, D and Hu, S and Li, X and Hong, J},
title = {Gene disruption via a transient hypercompact CRISPR-AsCas12f1 system in Kluyveromyces marxianus.},
journal = {Biotechnology letters},
volume = {47},
number = {4},
pages = {84},
pmid = {40742466},
issn = {1573-6776},
support = {202423m10050005//Anhui Provincial Science and Technology Innovation Program/ ; 231100110300//Major Special Science and Technology Project of Henan Province/ ; 32071469//National Natural Science Foundation of China/ ; },
mesh = {*Kluyveromyces/genetics ; *CRISPR-Cas Systems ; *Gene Knockout Techniques/methods ; RNA, Guide, CRISPR-Cas Systems/genetics ; Gene Editing/methods ; *CRISPR-Associated Proteins/genetics/metabolism ; RNA, Transfer/genetics ; },
abstract = {Kluyveromyces marxianus is an attractive chassis for microbial cell factories due to its rapid growth, thermotolerance, and wide substrate spectrum. However, gene disruption in this organism is challenging primarily due the prevalence of dominant nonhomologous recombination. AsCas12f1, a hypercompact CRISPR-associated protein consisting of 422 amino acids-approximately one-third the size of Cas9 or Cas12a-enables more efficient packaging into delivery vehicles than its larger counterparts. In this study, a gene disruption method using AsCas12f1 was established in K. marxianus through a transient targeting strategy. The integration of tRNA-gRNA into the gRNA construct increased gene disruption efficiency. Additionally, disrupting KmKU70 or KmLIG4 further increased this efficiency, achieving nearly 100%. By combining the disruption of KmKU70 with the AsCas12f1 system, the length of the homologous arm was shortened to 200 bp while maintaining a disruption efficiency of 87.5%. The implementation of the gRNA-tRNA-array system resulted in the successful generation of three single-gene knockout strains from a single transformation, resulting an overall efficiency of 86.4%. This approach leverages the transient transformation of fragments, eliminates the need for extensive time investment in constructing gRNA expression vectors and negates the requirement for the removal of the CRISPR-AsCas12f1 system after gene disruption. This study presents a novel strategy for gene disruption in K. marxianus and demonstrates the applicability of Cas12f in yeast systems.},
}
@article {pmid40741393,
year = {2025},
author = {Wei, J and Motawaa, M and Li, Y},
title = {Thermus thermophilus CRISPR Cas6 Heterologous Expression and Purification.},
journal = {Bio-protocol},
volume = {15},
number = {14},
pages = {e5382},
pmid = {40741393},
issn = {2331-8325},
abstract = {The CRISPR-Cas system of Thermus thermophilus has emerged as a potent biotechnological tool, particularly its Cas6 endonuclease, which plays a crucial role in CRISPR RNA (crRNA) maturation. This protocol details a robust and reproducible method for the high-level expression and purification of recombinant T. thermophilus Cas6 proteins (Cas6-1 and Cas6-2) in E. coli. We describe a streamlined approach encompassing plasmid construction using seamless assembly, optimized bacterial heterologous expression, and multi-step purification leveraging affinity and size-exclusion chromatography. The protocol outlines the generation of both His-tagged and GST-tagged Cas6 variants, enabling flexibility in downstream applications. Key steps, including primer design, PCR optimization, competent cell transformation, and chromatography strategies, are meticulously detailed with critical parameters and troubleshooting guidance to ensure experimental success and high yields of highly pure and active T. thermophilus Cas6 proteins. This protocol is useful for researchers requiring purified T. thermophilus Cas6 for structural studies, biochemical characterization, and the development of CRISPR-based biotechnological tools. Key features • Robust method for expressing and purifying Thermus thermophilus Cas6 proteins in E. coli. • Seamless assembly cloning and dual affinity tagging system: Offers options for both His-tag and GST-tag purification strategies for increased versatility. • Applicable for diverse heterologous expression and purification of well-folding thermostable proteins in mesophilic host chassis cells [E. coli BL21(DE3)].},
}
@article {pmid40739342,
year = {2025},
author = {Ruffolo, JA and Nayfach, S and Gallagher, J and Bhatnagar, A and Beazer, J and Hussain, R and Russ, J and Yip, J and Hill, E and Pacesa, M and Meeske, AJ and Cameron, P and Madani, A},
title = {Design of highly functional genome editors by modelling CRISPR-Cas sequences.},
journal = {Nature},
volume = {},
number = {},
pages = {},
pmid = {40739342},
issn = {1476-4687},
abstract = {Gene editing has the potential to solve fundamental challenges in agriculture, biotechnology and human health. CRISPR-based gene editors derived from microorganisms, although powerful, often show notable functional tradeoffs when ported into non-native environments, such as human cells[1]. Artificial-intelligence-enabled design provides a powerful alternative with the potential to bypass evolutionary constraints and generate editors with optimal properties. Here, using large language models[2] trained on biological diversity at scale, we demonstrate successful precision editing of the human genome with a programmable gene editor designed with artificial intelligence. To achieve this goal, we curated a dataset of more than 1 million CRISPR operons through systematic mining of 26 terabases of assembled genomes and metagenomes. We demonstrate the capacity of our models by generating 4.8× the number of protein clusters across CRISPR-Cas families found in nature and tailoring single-guide RNA sequences for Cas9-like effector proteins. Several of the generated gene editors show comparable or improved activity and specificity relative to SpCas9, the prototypical gene editing effector, while being 400 mutations away in sequence. Finally, we demonstrate that an artificial-intelligence-generated gene editor, denoted as OpenCRISPR-1, exhibits compatibility with base editing. We release OpenCRISPR-1 to facilitate broad, ethical use across research and commercial applications.},
}
@article {pmid40739316,
year = {2025},
author = {Callahan, A and Mojumdar, A and Hu, M and Wang, A and Griffith, AA and Huang, N and Chua, XY and Mroz, N and Puterbaugh, RZ and Reilly, SP and Salomon, A},
title = {The phosphatases TCPTP, PTPN22, and SHP1 play unique roles in T cell phosphotyrosine maintenance and feedback regulation of the TCR.},
journal = {Scientific reports},
volume = {15},
number = {1},
pages = {27747},
doi = {10.1038/s41598-025-12951-2},
pmid = {40739316},
issn = {2045-2322},
support = {R01AI083636/NH/NIH HHS/United States ; R01AI083636/NH/NIH HHS/United States ; R01AI083636/NH/NIH HHS/United States ; R01AI083636/NH/NIH HHS/United States ; R01AI083636/NH/NIH HHS/United States ; R01AI083636/NH/NIH HHS/United States ; R01AI083636/NH/NIH HHS/United States ; R01AI083636/NH/NIH HHS/United States ; R01AI083636/NH/NIH HHS/United States ; },
mesh = {Humans ; *Receptors, Antigen, T-Cell/metabolism ; *Protein Tyrosine Phosphatase, Non-Receptor Type 6/metabolism/genetics ; Jurkat Cells ; *T-Lymphocytes/metabolism/immunology ; *Protein Tyrosine Phosphatase, Non-Receptor Type 22/metabolism/genetics ; *Phosphotyrosine/metabolism ; Feedback, Physiological ; Signal Transduction ; CRISPR-Cas Systems ; Protein Tyrosine Phosphatase, Non-Receptor Type 1 ; },
abstract = {The protein tyrosine phosphatases (PTPs) TCPTP, PTPN22, and SHP1 are critical regulators of the activating phosphotyrosine (pY) site on the initiating T cell kinase, Lck[Y394]. Still, the broader implications of these phosphatases in T cell receptor (TCR) signalling and T cell biology remain unclear. By combining CRISPR/Cas9 gene editing and mass spectrometry, we evaluate the protein- and pY-level effects of TCPTP, PTPN22, and SHP1 in the Jurkat T cell model system. We find that deletion of each phosphatase corresponds to unique changes in the proteome of T cells, with few large-scale changes to TCR signalling proteins. Notably, PTPN22 and SHP1 deletions have opposing effects on pY abundance globally, while TCPTP deletion modestly elevates pY levels. Finally, we show that TCPTP is indirectly involved in Erk1/2 positive feedback to the TCR. Overall, our work provides evidence for alternative functions of three T cell phosphatases long thought to be redundant.},
}
@article {pmid40738762,
year = {2025},
author = {Gupta, PK and Kumar, S},
title = {Third-generation novel technologies for gene editing.},
journal = {Trends in biotechnology},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.tibtech.2025.07.012},
pmid = {40738762},
issn = {1879-3096},
abstract = {Gene editing technologies have revolutionized the field of biotechnology. CRISPR-Cas methods using RNA-guided enzymes are the most used gene editing tools and have produced gene-edited crops (rice, wheat, corn, etc.) and human therapeutics (Casgevy, approved for commercial use; Vertex Pharmaceuticals). However, these systems have some limitations, including the requirement of a protospacer adjacent motif sequence, generation of undesirable double-strand breaks (DSBs), and the inability to edit long genomic segments. Some of these limitations were partially addressed by the development of second-generation editors, including base editors (BEs) and prime editors (PEs). Third-generation gene editing technologies such as seekRNA and bridgeRNA can overcome most of these limitations and are the subject of this review.},
}
@article {pmid40690516,
year = {2025},
author = {Shigenobu, S and Yoda, S and Ohsawa, S and Suzuki, M},
title = {Refined CRISPR/Cas9 genome editing in the pea aphid uncovers the essential roles of Laccase2 in overwintering egg adaptation.},
journal = {PLoS genetics},
volume = {21},
number = {7},
pages = {e1011557},
doi = {10.1371/journal.pgen.1011557},
pmid = {40690516},
issn = {1553-7404},
mesh = {Animals ; *CRISPR-Cas Systems/genetics ; Gene Editing/methods ; *Aphids/genetics/physiology ; *Laccase/genetics/metabolism ; Pigmentation/genetics ; *Insect Proteins/genetics ; *Adaptation, Physiological/genetics ; Ovum ; Female ; Seasons ; Diapause/genetics ; Gene Knockout Techniques ; },
abstract = {The production of overwintering eggs is a critical adaptation for winter survival among many insects. Melanization contributes to eggshell pigmentation and hardening, consequently enhancing resistance to environmental stress. The complex life cycle of the pea aphid (Acyrthosiphon pisum), a model hemipteran insect with remarkable reproductive capacity, involves cyclical parthenogenesis. It enables the production of black overwintering eggs that undergo obligate diapause to survive under unfavorable conditions. Laccase2 (Lac2) is essential for cuticle sclerotization and pigmentation in other insects. We hypothesized that Lac2 plays a critical role in aphid eggshell pigmentation and survival during diapause. To test the hypothesis, we used CRISPR/Cas9 ribonucleoprotein microinjections and a novel Direct Parental CRISPR (DIPA-CRISPR) method to knockout Lac2. In Lac2 knockout (KO) crispants (G0), pigment-less eggs correlated with induced indel rates. Additionally, eggshell pigmentation was completely lost in homozygous Lac2 knockouts, leading to embryonic lethality. Observation of late-stage embryos in KO diapause eggs suggested that lethality occurred during late embryogenesis or hatching. Furthermore, eggshell stiffness was significantly reduced in Lac2 KOs, highlighting the role of this gene in eggshell hardening. Moreover, fungal growth was observed in KO eggs. These findings reveal the essential roles of Lac2 in eggshell pigmentation, hardening, late embryonic development, hatching, and fungal protection, which are critical for pea aphid survival during overwintering diapause. This study also advances CRISPR/Cas9-mediated genome editing in pea aphids by addressing the challenges associated with their unique biology, including complex life cycles, obligatory diapause, bacterial endosymbiosis, inbreeding depression, and high nuclease activity. Our optimized protocol achieved efficient targeted mutagenesis and germline transmission, thereby generating stable KO lines. Additionally, we successfully applied DIPA-CRISPR to aphids by inducing mutations via adult oviparous female injections in fertilized eggs. These robust genome-editing protocols will facilitate functional studies in aphids, a key model for research on evolution, ecology, development, and agriculture.},
}
@article {pmid40666904,
year = {2025},
author = {Pindi, C and Ahsan, M and Sinha, S and Palermo, G},
title = {Graph Attention Neural Networks Reveal TnsC Filament Assembly in a CRISPR-Associated Transposon.},
journal = {bioRxiv : the preprint server for biology},
volume = {},
number = {},
pages = {},
pmid = {40666904},
issn = {2692-8205},
abstract = {CRISPR-associated transposons (CAST) enable programmable, RNA-guided DNA integration, marking a transformative advancement in genome engineering. A central player in the type V-K CAST system is the AAA+ ATPase TnsC, which assembles into helical filaments on double-stranded DNA (dsDNA) to orchestrate target site recognition and transposition. Despite its essential role, the molecular mechanisms underlying TnsC filament nucleation and elongation remain poorly understood. Here, multiple-microsecond and free energy simulations are combined with deep learning-based Graph Attention Network (GAT) models to elucidate the mechanistic principles of TnsC filament formation and growth. Our findings reveal that ATP binding promotes TnsC nucleation by inducing DNA remodelling and stabilizing key protein-DNA interactions, particularly through conserved residues in the initiator-specific motif (ISM). Furthermore, GNN-based attention analyses identify a directional bias in filament elongation in the 5'→3' direction and uncover a dynamic compensation mechanism between incoming and bound monomers that facilitate directional growth along dsDNA. By leveraging deep learning-based graph representations, our GAT model provides interpretable mechanistic insights from complex molecular simulations and is readily adaptable to a wide range of biological systems. Altogether, these findings establish a mechanistic framework for TnsC filament dynamics and directional elongation, advancing the rational design of CAST systems with enhanced precision and efficiency.},
}
@article {pmid40570715,
year = {2026},
author = {Fang, T and Deng, Y and Chen, M and Luo, T and Ning, T and Chen, G},
title = {Nanoparticles-mediated intratumoral gene editing targeting PD-L1 and Galectin-9 for improved cancer immunotherapy.},
journal = {Biomaterials},
volume = {324},
number = {},
pages = {123511},
doi = {10.1016/j.biomaterials.2025.123511},
pmid = {40570715},
issn = {1878-5905},
mesh = {*Galectins/genetics ; Animals ; *Nanoparticles/chemistry ; *B7-H1 Antigen/genetics ; *Immunotherapy/methods ; Mice ; *Gene Editing/methods ; Humans ; Calcium Phosphates/chemistry ; Cell Line, Tumor ; *Neoplasms/therapy/immunology/genetics ; CRISPR-Cas Systems/genetics ; Female ; Mice, Inbred C57BL ; },
abstract = {PD-L1, a typical immune checkpoint expressed on tumor cells, reduces the effectiveness of T cell-mediated killing, which is further aggravated by Galectin-9 (Gal-9) co-expression through the TIM3/Gal-9 pathway. Although immune checkpoint inhibitors have shown promise in cancer therapy, limitations remain including low response rate, systemic toxicities, and the need of frequent treatments. Here, we described a dual knockout approach targeting PD-L1 and Gal-9 in tumor cells, achieved by nanoparticle-assisted CRISPR-Cas9 delivery, aimed at improved cancer immunotherapy. A calcium phosphate nanoparticle (CaP NP) was engineered for co-delivery of CRISPR-Cas9/sgRNA ribonucleoprotein (RNP) and initiation of anti-tumor immunity. Intratumoral administration of RNP-loaded CaP NPs effectively knocked out PD-L1 and Gal-9 in tumor cells, evoking robust anti-tumor immunity. Additionally, Ca[2+] overload due to the degradation of CaP NPs led to release of damage-associated molecular patterns (DAMPs) signals, further enhancing T-cell-mediated antitumor immune responses. Our results demonstrated that this treatment effectively evoked both local and systemic anti-tumor immune responses, significantly inhibiting the growth of primary and distant tumors in mouse models. Importantly, local treatment also altered the phenotypes of circulating tumor cells, as a substantial of circulating tumor cells originated from RNP-CaP-treated primary tumors and exhibited dual knockouts, which led to reduced lung metastasis.},
}
@article {pmid40550345,
year = {2025},
author = {Bedir, I and Ozturk, K and Telci, D},
title = {Impact of PLA2G2A rs11573156 C &gt; G Variant on Phospholipase Expression and Metastatic Behavior in Prostate Cancer.},
journal = {Gene},
volume = {964},
number = {},
pages = {149641},
doi = {10.1016/j.gene.2025.149641},
pmid = {40550345},
issn = {1879-0038},
mesh = {Male ; Humans ; *Prostatic Neoplasms/genetics/pathology ; *Group II Phospholipases A2/genetics/metabolism ; Neoplasm Metastasis ; Epithelial-Mesenchymal Transition/genetics ; Gene Expression Regulation, Neoplastic ; PC-3 Cells ; *Polymorphism, Single Nucleotide ; Cell Line, Tumor ; Cell Movement/genetics ; CRISPR-Cas Systems ; Alleles ; },
abstract = {Prostate cancer (PCa) is a major global health concern and a leading cause of cancer-related deaths in men. Despite available treatments, PCa frequently recurs and exhibits high metastatic potential. One key factor in its malignancy is PLA2G2A, a secretory phospholipase A2 enzyme with strong inflammatory properties. Elevated PLA2G2A levels in the bloodstream have been linked to tumor grade, potentially due to the rs11573156 C > G polymorphism in the gene's 5' untranslated region (5'UTR). Previously, we demonstrated that individuals carrying the G allele have a 2.5-fold lower risk of developing metastatic PCa compared to those with the C allele. To further investigate the functional impact of this polymorphism, we employed CRISPR-Cas9 base editing to convert the GC genotype of metastatic PC-3 prostate cancer cells into the homozygous GG genotype. Our findings indicate that the G allele enhances PLA2G2A expression while downregulating genes associated with epithelial-to-mesenchymal transition (EMT), an effect reversed upon siRNA-mediated PLA2G2A silencing. Furthermore, this genetic alteration impaired the migratory capacity of PC-3 cells following interferon-γ (IFN-γ) priming, simulating an inflammatory tumor microenvironment. Notably, siRNA-induced downregulation of PLA2G2A reversed the anti-migratory effects associated with the GG genotype. Our results suggest that increased PLA2G2A expression, driven by the G allele, negatively affects cell proliferation, EMT, and metastatic properties, underscoring its potential anti-tumor role in PCa.},
}
@article {pmid38489095,
year = {2025},
author = {Yu, S and Lei, X and Qu, C},
title = {MicroRNA Sensors Based on CRISPR/Cas12a Technologies: Evolution From Indirect to Direct Detection.},
journal = {Critical reviews in analytical chemistry},
volume = {55},
number = {5},
pages = {968-984},
doi = {10.1080/10408347.2024.2329229},
pmid = {38489095},
issn = {1547-6510},
mesh = {*MicroRNAs/analysis/genetics ; *CRISPR-Cas Systems/genetics ; *Biosensing Techniques/methods ; Humans ; },
abstract = {MicroRNA (miRNA) has emerged as a promising biomarker for disease diagnosis and a potential therapeutic targets for drug development. The detection of miRNA can serve as a noninvasive tool in diseases diagnosis and predicting diseases prognosis. CRISPR/Cas12a system has great potential in nucleic acid detection due to its high sensitivity and specificity, which has been developed to be a versatile tool for nucleic acid-based detection of targets in various fields. However, conversion from RNA to DNA with or without amplification operation is necessary for miRNA detection based on CRISPR/Cas12a system, because dsDNA containing PAM sequence or ssDNA is traditionally considered as the activator of Cas12a. Until recently, direct detection of miRNA by CRISPR/Cas12a system has been reported. In this review, we provide an overview of the evolution of biosensors based on CRISPR/Cas12a for miRNA detection from indirect to direct, which would be beneficial to the development of CRISPR/Cas12a-based sensors with better performance for direct detection of miRNA.},
}
@article {pmid40738541,
year = {2025},
author = {Choudhury, PR and Chakravarti, M and Banerjee, S and Baral, R and Bose, A},
title = {B-cell editing: An emerging alternative of precision oncotherapy.},
journal = {Advances in immunology},
volume = {166},
number = {},
pages = {103-135},
doi = {10.1016/bs.ai.2024.10.002},
pmid = {40738541},
issn = {1557-8445},
mesh = {Humans ; *Precision Medicine/methods ; *B-Lymphocytes/immunology/metabolism ; *Neoplasms/therapy/immunology/genetics ; *Gene Editing/methods ; Animals ; *Immunotherapy/methods ; *Cancer Vaccines/immunology/therapeutic use ; CRISPR-Cas Systems ; Antibodies, Monoclonal/therapeutic use/immunology ; },
abstract = {Lately, the urgency of precision medicine in cancer care through immunotherapy has reformed the arena of oncology. Although immunomodulatory therapeutics in cancer have been preliminarily concentrated on T-cells, emerging evidences have suggested that intra-tumoral B-cells and plasma cells have significant contributions in cancer prognosis primarily through the production of antibodies. B-cell oriented cancer vaccines have been used in early clinical trials of breast and other cancers after multiple preclinical studies. Passive immunotherapy via administration of monoclonal antibodies (mAbs) and emergence of anti-idiotypic antibodies have led to considerable advancement in oncotherapy. Endogenous production of mAbs would be of significant benefit in recurrent or residual malignancies and permanent infusion would help in the overcoming of issues related to pharmacodynamic variations observed in case of intravenous inoculations of bi or tri specific mAbs. This has directed towards the development of genome reprogrammed B-cells with the capability of yielding therapeutic mAbs independently. Genetic alteration through clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) nucleases have enabled the introduction of transgenes into B-cell genome thereby stimulating the plasma cells to produce exogenous remedial antibodies. It also facilitates ex vivo B-cell editing to elevate specificities of antigen receptors and generate target specific antibody responses which cannot normally be evoked in patient's immune system. Hence, genome-altered B-cells possess the potential of engineered therapeutics against certain malignancies. Co-operation of B-cells in T-cell based vaccines are ultimate need for vaccine success. In this chapter, the mechanisms, challenges and potential advantages of B-cell editing in cancer immune therapy shall be explored. The prospects of B-cell editing in onco-therapy will be clearly elucidated with all its strength and weaknesses.},
}
@article {pmid40737091,
year = {2025},
author = {Jain, S and Planells, J and Regadas, I and Barrett, D and von Euler, A and Sinha, I and Lindberg, BG and Hesson, JC and Swacha, P and Gekara, NO and Pelechano, V and Engström, Y and Mannervik, M and Visa, N},
title = {A U3 snoRNA is required for the regulation of chromatin dynamics and antiviral response in Drosophila melanogaster.},
journal = {Nucleic acids research},
volume = {53},
number = {14},
pages = {},
doi = {10.1093/nar/gkaf715},
pmid = {40737091},
issn = {1362-4962},
support = {2019-02335//Swedish Research Council/ ; 2020-01480//Swedish Research Council/ ; 2021-06112//Swedish Research Council/ ; KAW 2021.0167//Wallenberg Academy Fellowship/ ; //Karolinska Institute/ ; 2024-04173//Swedish Research Council/ ; 20 1044 Pj//The Swedish Cancer Society/ ; 23 2963 Pj//The Swedish Cancer Society/ ; 2022-01308_3//Swedish Research Council/ ; CAN 23 3096 Pj//The Swedish Cancer Society/ ; //Stockholm University/ ; //Stockholm University/ ; },
mesh = {Animals ; *Drosophila melanogaster/genetics/virology/immunology ; *RNA, Small Nucleolar/genetics/metabolism ; *Chromatin/genetics/metabolism ; Drosophila Proteins/genetics/metabolism ; Sindbis Virus/immunology/genetics ; Chromatin Assembly and Disassembly/genetics ; Larva/genetics/virology ; CRISPR-Cas Systems ; Trans-Activators ; Cell Cycle Proteins ; },
abstract = {Small nucleolar RNAs (snoRNAs) are prevailing components of the chromatin-associated transcriptome. Here we show that specific snoRNAs are required for the activation of immune response genes and for survival during viral infections in Drosophila melanogaster. We have studied snoRNA:U3:9B, a chromatin-associated snoRNA that binds to a large number of protein coding genes, including immune response genes. We have used CRISPR/Cas9 to delete snoRNA:U3:9B and study its function in vivo. SnoRNA:U3:9B-deficient larvae are viable but failed to develop into pupae when challenged by expression of a Sindbis virus replicon. SnoRNA:U3:9B is localized to immune genes in vivo and the chromatin decompaction and gene activation typically observed at immune genes following infection are abolished in snoRNA:U3:9B-deficient larvae, which suggests that this snoRNA acts locally to regulate chromatin accessibility. Mechanistically, snoRNA:U3:9B is required for the recruitment of the chromatin remodeler Brahma to a set of target immune genes. In summary, these results uncover an antiviral defense mechanism that relies on a snoRNA for the recruitment of a chromatin remodeling factor to immune genes to facilitate immune gene activation.},
}
@article {pmid40736862,
year = {2025},
author = {Mercer, GD and Ballios, BG and Kertes, PJ},
title = {Mutation Specific Treatments for Inherited Retinal Diseases.},
journal = {Advances in experimental medicine and biology},
volume = {1467},
number = {},
pages = {337-341},
pmid = {40736862},
issn = {0065-2598},
mesh = {Humans ; *Genetic Therapy/methods ; *Retinal Diseases/genetics/therapy ; *Gene Editing/methods ; *Mutation ; CRISPR-Cas Systems ; Animals ; Oligonucleotides, Antisense/therapeutic use/genetics ; RNA Interference ; RNA Editing ; },
abstract = {The next frontier in genetic therapy for IRDs is the correction or silencing of specific mutations. This is of relevance to conditions with dominant negative and gain-of-function disease mechanisms, or with causative genes that are too large for gene replacement using AAV vectors. We discuss two approaches that have reached the stage of human clinical trials: CRISPR-Cas9 based gene editing and post-transcriptional gene silencing using antisense oligonucleotides. Other mutation-specific treatment approaches in pre-clinical development include adenosine deaminases acting on RNA (ADAR)-based RNA editing, RNA interference, and translational read through inducing drugs (TRADs). These have been reviewed elsewhere (e.g., Martinez Velazquez and Ballios 2021).},
}
@article {pmid40736578,
year = {2025},
author = {Allard-Chamard, X and Rodríguez, EC and Brais, B and Armstrong, GAB},
title = {Loss of dcst2 expression in male zebrafish is not associated with muscle hypertrophy.},
journal = {Molecular genetics and genomics : MGG},
volume = {300},
number = {1},
pages = {74},
doi = {10.1007/s00438-025-02279-x},
pmid = {40736578},
issn = {1617-4623},
support = {Natural Sciences and Engineering Research Council of Canada//Natural Sciences and Engineering Research Council of Canada/ ; },
mesh = {Animals ; *Zebrafish/genetics ; *Zebrafish Proteins/genetics/metabolism ; Male ; Hypertrophy/genetics ; CRISPR-Cas Systems ; *Membrane Proteins/genetics/metabolism ; *Muscle, Skeletal/metabolism/pathology ; Frameshift Mutation ; },
abstract = {Recently, a large family of French-Canadians was found to possess above-average strength and muscle hypertrophy that segregated with a single variant in the gene encoding Dendritic Cell-specific Six Transmembrane domain containing protein 2 (DCST2). To investigate the potential role DCST2 has in muscle cell biology we used the CRISPR/Cas9 mutagenic system and generated a 2-nucleotide deletion in exon 3 of zebrafish dcst2 resulting in a frameshift mutation. Homozygous carriers of the mutation displayed reduced transcriptional expression of dcst2 suggesting that our mutation disrupted gene expression. Homozygous mutant dcst2 zebrafish developed normally to adulthood and displayed no differences in motor function using a free-swim and swim tunnel assays. Furthermore, histological examination of muscle cells revealed no differences in slow-twitch or fast-twitch muscle cell cross-sectional area in our mutants. We did observe that male dcst2[-/-] zebrafish were infertile. The data collected here, suggest that dcst2 does not play a role in zebrafish muscle cell biology.},
}
@article {pmid40735630,
year = {2025},
author = {Wu, L and Yu, Z and Li, P and Wang, Z and Li, Q and Bao, H and García, P and Letarov, A and Zhou, Y and Zhang, H},
title = {Genome MLST scheme for tracing genetic diversity and multidrug resistance of food animal-derived Clostridium perfringens.},
journal = {Current research in food science},
volume = {11},
number = {},
pages = {101149},
pmid = {40735630},
issn = {2665-9271},
abstract = {Clostridium perfringens is an important food-borne anaerobic bacterium that can cause chronic disease in humans and animals. The complex toxins that it produces can cause diarrhea, necrotizing enteritis, and even death. This study aimed to evaluate the presence of antibiotic resistance genes and determinants of toxin production in various strains of C. perfringens, derived from different geographical locations and animal sources, through whole-genome sequencing (WGS) and bioinformatics analysis. Antimicrobial resistance testing revealed a notable prevalence of resistance among isolates, with 39.5 % being resistant to clindamycin and 32.6 % resistant to tetracycline. Additionally, 29.1 % of the isolates were found to be resistant to at least two classes of commonly used antibiotics. TetA(P) and tetB(P), associated with tetracycline resistance, were the most frequently identified resistance genes, present in 93.0 % and 79.0 % of the isolates, respectively. Virulence genes encoding extracellular collagenases (colA and colSI), alpha-toxin (plc), and sialidase (nanH) were detected in 85 isolates, representing 99 % of all sequenced strains. Notably, the gene encoding C. perfringens enterotoxin (cpe) was identified in only one isolate, which originated from chicken. Multi-locus sequence typing revealed that the 86 representative isolates belonged to 49 sequence types (STs), including 33 unique, previously uncharacterized STs. Furthermore, 30.23 % of these STs were grouped into six clonal complexes (CCs). CgSNP analysis of globally circulating isolates demonstrated that the prevalence of the virulence gene cpe was higher in these isolates than in clinical strains. The identification of multi-drug resistance and toxin-encoding genes among the isolates underscores the concerning spread of antimicrobial resistance among C. perfringens affecting both animals and humans. The diversity of CRISPR/Cas system provided addition insight into complex genetic correlations and evolutionary dynamics of pathogenic C. perfringens. Notably, the phage SD72 exhibited a broad inhibitory effect against C. perfringens isolates, irrespective of STs types and antimicrobial resistance (74/86, 80.1 %). These findings highlight the urgent need for enhanced epidemic surveillance of C. perfringens in livestock settings to mitigate the risks of human transmission via environmental or food sources. Additionally, bacteriophages present a promising avenue as bio-antimicrobial agents for controlling the transmission of C. perfringens from animal sources to humans.},
}
@article {pmid40733833,
year = {2025},
author = {Cheng, Y and Wang, Z and Cao, M and Liu, S and Zhang, X and Cheng, C and Cai, C and Zhang, X and Zheng, J},
title = {Structure-activity relationships study on inhibition of CRISPR-Cas9 by glycosaminoglycans.},
journal = {Carbohydrate polymers},
volume = {366},
number = {},
pages = {123912},
doi = {10.1016/j.carbpol.2025.123912},
pmid = {40733833},
issn = {1879-1344},
mesh = {*CRISPR-Cas Systems/drug effects ; Structure-Activity Relationship ; *Heparin/chemistry/pharmacology ; *Chondroitin Sulfates/chemistry/pharmacology ; *Glycosaminoglycans/chemistry/pharmacology ; Molecular Dynamics Simulation ; Gene Editing ; *CRISPR-Associated Protein 9/antagonists &amp; inhibitors/metabolism ; Humans ; },
abstract = {The CRISPR-Cas9 system is a revolutionary genome editing system known for its precision, simplicity and efficiency, playing a crucial role in gene-editing. It has enabled applications ranging from biomedicine to agriculture. However, the uncontrollable activity of Cas9 has raised significant safety concerns in clinical settings, limiting its broader application. Consequently, regulating CRISPR-Cas9 activity holds substantial promise for enhancing the safety and efficacy of gene-editing technologies. In this study, we demonstrated that sulfated glycosaminoglycans (GAGs) exhibit inhibitory effects of Cas9. Specifically, both chondroitin sulfate (CS) and heparin (HP) can inhibit CRISPR/Cas9 activity, with heparin showing a stronger inhibitory effect that correlates positively with its concentration. Molecular dynamics simulations indicate that sulfated heparin residues might inhibit Cas9 function by binding to essential DNA-binding sites, which are crucial for functional interactions, potentially impairing activity. Additionally, higher molecular weight GAGs exhibit enhanced inhibitory effects under the same sulphation. Notably, the sulfation site also influenced activity. C6-sulfation of chondroitin sulfate is more favorable for Cas9 inhibition, and N-sulfation of heparin enhances its inhibitory effect on Cas9 activity. These findings provide valuable insights into the development of carbohydrate-based inhibitors for CRISPR-Cas9, offering a foundation for further exploration in this field.},
}
@article {pmid40733529,
year = {2025},
author = {Anastassopoulou, C and Tsakri, D and Panagiotopoulos, AP and Saldari, C and Sagona, AP and Tsakris, A},
title = {Armed Phages: A New Weapon in the Battle Against Antimicrobial Resistance.},
journal = {Viruses},
volume = {17},
number = {7},
pages = {},
doi = {10.3390/v17070911},
pmid = {40733529},
issn = {1999-4915},
mesh = {*Bacteriophages/genetics/physiology ; Humans ; *Phage Therapy/methods ; *Bacterial Infections/therapy/microbiology ; *Bacteria/virology/drug effects ; Drug Resistance, Multiple, Bacterial ; CRISPR-Cas Systems ; Animals ; Anti-Bacterial Agents/pharmacology ; },
abstract = {The increasing prevalence of multidrug-resistant (MDR) bacterial infections necessitates the exploration of alternative antimicrobial strategies, with phage therapy emerging as a viable option. However, the effectiveness of naturally occurring phages can be significantly limited by bacterial defense systems that include adsorption blocking, restriction-modification, CRISPR-Cas immunity, abortive infection, and NAD+ depletion defense systems. This review examines these bacterial defenses and their implications for phage therapy, while highlighting the potential of phages' bioengineering to overcome these barriers. By leveraging synthetic biology, genetically engineered phages can be tailored to evade bacterial immunity through such modifications as receptor-binding protein engineering, anti-CRISPR gene incorporation, methylation pattern alterations, and enzymatic degradation of bacterial protective barriers. "Armed phages", enhanced with antimicrobial peptides, CRISPR-based genome-editing tools, or immune-modulating factors, offer a novel therapeutic avenue. Clinical trials of bioengineered phages, currently SNIPR001 and LBP-EC01, showcase their potential to safely and effectively combat MDR infections. SNIPR001 has completed a Phase I clinical trial evaluating safety in healthy volunteers, while LBP-EC01 is in Phase II trials assessing its performance in the treatment of Escherichia coli-induced urinary tract infections in patients with a history of drug-resistant infections. As "armed phages" progress toward clinical application, they hold great promise for precision-targeted antimicrobial therapies and represent a critical innovation in addressing the global antibiotic resistance crisis.},
}
@article {pmid40733516,
year = {2025},
author = {Lin, L and Lui, WY and Ong, CP and Yau, MY and Jin, DY and Yuen, KS},
title = {CRISPR/Cas13-Mediated Inhibition of EBNA1 for Suppression of Epstein-Barr Virus Transcripts and DNA Load in Nasopharyngeal Carcinoma Cells.},
journal = {Viruses},
volume = {17},
number = {7},
pages = {},
doi = {10.3390/v17070899},
pmid = {40733516},
issn = {1999-4915},
support = {UGC/FDS17/M07/22//Research Grants Council of the Hong Kong Special Administrative Region/ ; },
mesh = {Humans ; *Herpesvirus 4, Human/genetics/drug effects ; *Epstein-Barr Virus Nuclear Antigens/genetics/metabolism ; *CRISPR-Cas Systems ; *Nasopharyngeal Carcinoma/virology ; Cell Line, Tumor ; *Nasopharyngeal Neoplasms/virology ; *Viral Load ; *Epstein-Barr Virus Infections/virology/therapy ; DNA, Viral/genetics ; },
abstract = {Epstein-Barr virus (EBV), a double-stranded DNA virus, is implicated in nasopharyngeal carcinoma (NPC), with particularly high incidence in regions such as southern China and Hong Kong. Although NPC is typically treated with radio- and chemotherapy, outcomes remain poor for advanced-stage diagnoses, highlighting the need for targeted therapies. This study explores the potential of CRISPR/CRISPR-associated protein 13 (Cas13) technology to target essential EBV RNA in NPC cells. Previous research demonstrated that CRISPR/Cas9 could partially reduce EBV load, but suppression was incomplete. Here, the combination of CRISPR/Cas13 with CRISPR/Cas9 shows enhanced viral clearance. Long-term EBNA1 suppression via CRISPR/Cas13 reduced the EBV genome, improved CRISPR/Cas9 effectiveness, and identified suitable AAV serotypes for delivery. Furthermore, cotreatment increased NPC cell sensitivity to 5-fluorouracil and cisplatin. These findings underscore the potential of CRISPR/Cas13 as an anti-EBV therapeutic approach, effectively targeting latent EBV transcripts and complementing existing treatments. The study suggests a promising new direction for developing anti-EBV strategies, potentially benefiting therapies for NPC and other EBV-associated malignancies.},
}
@article {pmid40733494,
year = {2025},
author = {Wo, L and Qi, S and Guo, Y and Sun, C and Yin, X},
title = {TRIM5α/Cyclophilin A-Modified MDBK Cells for Lentiviral-Based Gene Editing.},
journal = {Viruses},
volume = {17},
number = {7},
pages = {},
doi = {10.3390/v17070876},
pmid = {40733494},
issn = {1999-4915},
support = {32370162//National Natural Science Foundation of China/ ; },
mesh = {Animals ; *Gene Editing/methods ; *Cyclophilin A/genetics/metabolism ; Cattle ; *Lentivirus/genetics ; Cell Line ; CRISPR-Cas Systems ; *Tripartite Motif Proteins/genetics/metabolism ; HIV-1/genetics ; Ubiquitin-Protein Ligases/genetics ; Humans ; },
abstract = {The human immunodeficiency virus 1 (HIV-1)-based lentivirus has been widely used for genetic modification. However, the efficiency of lentiviral-based gene modification in Madin-Darby bovine kidney (MDBK) cells is considerably limited. In this study, we have shown that siRNA-mediated depletion of TRIM5α, a restriction factor in HIV-1 infection, can dramatically enhance HIV-1 infection in MDBK cells. Furthermore, we generated a doxycycline-inducible Cas9-overexpressing MDBK cell line (MDBK-iCas9) suitable for CRISPR/Cas9-mediated editing. On this basis, we created a TRIM5α knock-out MDBK-iCas9 cell line MDBK-iCas9[TRIM5α-/-] without additional genome insertions by combining sgRNA transfection and single-cell cloning. We found that MDBK-iCas9[TRIM5α-/-] displayed greater permissiveness to lentivirus infection compared with MDBK-WT cells. Notably, we found that treatment with the chemical compound cyclosporine A, which directly interacts with cell factor cyclophilin A (CypA), could markedly increase the infectivity of lentivirus in both MDBK-iCas9[TRIM5α-/-] and MDBK-WT cell lines, suggesting that CypA functions independently with TRIM5α as an inhibitor of the lentivirus in bovine cells. Therefore, combining bovine TRIM5α and CypA targeting could remarkably enhance lentivirus infection. In conclusion, our findings highlight a promising gene engineering strategy for bovine cells that can surmount the significant barriers to investigating the interplay between bovine viruses and their host cells.},
}
@article {pmid40733490,
year = {2025},
author = {Jiang, X and Huang, Y and Jiang, Y and Yang, G and Zheng, X and Gao, S},
title = {CRISPR-Cas12a/RPA Dual-Readout Assay for Rapid Field Detection of Porcine Rotavirus with Visualization.},
journal = {Viruses},
volume = {17},
number = {7},
pages = {},
doi = {10.3390/v17070872},
pmid = {40733490},
issn = {1999-4915},
support = {Grant No: SCKJ-JYRC-2023-29//the Project of Sanya Yazhou Bay Science and Technology City/ ; Grant No: 324QN343.//the Youth Fund Project of Hainan Provincial Natural Science Foundation/ ; },
mesh = {Animals ; Swine ; *Swine Diseases/virology/diagnosis ; *Rotavirus/genetics/isolation &amp; purification ; *CRISPR-Cas Systems ; *Rotavirus Infections/diagnosis/veterinary/virology ; *Nucleic Acid Amplification Techniques/methods ; Sensitivity and Specificity ; Recombinases/metabolism/genetics ; Capsid Proteins/genetics ; Endodeoxyribonucleases/genetics ; Antigens, Viral/genetics ; CRISPR-Associated Proteins/genetics ; Bacterial Proteins ; },
abstract = {PoRV is a significant etiological agent of neonatal diarrhea in piglets, resulting in substantial economic losses within the global swine industry due to elevated mortality rates and reduced productivity. To address the urgent need for accessible and rapid diagnostics in resource-limited settings, we have developed a CRISPR/Cas12a-based assay integrated with recombinase polymerase amplification (RPA) for the visual detection of PoRV. This platform specifically targets the conserved VP6 gene using optimized RPA primers and crRNA, harnessing Cas12a's collateral cleavage activity to enable dual-readout via fluorescence or lateral flow dipsticks (LFDs). The assay demonstrates a detection limit of 10[2] copies/μL within 1 h, exhibiting no cross-reactivity with phylogenetically related pathogens such as Transmissible Gastroenteritis Virus (TGEV). By eliminating reliance on thermal cyclers or specialized equipment, this method is fully deployable in swine farms, veterinary clinics, or field environments. The lateral flow format provides immediate colorimetric results that require minimal technical expertise, while the fluorescence mode allows for semi-quantitative analysis. This study presents a robust and cost-effective platform for decentralized PoRV surveillance in swine populations, addressing the critical need for portable diagnostics in resource-limited settings and enhancing veterinary health management.},
}
@article {pmid40732725,
year = {2025},
author = {Naveed, S and Brown, JK and Mubin, M and Javed, N and Nawaz-Ul-Rehman, MS},
title = {Potential for Duplexed, In-Tandem gRNA-Mediated Suppression of Two Essential Genes of Tomato Leaf Curl New Delhi Virus in Crop Plants.},
journal = {Pathogens (Basel, Switzerland)},
volume = {14},
number = {7},
pages = {},
doi = {10.3390/pathogens14070679},
pmid = {40732725},
issn = {2076-0817},
support = {6427//Higher Education Commission of Pakistan/ ; },
mesh = {*Begomovirus/genetics ; *Plant Diseases/virology ; *Solanum lycopersicum/virology ; Capsicum/virology ; Nicotiana/virology ; CRISPR-Cas Systems ; Genome, Viral ; Capsid Proteins/genetics ; },
abstract = {Tomato leaf curl New Delhi virus (ToLCNDV) is among the most prevalent and widely distributed begomovirus infecting chili pepper (Capsicum annuum) and tomato in the Indian subcontinent. In this study, a guide RNA (gRNA) sequence-CRISPR-Cas9 approach was used to target and cleave two essential coding regions in the begomovirus genome. The gRNAs were designed to target conserved regions of the ToLCNDV replication-associated protein (rep) gene or ORF AC1, and/or the coat protein (cp) gene or AV1 ORF, respectively. Based on an alignment of 346 representative ToLCNDV genome sequences, all predicted single nucleotide polymorphisms off-target sites were identified and eliminated as potential gRNA targets. Based on the remaining genome regions, four candidate gRNAs were designed and used to build gRNA-Cas9 duplexed constructs, e.g., containing two gRNAs cloned in tandem, in different combinations (1-4). Two contained two gRNAs that targeted the coat protein gene (cp; AV1 ORF), while the other two constructs targeted both the cp and replication-associated protein gene (rep; AC1 ORF). These constructs were evaluated for the potential to suppress ToLCNDV infection in Nicotiana benthamiana plants in a transient expression-transfection assay. Among the plants inoculated with the duplexed gRNA construct designed to cleave ToLCNDV-AV1 or AC1-specific nucleotides, the construct designed to target both the cp (293-993 nt) and rep (1561-2324) showed the greatest reduction in virus accumulation, based on real-time quantitative PCR amplification, and attenuated disease symptoms, compared to plants inoculated with the DNA-A component alone or mock-inoculated, e.g., with buffer. The results demonstrate the potential for gRNA-mediated suppression of ToLCNDV infection in plants by targeting at least two viral coding regions, underscoring the great potential of CRISPR-Cas-mediated abatement of begomovirus infection in numerous crop species.},
}
@article {pmid40732701,
year = {2025},
author = {Hanssens, C and Van Cleemput, J},
title = {Applying CRISPR Technologies for the Treatment of Human Herpesvirus Infections: A Scoping Review.},
journal = {Pathogens (Basel, Switzerland)},
volume = {14},
number = {7},
pages = {},
doi = {10.3390/pathogens14070654},
pmid = {40732701},
issn = {2076-0817},
support = {202409/004//Ghent University/ ; },
mesh = {Humans ; *CRISPR-Cas Systems ; *Herpesviridae Infections/therapy/virology/genetics ; Gene Editing/methods ; *Herpesviridae/genetics ; Antiviral Agents/therapeutic use ; *Genetic Therapy/methods ; Virus Latency/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {BACKGROUND: Human herpesviruses are double-stranded DNA viruses of which eight types have been identified at present. Herpesvirus infection comprises an active lytic phase and a lifelong latency phase with the possibility of reactivation. These infections are highly prevalent worldwide and can lead to a broad spectrum of clinical manifestations, ranging from mild symptoms to severe disease, particularly in immunocompromised individuals. Clustered regularly interspaced palindromic repeats (CRISPR)-based therapy is an interesting alternative to current antiviral drugs, which fail to cure latent infections and are increasingly challenged by viral resistance.<br><br>OBJECTIVE: This scoping review aimed to summarize the current state of CRISPR-based antiviral strategies against herpesvirus infections, highlighting the underlying mechanisms, study design and outcomes, and challenges for clinical implementation.<br><br>DESIGN: A literature search was conducted in the databases PubMed and Web of Science, using both a general and an individual approach for each herpesvirus.<br><br>RESULTS: This scoping review identified five main mechanisms of CRISPR-based antiviral therapy against herpesvirus infections in vitro and/or in vivo. First, CRISPR systems can inhibit the active lytic replication cycle upon targeting viral lytic genes or host genes. Second, CRISPR technologies can remove latent viral genomes from infected cells by targeting viral genes essential for latency maintenance or destabilizing the viral genome. Third, reactivation of multiple latent herpesvirus infections can be inhibited by CRISPR-Cas-mediated editing of lytic viral genes, preventing a flare-up of clinical symptoms and reducing the risk of viral transmission. Fourth, CRISPR systems can purposefully induce viral reactivation to enhance recognition by the host immune system or improve the efficacy of existing antiviral therapies. Fifth, CRISPR technology can be applied to develop or enhance the efficiency of cellular immunotherapy.<br><br>CONCLUSIONS: Multiple studies demonstrate the potential of CRISPR-based antiviral strategies to target herpesvirus infections through various mechanisms in vitro and in vivo. However, aspects regarding the delivery and biosafety of CRISPR systems, along with the time window for treatment, require further investigation before broad clinical implementation can be realized.},
}
@article {pmid40732192,
year = {2025},
author = {Yang, K and Wu, X and Ding, H and Ma, B and Li, Z and Wang, Y and Yang, Z and Yao, X and Luo, Y},
title = {Isolation, Identification, and Antibiotic Resistance, CRISPR System Analysis of Escherichia coli from Forest Musk Deer in Western China.},
journal = {Microorganisms},
volume = {13},
number = {7},
pages = {},
doi = {10.3390/microorganisms13071683},
pmid = {40732192},
issn = {2076-2607},
abstract = {Escherichia coli (E. coli) is an opportunistic pathogen widely distributed in nature, and multi-drug resistance (MDR) E. coli has been widely recognized as a critical reservoir of resistance genes, posing severe health threats to humans and animals. A total of 288 E. coli strains were isolated and purified from fresh fecal samples of forest musk deer collected from farms in Sichuan, Shaanxi, and Yunnan Provinces of China between 2013 and 2023. This study aimed to conduct antibiotic susceptibility testing and resistance gene detection on the isolated forest musk deer-derived E. coli, analyze the correlations between them, investigate the presence of CRISPR systems within the strains, and perform bioinformatics analysis on the CRISPR systems carried by the strains. Results showed that 138 out of 288 E. coli strains were MDR, with the highest resistance to tetracycline (48.3%), cefalexin (45.1%), and doxycycline (41.7%). Prevalent genes were tetA (41.0%), sul2 (30.2%), blaTEM (27.1%), with 29 gene-phenotype pairs correlated. CRISPR system-negative strains had higher resistance rates to 16 antibiotics and lower detection rates only for aac (6')-Ib-cr, qnrA, and qnrB compared to CRISPR system-positive strains. Regional analysis showed that the problem of drug resistance in Sichuan and Shaanxi was more serious, and that the detection rate of antibiotic resistance genes was relatively high. This study guides E. coli infection control in forest musk deer and enriches resistance research data.},
}
@article {pmid40732175,
year = {2025},
author = {Di, T and Zhang, H and Zhang, C and Tian, L and Chang, M and Han, W and Qiao, R and Li, M and Zhang, S and Yang, G},
title = {Complete Genome and Characterization Analysis of a Bifidobacterium animalis Strain Isolated from Wild Pigs (Sus scrofa ussuricus).},
journal = {Microorganisms},
volume = {13},
number = {7},
pages = {},
doi = {10.3390/microorganisms13071666},
pmid = {40732175},
issn = {2076-2607},
support = {092102110088, 212102110001, 22210320010//This study was supported by the Key R&amp;D and Promotion Program in Henan Province of China/ ; },
abstract = {Bifidobacterium is a predominant probiotic in animals that is associated with host intestinal health. The protective mechanisms of the Bifidobacterium animalis (B. animalis) strain, specifically those related to functional gene-host interactions in intestinal homeostasis, remain poorly elucidated. This study reports the complete genome sequence and characterization of a B. animalis strain isolated from wild pig feces, which comprised a single circular chromosome (1,944,022 bp; GC content 60.49%) with 1567 protein-coding genes, and the B. animalis strain had certain acid resistance, bile salt resistance, gastrointestinal fluid tolerance, and antibacterial characteristics. Genomic annotation revealed three putative genomic islands and two CRISPR-Cas systems. Functional characterization identified genes encoding carbohydrate-active enzymes (CAZymes) and associated metabolic pathways, indicating that this strain can degrade complex dietary carbohydrates and synthesize bioactive metabolites for gut homeostasis. Although the antibiotic resistance genes were predicted, phenotypic assays demonstrated discordant resistance patterns, indicating complex regulatory networks. This study indicated the genomic basis of Bifidobacterium-host crosstalk in intestinal protection, providing a framework for developing novel probiotic interventions.},
}
@article {pmid40731998,
year = {2025},
author = {Gagaletsios, LA and Kikidou, E and Galbenis, C and Bitar, I and Papagiannitsis, CC},
title = {Exploring Virulence Characteristics of Clinical Escherichia coli Isolates from Greece.},
journal = {Microorganisms},
volume = {13},
number = {7},
pages = {},
doi = {10.3390/microorganisms13071488},
pmid = {40731998},
issn = {2076-2607},
abstract = {The aim of this study was to examine the genetic characteristics that could be associated with the virulence characteristics of Escherichia coli collected from clinical samples. A collection of 100 non-repetitive E. coli isolates was analyzed. All isolates were typed by MLST. String production, biofilm formation and serum resistance were examined for all isolates. Twenty E. coli isolates were completely sequenced Illumina platform. The results showed that the majority of E. coli isolates (87%) produced significant levels of biofilm, while none of the isolates were positive for string test and resistance to serum. Additionally, the presence of CRISPR/Cas systems (type I-E or I-F) was found in 18% of the isolates. Analysis of WGS data found that all sequenced isolates harbored a variety of virulence genes that could be implicated in adherence, invasion, iron uptake. Also, WGS data confirmed the presence of a wide variety of resistance genes, including ESBL- and carbapenemase-encoding genes. In conclusion, an important percentage (87%) of the E. coli isolates had a significant ability to form biofilm. Biofilms, due to their heterogeneous nature and ability to make microorganisms tolerant to multiple antimicrobials, complicate treatment strategies. Thus, in combination with the presence of multidrug resistance, expression of virulence factors could challenge antimicrobial therapy of infections caused by such bacteria.},
}
@article {pmid40731176,
year = {2025},
author = {Wang, Y and Zhu, K and Wang, Y and Wang, Y and Xu, J and Li, Y and Liu, F and Long, J and Yang, H},
title = {The application of the CRISPR-Cas system in Klebsiella pneumoniae infections.},
journal = {Molecular biology reports},
volume = {52},
number = {1},
pages = {766},
pmid = {40731176},
issn = {1573-4978},
support = {2023YFC2605603//National Key Research and Development Project/ ; 82273696//National Natural Science Foundation of China/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; *Klebsiella pneumoniae/genetics/drug effects/pathogenicity ; Humans ; Gene Editing/methods ; *Klebsiella Infections/genetics/microbiology/therapy/drug therapy ; Drug Resistance, Multiple, Bacterial/genetics ; Anti-Bacterial Agents/pharmacology ; },
abstract = {Klebsiella pneumoniae (Kp) is an important conditional pathogen that causes bacteremia, pneumonia, sepsis, urinary tract infections, and liver abscesses. The emergence of multidrug-resistant Kp, especially carbapenem-resistant Kp (CRKP), has become a major challenge to global public health. There is a need for early identification and diagnosis of Kp pathogens as well as precise treatment of Kp infections. The CRISPR-Cas (Clustered regularly interspaced short palindromic repeats and associated Cas proteins) system is an adaptive immune mechanism for bacteria. Studies have shown that the presence of the CRISPR-Cas system is negatively correlated with drug resistance in Kp strains, and CRISPR-Cas system-based technologies have been developed for the detection, gene editing, the development of therapeutic drugs and vaccines in Kp infections. However, no review has been published on this area; therefore, this review describes the role of the CRISPR-Cas system in the development of antibiotic resistance in Kp, the application of CRISPR-Cas system-based technology in detection and gene editing, and the therapeutic potential of CRISPR-Cas system in Kp infections.},
}
@article {pmid40731099,
year = {2025},
author = {Santinha, AJ and Strano, A and Platt, RJ},
title = {Methods and applications of in vivo CRISPR screening.},
journal = {Nature reviews. Genetics},
volume = {},
number = {},
pages = {},
doi = {10.1038/s41576-025-00873-8},
pmid = {40731099},
issn = {1471-0064},
abstract = {A fundamental goal in genetics is to understand the connection between genotype and phenotype in health and disease. Genetic screens in which dozens to thousands of genetic elements are perturbed in a pooled fashion offer the opportunity to generate large-scale, information-rich and unbiased genotype-phenotype maps. Although typically applied in reductionist in vitro settings, methods enabling pooled CRISPR-Cas perturbation screening in vivo are gaining attention as they have the potential to accelerate the discovery and annotation of gene function across cells, tissues, developmental stages, disease states and species. In this Review, we discuss essential criteria for understanding, designing and implementing in vivo screening experiments, with a focus on pooled CRISPR-based screens in mice. We also highlight how the resulting datasets, combined with advances in multi-omics and artificial intelligence, will accelerate progress and enable fundamental discoveries across basic and translational sciences.},
}
@article {pmid40731026,
year = {2025},
author = {Geng, XL and Li, JY and Xu, HY and Wu, JP and Tao, DL and Chen, JM and Fan, YY and Yang, X and Song, JK and Zhao, GH},
title = {Proline dehydrogenase, a rate-limiting catabolic enzyme, affecting the growth and pathogenicity of Toxoplasma gondii tachyzoites by regulating the proline metabolism and mitochondrial function of the parasite.},
journal = {Parasites &amp; vectors},
volume = {18},
number = {1},
pages = {309},
doi = {10.1186/s13071-025-06966-x},
pmid = {40731026},
issn = {1756-3305},
mesh = {*Toxoplasma/pathogenicity/growth &amp; development/enzymology/genetics/metabolism ; *Proline Oxidase/genetics/metabolism ; *Proline/metabolism ; *Mitochondria/metabolism ; Animals ; Virulence ; Mice ; Reactive Oxygen Species/metabolism ; Membrane Potential, Mitochondrial ; Mice, Inbred BALB C ; Protozoan Proteins/genetics/metabolism ; CRISPR-Cas Systems ; Female ; },
abstract = {BACKGROUND: The pathogenicity of Toxoplasma gondii is closely associated with its intracellular lytic cycle in host cells. Currently, the mechanisms by which T. gondii completes the lytic cycle remain unclear. The proline metabolism has been reported to be crucial for intracellular growth of pathogens by providing energy and nutrients. However, it remains unclear whether the intracellular growth and pathogenicity of T. gondii are related to proline metabolism.<br><br>METHODS: The gene-edited strains of proline dehydrogenase (Tgprodh) were constructed by using clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR-Cas9) technology. The effects of the Tgprodh gene on the growth in vitro and pathogenicity in vivo of the tachyzoites for T. gondii were studied through proliferation, plaque, invasion, egress and virulence assays. The effects of the Tgprodh gene on mitochondrial function were studied by using reactive oxygen species (ROS), mitochondrial membrane potential (&#x2206;&#x3a8;m), adenosine triphosphate (ATP) assay kits, mitochondrial DNA (mtDNA) copy numbers, transmission electron microscopy (TEM) analysis, and reverse transcriptase quantitative polymerase chain reaction (RT-qPCR). The effects of the Tgprodh gene on proline metabolism were studied by using L-proline (L-Pro), L-glutamic acid (L-Glu), L-glutamine (L-Gln) assay kits, and RT-qPCR.<br><br>RESULTS: TgPRODH, the first rate-limiting enzyme in proline metabolism, was identified to be encoded by T. gondii and localized in the cytoplasm of T. gondii. Deletion of the Tgprodh gene resulted in significant growth inhibition in vitro and reduced pathogenicity in vivo of T. gondii. Further study found that deletion of the Tgprodh gene caused damage to the mitochondrial morphology, decreased membrane potential, mtDNA copy numbers, and the production of ATP and ROS. The expression of genes for maintaining mitochondrial integrity was downregulated in the Tgprodh-knockout strain of T. gondii, while complementation of the Tgprodh gene restored these defects in this parasite. Meantime, the deletion of the Tgprodh gene resulted in the accumulation of proline, reduced the contents of glutamate and glutamine, and affected the expression of genes related to proline catabolism in T. gondii.<br><br>CONCLUSIONS: The present study found the significance of the Tgprodh gene for the intracellular growth and pathogenicity of T. gondii through regulating mitochondrial function and the proline metabolism and provided a novel insight to reveal intracellular survival strategies of T. gondii.},
}
@article {pmid40730819,
year = {2025},
author = {Schertzer, MD and Stirn, A and Isaev, K and Pereira, L and Park, SH and Das, A and Réal, A and Jeffery, ED and Harbison, C and Wessels, HH and Sheynkman, GM and Sanjana, NE and Knowles, DA},
title = {Cas13d-mediated isoform-specific RNA knockdown with a unified computational and experimental toolbox.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {6948},
doi = {10.1038/s41467-025-62066-5},
pmid = {40730819},
issn = {2041-1723},
support = {F32GM142213//U.S. Department of Health &amp; Human Services | NIH | National Institute of General Medical Sciences (NIGMS)/ ; DP2HG010099//U.S. Department of Health &amp; Human Services | NIH | National Human Genome Research Institute (NHGRI)/ ; R21CA272345//U.S. Department of Health &amp; Human Services | NIH | National Cancer Institute (NCI)/ ; R01AI176601//U.S. Department of Health &amp; Human Services | NIH | National Cancer Institute (NCI)/ ; },
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Gene Knockdown Techniques/methods ; RNA, Guide, CRISPR-Cas Systems/genetics ; Exons/genetics ; Protein Isoforms/genetics/metabolism ; *Computational Biology/methods ; Alternative Splicing ; HEK293 Cells ; *RNA/genetics/metabolism ; },
abstract = {Pre- and post-transcriptional mechanisms, including alternative promoters, termination signals, and splicing, play essential roles in diversifying protein output by generating distinct RNA and protein isoforms. Two major challenges in characterizing the cellular function of alternative isoforms are the lack of experimental methods to specifically and efficiently modulate isoform expression and computational tools for complex experimental design and analysis. To address these gaps, we develop and methodically test an isoform-specific knockdown strategy which pairs the RNA-targeting CRISPR/Cas13d system with guide RNAs that span exon-exon junctions. In parallel, we provide computational tools for experimental design and analysis. In this study, we demonstrate that junction-targeting achieves robust and isoform-specific RNA knockdown across diverse alternative isoform events, genes, and cell types.},
}
@article {pmid40730529,
year = {2025},
author = {Wang, P and Wu, M and Du, J},
title = {[Generation of a dense granule protein 3 gene-deficient strain of Toxoplasma gondii and its virulence testing].},
journal = {Zhongguo xue xi chong bing fang zhi za zhi = Chinese journal of schistosomiasis control},
volume = {37},
number = {3},
pages = {304-309},
doi = {10.16250/j.32.1915.2024293},
pmid = {40730529},
issn = {1005-6661},
support = {82472313//National Natural Science Foundation of China/ ; AHWJ2023A30013//Scientific Research Project of Anhui Provincial Health Commission/ ; },
mesh = {*Toxoplasma/genetics/pathogenicity ; *Protozoan Proteins/genetics/metabolism ; Virulence ; Animals ; Mice ; Humans ; CRISPR-Cas Systems ; *Toxoplasmosis/parasitology ; *Antigens, Protozoan/genetics ; },
abstract = {OBJECTIVE: To generate a dense granule protein 3 (GRA3) gene-deficient mutant of the Toxoplasma gondii ME49 strain and to test the virulence of the mutant.<br><br>METHODS: Gene-deficient parasites were generated with the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (CRISPR/Cas9) system. Guide RNA (gRNA) was designed using the E-CRISPR software, and mutated on the pSAG1::Cas9-U6::sgUPRT plasmid using the Q5 site-directed mutagenesis kit to generate the pSAG1::Cas9-U6::sgGRA3 plasmid. A GRA3 donor plasmid containing GRA3 gene upstream sequences, pyrimethamine resistant gene dihydrofolate reductase-thymidylate synthase (DHFR-TS) and GRA3 gene downstream sequence was generated, and GRA3 donor DNA was amplified using PCR assay. The pSAG1::Cas9-U6::sgGRA3 plasmid and GRA3 donor DNA were electroporated into tachyzoites of the wild-type T. gondii ME49 strain. Then, parasite suspensions were inoculated into human foreskin fibroblast (HFF) cells and screened with pyrimethamine to yield pyrimethamine-resistant parasites for monoclonal screening. The GRA3 gene deficient monoclonal strain (ME49&#x394;gra3) of T. gondii was identified using PCR and Western blotting assays, and the expression of GRA3 protein was determined in the T. gondii ME49&#x394;gra3 strain using Western blotting. Subsequently, 1 000 freshly lysed tachyzoites of T. gondii ME49 and ME49&#x394;gra3 strains were transferred to 12-well plates seeded with HFF cells, and incubated at 37 &#xb0;C containing 5% CO2 for 7 days, and the number of plaques was counted by staining with crystal violet solutions. HFF cells infected with tachyzoites of T. gondii ME49 and ME49&#x394;gra3 strains were stained using Giemsa solutions, and the numbers of cells containing 1, 2, 4, and > 4 T. gondii parasitophorous vacuoles were counted. In addition, the survival rates of C57BL/6 mice infected with T. gondii ME49 and ME49&#x394;gra3 strains were compared 35 days post-infection.<br><br>RESULTS: PCR assay revealed successful amplification of both the upstream and downstream homologous arm bands of the DHFR-TS gene in the T. gondii ME49&#x394;gra3 strain, and no corresponding bands were amplified in the ME49 strain. The GRA3 band was amplified in the ME49 strain, and the DHFR-TS band, rather than GRA3 band, was amplified in the ME49&#x394;gra3 strain. Western blotting determined absence of GRA3 protein expression in the ME49&#x394;gra3 strain. Crystal violet staining showed that the T. gondii ME49 strain produced more plaques than the ME49&#x394;gra3 strain [(352.67 &#xb1; 26.39) plaques vs. (235.00 &#xb1; 26.29) plaques; t = 5.472, P < 0.01], and Giemsa staining revealed that the proportion of T. gondii parasitophorous vacuoles containing at least four T. gondii tachyzoites was higher in HFF cells infected with the ME49 strain than in those infected with the T. gondii ME49&#x394;gra3 strain [(75.67 &#xb1; 2.52)% vs. (59.67 &#xb1; 2.31)%; t = 8.113, P < 0.01], and the proportion of T. gondii parasitophorous vacuoles containing at least 1 or 2 T. gondii tachyzoites was higher in HFF cells infected with the T. gondii ME49 strain than in those infected with the T. gondii ME49&#x394;gra3 strain [(24.33 &#xb1; 2.52)% vs. (40.33 &#xb1; 2.31)%; t = -8.113, P < 0.01]. In addition, mice infected with the T. gondii ME49 and ME49&#x394;gra3 strains started to die 8 and 9 days post-infection, and the 35-day mortality rates of mice infected with T. gondii ME49 and ME49&#x394;gra3 strains were 10.00% and 70.00% post-infection (&#x3c7;[2] = 6.762, P < 0.01).<br><br>CONCLUSIONS: The T. gondii ME49&#x394;gra3 strain has been successfully generated, and GRA3 protein may increase the virulence of the T. gondii ME49 strain.},
}
@article {pmid40728780,
year = {2025},
author = {Irum, S and Biswas, S and Cilkiz, M and Tsakirpaloglou, N and Thomson, MJ and Septiningsih, EM},
title = {Multiplex CRISPR-Cas9 editing of chlorophyll biosynthesis genes in chickpea via protoplast and Agrobacterium-mediated transformation.},
journal = {Functional &amp; integrative genomics},
volume = {25},
number = {1},
pages = {163},
pmid = {40728780},
issn = {1438-7948},
mesh = {*Cicer/genetics/metabolism ; *CRISPR-Cas Systems ; *Chlorophyll/biosynthesis/genetics ; *Gene Editing/methods ; Protoplasts/metabolism ; Agrobacterium/genetics ; Transformation, Genetic ; Plant Proteins/genetics/metabolism ; },
abstract = {Chickpea is an important legume consumed worldwide and a rich source of protein. Chickpea is less amenable to recent gene editing techniques despite its economic significance. Accelerating the improvement process and enabling novel trait development in chickpea will require new approaches for genetic intervention. The CRISPR system has been used in different plant species to generate genetic variation and manipulate gene functions, facilitating studies on gene function and crop improvement. To implement genome editing in chickpea, genes involved in the chlorophyll biosynthesis pathway were selected as targets for gene editing. A construct (pTrans_100-Chbio) carrying gRNAs for chlorophyllide a oxygenase (CAO) and chlorophyll synthase (CHLG), along with the Cas9 protein, was introduced into chickpea protoplasts via PEG-mediated transformation. Multiple edits containing deletions and base insertions were identified after protoplast transformation, as confirmed by Sanger sequencing. Afterward, Agrobacterium transformation of explants was performed, resulting in the successful regeneration of pale and chimeric yellow tissues, subsequently confirmed as containing largely substitutions, as detected through deep amplicon sequencing. Edited plants showed yellowish leaves and lower chlorophyll content. Our results indicated that chlorophyll biosynthesis pathway genes played an essential role in chlorophyll degradation and ROS scavenging to regulate both natural and induced chickpea senescence. We established an efficient and feasible CRISPR/Cas9-based editing system in chickpea that successfully generates allelic mutations and phenotypic variation. The established platform can be a foundation for future functional studies and precise genome editing of additional agronomic traits, ultimately contributing to chickpea crop improvement and sustainable agriculture.},
}
@article {pmid40728527,
year = {2025},
author = {Pelea, O and Mayes, S and Ferry, QRV and Fulga, TA and Sauka-Spengler, T},
title = {Specific modulation of CRISPR transcriptional activators through RNA-sensing guide RNAs in mammalian cells and zebrafish embryos.},
journal = {eLife},
volume = {12},
number = {},
pages = {},
pmid = {40728527},
issn = {2050-084X},
support = {grant EP/L016494/1//UKERC/ ; 10.35802/215615/WT_/Wellcome Trust/United Kingdom ; },
mesh = {*Zebrafish/embryology/genetics ; Animals ; *RNA, Guide, CRISPR-Cas Systems/genetics/metabolism ; HEK293 Cells ; Humans ; *CRISPR-Cas Systems ; Embryo, Nonmammalian/metabolism ; *Clustered Regularly Interspaced Short Palindromic Repeats ; *RNA/metabolism ; Streptococcus pyogenes/genetics/enzymology ; Gene Editing/methods ; *Transcriptional Activation ; },
abstract = {Cellular transcripts encode important information regarding cell identity and disease status. The activation of CRISPR in response to RNA biomarkers holds the potential for controlling CRISPR activity with spatiotemporal precision. This would enable the restriction of CRISPR activity to specific cell types expressing RNA biomarkers of interest while preventing unwanted activity in other cells. Here, we present a simple and specific platform for modulating CRISPR activity in response to RNA detection through engineering Streptococcus pyogenes Cas9 single-guide RNAs (sgRNAs). sgRNAs are engineered to fold into complex secondary structures that, in the ground state, inhibit their activity. Engineered sgRNAs become activated upon recognising complementary RNAs, thus enabling Cas9 to perform its function. Our approach enables CRISPR activation in response to RNA detection in both HEK293T cells and zebrafish embryos. Iterative design optimisations allowed the development of computational tools for generating sgRNAs capable of detecting RNA sequences of choice. Mechanistic investigations reveal that engineered sgRNAs are cleaved during RNA detection, and we identify key positions that benefit from chemical modifications to improve the stability of engineered sgRNAs in vivo. Our sensors open up novel opportunities for developing new research and therapeutic applications using CRISPR activation in response to endogenous RNA biomarkers.},
}
@article {pmid40725091,
year = {2025},
author = {Bi, M and Wang, Z and Li, K and Ren, Y and Ma, D and Mo, X},
title = {CRISPR/Cas12a-Based One-Tube RT-RAA Assay for PoRV Genotyping.},
journal = {International journal of molecular sciences},
volume = {26},
number = {14},
pages = {},
pmid = {40725091},
issn = {1422-0067},
support = {32071476//National Natural Science Foundation in China/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; Animals ; Swine ; *Rotavirus/genetics ; Genotype ; *Genotyping Techniques/methods ; *Swine Diseases/virology/diagnosis ; *Rotavirus Infections/virology/veterinary/diagnosis ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {Porcine rotavirus (PoRV), a primary etiological agent of viral diarrhea in piglets, frequently co-infects with other enteric pathogens, exacerbating disease severity and causing substantial economic losses. Its genetic recombination capability enables cross-species transmission potential, posing public health risks. Globally, twelve G genotypes and thirteen P genotypes have been identified, with G9, G5, G3, and G4 emerging as predominant circulating strains. The limited cross-protective immunity between genotypes compromises vaccine efficacy, necessitating genotype surveillance to guide vaccine development. While conventional molecular assays demonstrate sensitivity, they lack rapid genotyping capacity and face technical limitations. To address this, we developed a novel diagnostic platform integrating reverse transcription recombinase-aided amplification (RT-RAA) with CRISPR-Cas12a. This system employs universal primers for the simultaneous amplification of G4/G5/G9 genotypes in a single reaction, coupled with sequence-specific CRISPR recognition, achieving genotyping within 50 min at 37 °C with 10[0] copies/μL sensitivity. Clinical validation showed a high concordance with reverse transcription quantitative polymerase chain reaction (RT-qPCR). This advancement provides an efficient tool for rapid viral genotyping, vaccine compatibility evaluation, and optimized epidemic control strategies.},
}
@article {pmid40685572,
year = {2025},
author = {Liang, Z and Yu, Y and Chen, X and Qin, L and Xu, G and Wei, F and Yang, J and Hu, Q and Cen, Y},
title = {Breakthrough in LbCas12a Chemical Inhibitor Screening: The Firefly Probe and Its Application in Dual-Mode Biosensors.},
journal = {Analytical chemistry},
volume = {97},
number = {29},
pages = {16050-16057},
doi = {10.1021/acs.analchem.5c03437},
pmid = {40685572},
issn = {1520-6882},
mesh = {*Biosensing Techniques/methods ; *Fluorescent Dyes/chemistry ; Hydrogen Peroxide/pharmacology/chemistry ; *CRISPR-Associated Proteins/antagonists &amp; inhibitors/metabolism ; Glucose/analysis ; Molecular Docking Simulation ; Catechols/pharmacology/chemistry ; Catalase/analysis/metabolism ; Hydroquinones/pharmacology/chemistry ; CRISPR-Cas Systems/drug effects ; Bacterial Proteins ; Endodeoxyribonucleases ; },
abstract = {Biosensors employing anti-CRISPR (Acr) proteins have been designed, and prohibitive manufacturing expenses and unfavorable storage conditions still restrict Acr protein applications. Since chemical inhibitors have lower production costs and are relatively insensitive to storage conditions, they are more accessible and easier to store and transport than protein inhibitors. Furthermore, they could be efficiently manufactured industrially for future applications. To screen the chemical inhibitors of CRISPR/Cas12a, we constructed a label-free fluorescent probe, dubbed the firefly probe. Then, three highly effective chemical inhibitors (H2O2, catechol, and hydroquinone) against LbCas12a were discovered based on this probe. These small molecule inhibitors could preferentially bind with LbCas12a to interrupt the assembly of the LbCas12a/crRNA binary complex. Thus, the cis- and trans-cleavage activities of LbCas12a were simultaneously inhibited. The inhibitory mechanisms were also explored through electrophoresis and molecular docking experiments. Subsequently, a universal sensing platform based on the inhibitor H2O2 was built, which enabled fluorescence analysis of catalase and glucose. The limits of detection for catalase and glucose in the fluorescence mode were as low as 0.080 U/mL and 0.0059 mM, respectively. Furthermore, the platform supported visual analysis by integrating smartphones with the RGB measurement software. In summary, small molecule inhibitors of LbCas12a were found, and their inhibition mechanism was identified in this research. These inhibitors can serve as efficient tools for blocking the CRISPR/Cas12a system and facilitate the application of LbCas12a inhibitors in biosensors.},
}
@article {pmid40682549,
year = {2025},
author = {Yang, L and Liu, R and Meng, Y and Deng, Z and Bu, S and Liu, J and Huang, A and Wu, S and Kan, X},
title = {Fruit Phenotype Analysis of SlSAHH2-CRISPR Tomato and Methylation Mechanism of SlSAHH2 Promoting Fruit Ripening.},
journal = {Journal of agricultural and food chemistry},
volume = {73},
number = {30},
pages = {18691-18705},
doi = {10.1021/acs.jafc.5c05207},
pmid = {40682549},
issn = {1520-5118},
mesh = {*Solanum lycopersicum/genetics/growth &amp; development/metabolism/enzymology ; *Fruit/genetics/growth &amp; development/metabolism/enzymology ; *Plant Proteins/genetics/metabolism ; Gene Expression Regulation, Plant ; Ethylenes/biosynthesis/metabolism ; *Adenosylhomocysteinase/genetics/metabolism ; *Plants, Genetically Modified/genetics/metabolism/growth &amp; development/enzymology ; DNA Methylation ; Methylation ; Phenotype ; CRISPR-Cas Systems ; },
abstract = {S-adenosyl-l-homocysteine hydrolase (SAHH EC 3.3.1.1) is a key enzyme that maintains methylation homeostasis. In this study, the knockout of SlSAHH2 delayed tomato fruit ripening. Compared with the wild type (WT), most ripening-related biochemical characteristics were significantly reduced in SlSAHH2-CRISPR fruits. Additionally, the expression of genes related to ethylene synthesis and ripening was generally down-regulated. In transcriptome data, a total of 3701 up-regulated and 3134 down-regulated differentially expressed genes (DEGs) were identified and analyzed. Bisulfite sequencing PCR (BSP) results showed that the methylation levels of the promoters of ethylene-related genes in SlSAHH2-OE fruits were higher. Furthermore, in the presence of a methylation inhibitor, the expression of E4, E8, ACO1, ACO3, and ACS2 was generally up- or down-regulated in SlSAHH2-OE or SlSAHH2-CRISPR fruits, respectively. In summary, it can be inferred that in the balance between methylation and ethylene synthesis, SlSAHH2 was more inclined to promote the latter process during tomato fruit ripening.},
}
@article {pmid40680452,
year = {2025},
author = {Yang, T and Zhang, S and Nie, K and Cheng, C and Peng, X and Huo, J and Zhang, Y},
title = {ZNF207-driven PRDX1 lactylation and NRF2 activation in regorafenib resistance and ferroptosis evasion.},
journal = {Drug resistance updates : reviews and commentaries in antimicrobial and anticancer chemotherapy},
volume = {82},
number = {},
pages = {101274},
doi = {10.1016/j.drup.2025.101274},
pmid = {40680452},
issn = {1532-2084},
mesh = {Humans ; *NF-E2-Related Factor 2/metabolism/genetics ; *Ferroptosis/drug effects ; *Drug Resistance, Neoplasm/genetics/drug effects ; *Peroxiredoxins/metabolism/genetics ; *Liver Neoplasms/drug therapy/pathology/genetics ; *Carcinoma, Hepatocellular/drug therapy/pathology/genetics ; *Phenylurea Compounds/pharmacology ; *Pyridines/pharmacology ; Cell Line, Tumor ; Antineoplastic Agents/pharmacology ; CRISPR-Cas Systems ; },
abstract = {Regorafenib (RGF) is a critical second-line therapy for advanced hepatocellular carcinoma (HCC) following disease progression on sorafenib; however, the rapid onset of RGF resistance poses a significant barrier to enhancing patient outcomes. In this study, CRISPR/Cas9 screening in RGF-treated HCC cells identified Zinc Finger Protein 207 (ZNF207) as a primary driver of resistance. Further analysis revealed that ZNF207 promotes resistance by inducing antioxidant responses that inhibit ferroptosis, a form of iron-dependent cell death. Mechanistically, ZNF207 facilitates the lactylation of peroxiredoxin 1 (PRDX1) at lysine 67, enhancing nuclear translocation and activation of nuclear factor erythroid 2-related factor 2 (NRF2), a master regulator of antioxidant pathways. This ZNF207-PRDX1-NRF2 pathway creates a ferroptosis-resistant, pro-survival environment under RGF treatment, enabling HCC cells to evade cell death. Functional assays demonstrated that ZNF207 knockdown significantly enhances RGF sensitivity by restoring ferroptosis, with additional findings showing that disrupting PRDX1 lactylation or NRF2 activity similarly reverses resistance. Together, these findings establish a critical link between protein lactylation and RGF resistance, positioning the ZNF207-PRDX1-NRF2 axis as a promising therapeutic target to enhance treatment efficacy in HCC. The implications of this research extend beyond HCC, indicating that targeting ferroptosis-suppressive pathways may offer a broader approach to overcoming resistance in various cancers.},
}
@article {pmid40588867,
year = {2025},
author = {Rahimi, A and Karimipoor, M and Mahdian, R and Alipour, A and Hosseini, S and Mohammadi, M and Kaghazian, H and Shahsavarani, H and Shokrgozar, MA},
title = {Engineering of the Caspase-3 Gene in Recombinant CHO Cells Caused More Apoptosis Resistance and enhanced Recombinant Protein Production Than the BAX Gene.},
journal = {Iranian biomedical journal},
volume = {29},
number = {3},
pages = {1-10},
doi = {10.61186/ibj.4934},
pmid = {40588867},
issn = {2008-823X},
mesh = {Animals ; *Apoptosis/genetics ; *Caspase 3/genetics/metabolism ; *bcl-2-Associated X Protein/genetics/metabolism ; CHO Cells ; Cricetulus ; *Recombinant Proteins/biosynthesis/genetics ; Erythropoietin/biosynthesis/genetics ; CRISPR-Cas Systems/genetics ; *Genetic Engineering ; Cell Survival/genetics ; },
abstract = {BACKGROUND: BAX and caspase-3 are essential genes in the apoptotic pathway of cells, promoting the apoptotic cascade through different mechanisms. Inhibition of these genes can increase the longevity of cells in cell culture. This study aimed to compare the effects of CRISPR-Cas9-mediated knockdown of BAX and caspase-3 genes on apoptosis inhibition, cell lifespan, and EPO production in CHO cell lines.<br><br>METHODS: The BAX and caspase-3 gene expression was evaluated in the rCHO cell lines producing EPO using the CRISPR-Cas9 method. Their anti-apoptotic effects and the level of EPO expression were also compared. In addition, OP as an apoptosis inducer, was introduced to the manipulated cell line to assess the stability and viability of the manipulated cell lines.<br><br>RESULTS: The rCHO cells with the manipulated BAX gene exhibited a higher cell density than those with the manipulated caspase-3 gene (152% vs. 142%). Despite the increased cell density in the cells with the BAX gene manipulation, EPO production was higher in the cells with the manipulated caspase-3 gene (1.58-fold increase in the BAX-manipulated cells compared to a 1.70-fold increase in the caspase-3-manipulated cells).<br><br>CONCLUSION: Our observations suggest that the downregulation of the BAX and caspase-3 genes using the CRISPR method, inhibits apoptosis and enhances the yield of recombinant EPO, even in the presence of an apoptosis inducer. Additionally, reduction of caspase-3 expression was proved to be more effective than BAX in extending the lifespan of cells and producing heterologous recombinant proteins.},
}
@article {pmid40460025,
year = {2025},
author = {Solayappan, M and Azlan, A and Khor, KZ and Yik, MY and Jaishanker, A and Ramesh, T and Saleem, M and Yusoff, NM and Moses, EJ},
title = {Multifaceted roles of CRISPR technology in blood cancer research.},
journal = {Current opinion in hematology},
volume = {32},
number = {5},
pages = {287-299},
doi = {10.1097/MOH.0000000000000855},
pmid = {40460025},
issn = {1531-7048},
mesh = {Humans ; *Hematologic Neoplasms/genetics/therapy ; *Gene Editing/methods ; *CRISPR-Cas Systems ; Animals ; Genetic Therapy/methods ; Genomics/methods ; },
abstract = {PURPOSE OF REVIEW: Blood cancers are one of the most common cancers worldwide. These diseases stem from defects in blood components having cytogenetic aberrations and genetic mutations. There have been vast improvements in terms of treatment options and survival outcomes. Nevertheless, due to the clonal nature and heterogeneity of the diseases, the number of cases reported exhibit a rising pattern due to chemoresistance and disease relapse thus posing a healthcare burden. Therefore, the need for more specific forms of targeted therapies is ever-present.<br><br>RECENT FINDINGS: CRISPR has emerged as a key player and is the epitome of gene editing technology in this post genomic era. In line with the current trend, numerous studies in blood cancer research have extensively utilized CRISPR-based applications to understand the functional genomics of hematologic malignancies and identify potential therapeutic targets for development of novel therapeutic applications.<br><br>SUMMARY: The importance of comprehending the utilities of state-of-the-art technologies such as CRISPR for studying hematologic malignancies has never been more apparent and timelier. Therefore, this review attempts to scrutinize the versatility of CRISPR applications which range from functional genomics to immunotherapeutic applications.},
}
@article {pmid40449371,
year = {2025},
author = {Wang, M and Zhang, X and Fan, J and Zhang, C and Xian, Y},
title = {AND logic gate-based alternating PER-Cas12a signal amplification system for ultrasensitive detection of sEVs.},
journal = {Talanta},
volume = {295},
number = {},
pages = {128411},
doi = {10.1016/j.talanta.2025.128411},
pmid = {40449371},
issn = {1873-3573},
mesh = {Humans ; *Breast Neoplasms/diagnosis/pathology ; *Extracellular Vesicles/metabolism/chemistry ; Female ; *Biomarkers, Tumor ; Mucin-1/metabolism/genetics ; *CRISPR-Associated Proteins/metabolism/genetics ; *Endodeoxyribonucleases/metabolism/genetics ; Limit of Detection ; Epithelial Cell Adhesion Molecule/metabolism ; *Nucleic Acid Amplification Techniques/methods ; *Logic ; Biosensing Techniques/methods ; CRISPR-Cas Systems ; Bacterial Proteins ; },
abstract = {Protein biomarkers on breast cancer-derived small extracellular vesicles (BC-sEVs) hold great promise in liquid biopsy. However, it remains challenging due to their inherent heterogeneity and low abundance. Herein, we developed an AND logic gate-based DNA cascade signal amplification strategy, termed Alternating Primer Exchange Reaction-activated Cas12a (Alt-PER-Cas12a), for the ultrasensitive detection of BC-sEVs in clinic samples. This dual-protein recognition system employs EpCAM/MUC1-specific capture probes to release two DNA hairpins (Hep and Hmu) as AND gate inputs in Alt-PER. The corresponding Hep and Hmu hairpins can initiate the Alt-PER with a large amount of primers to generate long single-stranded DNA products with alternating repeat units. Each repeating unit serves as a CRISPR activator, inducing the trans-cleavage activity of Cas12a and enabling cascade signal amplification. The as-constructed strategy exhibits excellent sensitivity with LOD of 2.6 × 10[3] particles/mL. It has been successfully used to discriminate breast cancer patients from healthy donors (AUC = 0.992) in clinical validation, and shows great potential for liquid biopsy.},
}
@article {pmid40393239,
year = {2025},
author = {Lin, G and Li, J and Zhang, K},
title = {Multiple CRISPR zones-driven ultrasensitive detection of DNA via CRISPR-Cas12a and ligation-rolling circle amplification.},
journal = {Talanta},
volume = {295},
number = {},
pages = {128336},
doi = {10.1016/j.talanta.2025.128336},
pmid = {40393239},
issn = {1873-3573},
mesh = {*CRISPR-Cas Systems/genetics ; *DNA/analysis/genetics ; *Nucleic Acid Amplification Techniques/methods ; Limit of Detection ; *CRISPR-Associated Proteins/metabolism/genetics ; Humans ; Polymorphism, Single Nucleotide ; *Endodeoxyribonucleases/genetics/metabolism ; Bacterial Proteins ; },
abstract = {The ability to detect specific DNA, including single nucleotide variants (SNVs), with high sensitivity is essential for advancing genetic research, diagnostics, and personalized medicine. This study presents a novel method for ultrasensitive DNA detection, combining ligation-rolling circle amplification (L-RCA) with CRISPR-Cas12a. While L-RCA systems have been widely used for nucleic acid detection, the sensitivity of conventional L-RCA generally reaches approximately 100 pM. Here, we demonstrate that the sensitivity of RCA-Cas12a systems can be markedly enhanced by incorporating multiple CRISPR target regions into the padlock probe. This method achieves remarkable sensitivity, detecting DNA at concentrations as low as 1 aM (6 copies per reaction), and is capable of identifying single nucleotide variants (SNVs) with allele fractions as low as 1 %. Unlike many current complex RCA-Cas12a strategies, this approach is simple and does not require advanced labeling or instrumentation, making it a promising tool for ultrasensitive DNA detection in various applications.},
}
@article {pmid39998382,
year = {2025},
author = {Lin, K and Zou, C and Hubbard, A and Sengelmann, S and Goudy, L and Wang, IC and Sharma, R and Pak, J and Foster, K and Ozawa, T and de Groot, JF and Phillips, J and Vasudevan, HN and Raleigh, DR and Marson, A and Murthy, N and Gilbert, LA and Berger, MS and Liu, SJ},
title = {Multiplexed epigenetic memory editing using CRISPRoff sensitizes glioblastoma to chemotherapy.},
journal = {Neuro-oncology},
volume = {27},
number = {6},
pages = {1443-1457},
doi = {10.1093/neuonc/noaf055},
pmid = {39998382},
issn = {1523-5866},
support = {P50 CA097257/CA/NCI NIH HHS/United States ; //CRISPR/ ; P50 CA097257/GF/NIH HHS/United States ; },
mesh = {*Glioblastoma/genetics/drug therapy/pathology ; Humans ; Animals ; *Brain Neoplasms/genetics/drug therapy/pathology ; Mice ; *Epigenesis, Genetic ; Temozolomide/pharmacology ; Xenograft Model Antitumor Assays ; *Gene Editing/methods ; Lomustine/pharmacology ; Tumor Cells, Cultured ; *Drug Resistance, Neoplasm/genetics ; DNA Repair Enzymes/genetics ; *CRISPR-Cas Systems ; Tumor Suppressor Proteins/genetics ; DNA Modification Methylases/genetics ; Mice, Nude ; Epigenetic Memory ; },
abstract = {BACKGROUND: Glioblastoma (GBM) carries a poor prognosis, and new therapeutic strategies are necessary to improve outcomes for patients with this disease. Alkylating chemotherapies including temozolomide (TMZ) and lomustine (CCNU) are critical for treating GBM, but resistance mechanisms, including hypomethylation of O6-methylguanine-DNA methyltransferase (MGMT) promoter, undermine treatment. CRISPRoff is a programmable epigenetic memory editor that can induce stable and heritable gene silencing after transient delivery, and we hypothesize that CRISPRoff could potentiate the activity of TMZ and CCNU through long-term suppression of target genes.<br><br>METHODS: We transiently delivered CRISPRoff mRNA along with sgRNAs against target genes using both electroporation and lipid nanoparticles (LNPs) into established GBM cell lines, patient-derived primary GBM cultures, and orthotopic GBM xenografts. Gene repression, specificity, and stability were measured by RT-qPCR, Western blot, bisulfite sequencing, and RNA sequencing. Sensitivity to chemotherapies was measured by cell viability dose-response, microscopy, and bioluminescence imaging. Genome-wide mapping of CCNU sensitizers was performed using CRISPRi screens.<br><br>RESULTS: CRISPRoff induced complete suppression of MGMT and sensitization to TMZ that was stable for over 8 months of continuous cell propagation. GBM orthotopic tumors treated with CRISPRoff against MGMT demonstrated sensitivity to TMZ in vivo, and CRISPRoff delivery resulted in chemosensitivity in patient-derived primary GBM. Genome-wide CRISPRi screens identified combinatorial genetic vulnerabilities (BRIP1, FANCE) that were targetable by multiplexed CRISPRoff to achieve sensitization to CCNU.<br><br>CONCLUSION: Transient delivery of a site-specific epigenetic memory can induce stable, complete, and multiplexed suppression of target genes for therapeutic application in GBM.},
}
@article {pmid40727907,
year = {2025},
author = {Arifah, AQ and Vento, JM and Kurrer, I and Achmedov, T and Beisel, CL},
title = {Cas9-independent tracrRNA cytotoxicity in Lacticaseibacillus paracasei.},
journal = {microLife},
volume = {6},
number = {},
pages = {uqaf013},
pmid = {40727907},
issn = {2633-6693},
abstract = {CRISPR-Cas9 systems are widely used for bacterial genome editing, yet their heterologous expression has been associated with cytotoxicity. The Cas9 nuclease from Streptococcus pyogenes (SpyCas9) has been one common source, with reports of cytotoxicity with the nuclease alone or in combination with a single-guide RNA observed in some bacteria. However, the potential cytotoxic effects of other components of the CRISPR-Cas9 system remain unknown. Here, we report that expression of the short isoform of the trans-activating CRISPR RNA (tracr-S) from the S. pyogenes CRISPR-Cas locus is cytotoxic in Lacticaseibacillus paracasei, even in the absence of SpyCas9. Deleting a putative transcription regulator in L. paracasei alleviates tracr-S cytotoxicity and leads to expression of the long isoform of the trans-activating CRISPR RNA (tracr-L). Furthermore, cytotoxicity was specific to the tracr-S sequence and was linked to direct interactions with host RNAs. This work thus reveals that additional CRISPR components beyond Cas9 can interfere with the use of heterologous CRISPR-Cas systems in bacteria, with potential implications for the evolution of CRISPR immunity.},
}
@article {pmid40726316,
year = {2025},
author = {Alary, B and Mortada, M and Mas, P},
title = {Gene editing of clock components in Solanum lycopersicum: Effects on gene expression, development, and productivity.},
journal = {The Plant journal : for cell and molecular biology},
volume = {123},
number = {2},
pages = {e70383},
doi = {10.1111/tpj.70383},
pmid = {40726316},
issn = {1365-313X},
support = {2021-SGR-01131//Ag&#xe8;ncia de Gesti&#xf3; d'Ajuts Universitaris i de Recerca/ ; PID2022-137770NB-I00//Ministerio de Ciencia, Innovaci&#xf3;n y Universidades/ ; V6423//Fundaci&#xf3;n Ram&#xf3;n Areces/ ; CEX2019-000902-S//Ministerio de Econom&#xed;a y Competitividad/ ; },
mesh = {*Solanum lycopersicum/genetics/growth &amp; development/physiology ; *Gene Editing ; *Circadian Clocks/genetics ; Gene Expression Regulation, Plant ; Plant Leaves/genetics/physiology/growth &amp; development ; CRISPR-Cas Systems ; Circadian Rhythm/genetics ; Arabidopsis/genetics ; Plant Proteins/genetics/metabolism ; Fruit/genetics/growth &amp; development/physiology ; },
abstract = {The circadian clock plays a crucial role in regulating key biological processes, including growth and development. While studies in the model plant Arabidopsis thaliana have significantly advanced our understanding of circadian function, recent research has also focused on crop species for improved yield and quality. In this study, we examined the rhythmic behavior and regulatory function of circadian clock components in tomato (Solanum lycopersicum). Time course analyses of gene expression over the circadian cycle revealed robust rhythmic oscillations in tomato leaves under free-running conditions. Comparative analyses showed similar peak phases for several clock genes in Arabidopsis and tomato, suggesting functional conservation. Rhythms in tomato fruits, however, showed reduced amplitude, slight phase changes, or arrhythmia, indicating organ-specific circadian variations. By using CRISPR-Cas9 gene editing strategies (clock[crispr]), we also showed that proper clock gene expression is essential for setting the phase in tomato plants. Leaf movement analyses also showed a phase change in the clock[crispr] lines, correlating with shorter or longer periods. The clock[crispr] lines also displayed distinct growth and developmental phenotypes that differ from those reported in the Arabidopsis clock mutant counterparts. Our transcriptomic analyses identified species-specific regulation of key target genes. The results offer mechanistic insights into the conserved and divergent molecular pathways governing circadian phenotypic variations between Arabidopsis and tomato plants.},
}
@article {pmid40725481,
year = {2025},
author = {Cosiquien, RJS and Whalen, IJ and Wong, P and Sorensen, RJ and Shetty, AV and Liang, SQ and Steer, CJ},
title = {Detecting Methylation Changes Induced by Prime Editing.},
journal = {Genes},
volume = {16},
number = {7},
pages = {},
doi = {10.3390/genes16070825},
pmid = {40725481},
issn = {2073-4425},
mesh = {Humans ; *DNA Methylation/genetics ; *Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; CpG Islands/genetics ; *Epigenesis, Genetic ; },
abstract = {While prime editing offers improved precision compared to traditional CRISPR-Cas9 systems, concerns remain regarding potential off-target effects, including epigenetic changes such as DNA methylation. In this study, we investigated whether prime editing induces aberrant CpG methylation patterns. Whole-genome bisulfite sequencing revealed overall methylation similarity between Cas9-edited, and PE2-edited cells. However, localized epigenetic changes were observed, particularly in CpG islands and exon regions. The PE2-edited group showed a higher proportion of differentially methylated regions (DMRs) in some coding sequences compared to controls and Cas9-edited samples. Notably, CpG island methylation reached 0.18% in the PE2 vs. Cas9 comparison, indicating a higher susceptibility of these regulatory elements to epigenetic alterations by prime editing. Molecular function analyses including Gene Ontology and KEGG pathway analyses further revealed enrichment in molecular functions related to transcriptional regulation and redox activity in PE2-edited cells. These findings suggest that prime editing, while precise, may introduce subtle but functionally relevant methylation changes that could influence gene expression and cellular pathways. In summary, prime editing can induce localized DNA methylation changes in human cells, particularly within regulatory and coding regions. Understanding these epigenetic consequences is critical for the development of safer and more effective therapeutic applications of genome editing technologies.},
}
@article {pmid40725400,
year = {2025},
author = {Kim, JY and Jung, YJ and Kim, DH and Kang, KK},
title = {Transcriptomic Insights into GABA Accumulation in Tomato via CRISPR/Cas9-Based Editing of SlGAD2 and SlGAD3.},
journal = {Genes},
volume = {16},
number = {7},
pages = {},
doi = {10.3390/genes16070744},
pmid = {40725400},
issn = {2073-4425},
support = {2021R1I1A4A01057295//the National Research Foundation of Korea (NRF)/ ; },
mesh = {*Solanum lycopersicum/genetics/metabolism/growth &amp; development ; *gamma-Aminobutyric Acid/metabolism/genetics ; *CRISPR-Cas Systems ; *Glutamate Decarboxylase/genetics/metabolism ; Gene Editing/methods ; *Transcriptome/genetics ; *Plant Proteins/genetics/metabolism ; Gene Expression Regulation, Plant ; Fruit/genetics/metabolism ; Plant Leaves/genetics/metabolism ; Plants, Genetically Modified/genetics ; },
abstract = {BACKGROUND: γ-Aminobutyric acid (GABA) is a non-proteinogenic amino acid with key roles in plant metabolism, stress responses, and fruit nutritional quality. In tomato (Solanum lycopersicum), GABA levels are dynamically regulated during fruit development but decline in the late ripening stages.<br><br>METHODS: To enhance GABA accumulation, we used CRISPR/Cas9 to edit the calmodulin-binding domain (CaMBD) of SlGAD2 and SlGAD3, which encode glutamate decarboxylases (GADs). The resulting truncated enzymes were expected to be constitutively active. We quantified GABA content in leaves and fruits and performed transcriptomic analysis on edited lines at the BR+7 fruit stage.<br><br>RESULTS: CaMBD truncation significantly increased GABA levels in both leaves and fruits. In gad2 sg1 lines, GABA levels increased by 3.5-fold in leaves and 3.2-fold in BR+10 fruits; in gad3 sg3 lines, increases of 2.8- and 2.5-fold were observed, respectively. RNA-seq analysis identified 1383 DEGs in gad2 #1-5 and 808 DEGs in gad3 #3-8, with 434 DEGs shared across both lines. These shared DEGs showed upregulation of GAD, GABA-T, and SSADH, and downregulation of stress-responsive transcription factors including WRKY46, ERF, and NAC. Notably, total free amino acid content and fruit morphology remained unchanged despite elevated GABA.<br><br>CONCLUSIONS: CRISPR/Cas9-mediated editing of the CaMBD in SlGAD genes selectively enhances GABA biosynthesis in tomato without adverse effects on development or fruit quality. These lines offer a useful platform for GABA-centered metabolic engineering and provide insights into GABA's role in transcriptional regulation during ripening.},
}
@article {pmid40725376,
year = {2025},
author = {Kumar, H and Gal'chinsky, N and Sweta, V and Negi, N and Filatov, R and Chandel, A and Ali, J and Oberemok, V and Laikova, K},
title = {Perspectives of RNAi, CUADb and CRISPR/Cas as Innovative Antisense Technologies for Insect Pest Control: From Discovery to Practice.},
journal = {Insects},
volume = {16},
number = {7},
pages = {},
doi = {10.3390/insects16070746},
pmid = {40725376},
issn = {2075-4450},
support = {FZEG-2024-0001//This research results obtained within the framework of a state assignment V.I. Vernadsky Cri-mean Federal University for 2024 and the planning period of 2024-2026/ ; },
abstract = {Pest management is undergoing a transformative shift with the development of the cutting-edge antisense technologies: RNA interference (RNAi), contact unmodified antisense DNA biotechnology (CUADb), and the CRISPR-associated proteins (CRISPR/Cas). These approaches function by facilitating sequence-specific pairing of nucleic acids followed by nuclease-mediated cleavage, offering exceptional precision for targeted pest control. While RNA-guided mechanisms such as RNAi and CRISPR/Cas were initially characterized in non-insect systems, primarily as innate defenses against viral infections, the DNA-guided CUADb pathway was first identified in insect pests as a functional pest control strategy. Its broader role in ribosomal RNA (rRNA) biogenesis was recognized later. Together, these discoveries have revealed an entirely new dimension of gene regulation, with profound implications for sustainable pest management. Despite sharing a common principle of sequence-specific targeting RNAi, CUADb, and CRISPR/Cas differ in several key aspects, including their mechanisms of action, target specificity, and applicability. Rather than serving as universal solutions, each technology is likely to be optimally effective against specific pest groups. Moreover, these technologies allow for rapid adaptation of control strategies to overcome target-site resistance, ensuring long-term efficacy. This review summarizes the core functional characteristics, potential applications, and current limitations of each antisense technology, emphasizing their complementary roles in advancing environmentally sustainable pest control. By integrating foundational biological discoveries with applied innovations, this work provides a new perspectives on incorporating antisense-based strategies into next-generation integrated pest management systems.},
}
@article {pmid40725259,
year = {2025},
author = {Wolny, E and Mur, LAJ and Ohmido, N and Yin, Z and Wang, K and Hasterok, R},
title = {Thriving or Withering? Plant Molecular Cytogenetics in the First Quarter of the 21st Century.},
journal = {International journal of molecular sciences},
volume = {26},
number = {14},
pages = {},
doi = {10.3390/ijms26147013},
pmid = {40725259},
issn = {1422-0067},
mesh = {*Cytogenetic Analysis/methods ; Genome, Plant ; Chromosomes, Plant/genetics ; *Plants/genetics ; In Situ Hybridization, Fluorescence/methods ; *Cytogenetics/methods/trends ; Polyploidy ; Genomics/methods ; },
abstract = {Nearly four decades have passed since fluorescence in situ hybridisation was first applied in plants to support molecular cytogenetic analyses across a wide range of species. Subsequent advances in DNA sequencing, bioinformatic analysis, and microscopy, together with the immunolocalisation of various nuclear components, have provided unprecedented insights into the cytomolecular organisation of the nuclear genome in both model and non-model plants, with crop species being perhaps the most significant. The ready availability of sequenced genomes is now facilitating the application of state-of-the-art cytomolecular techniques across diverse plant species. However, these same advances in genomics also pose a challenge to the future of plant molecular cytogenetics, as DNA sequence analysis is increasingly perceived as offering comparable insights into genome organisation. This perception persists despite the continued relevance of FISH-based approaches for the physical anchoring of genome assemblies to chromosomes. Furthermore, cytogenetic approaches cannot currently rival purely genomic methods in terms of throughput, standardisation, and automation. This review highlights the latest key topics in plant cytomolecular research, with particular emphasis on chromosome identification and karyotype evolution, chromatin and interphase nuclear organisation, chromosome structure, hybridisation and polyploidy, and cytogenetics-assisted crop improvement. In doing so, it underscores the distinctive contributions that cytogenetic techniques continue to offer in genomic research. Additionally, we critically assess future directions and emerging opportunities in the field, including those related to CRISPR/Cas-based live-cell imaging and chromosome engineering, as well as AI-assisted image analysis and karyotyping.},
}
@article {pmid40725213,
year = {2025},
author = {Covache-Busuioc, RA and Toader, C and Rădoi, MP and Șerban, M},
title = {Precision Recovery After Spinal Cord Injury: Integrating CRISPR Technologies, AI-Driven Therapeutics, Single-Cell Omics, and System Neuroregeneration.},
journal = {International journal of molecular sciences},
volume = {26},
number = {14},
pages = {},
doi = {10.3390/ijms26146966},
pmid = {40725213},
issn = {1422-0067},
mesh = {*Spinal Cord Injuries/therapy/genetics/physiopathology ; Humans ; *Artificial Intelligence ; Animals ; Gene Editing/methods ; *Nerve Regeneration ; Single-Cell Analysis/methods ; *CRISPR-Cas Systems ; Recovery of Function ; Regenerative Medicine/methods ; Precision Medicine/methods ; },
abstract = {Spinal cord injury (SCI) remains one of the toughest obstacles in neuroscience and regenerative medicine due to both severe functional loss and limited healing ability. This article aims to provide a key integrative, mechanism-focused review of the molecular landscape of SCI and the new disruptive therapy technologies that are now evolving in the SCI arena. Our goal is to unify a fundamental pathophysiology of neuroinflammation, ferroptosis, glial scarring, and oxidative stress with the translation of precision treatment approaches driven by artificial intelligence (AI), CRISPR-mediated gene editing, and regenerative bioengineering. Drawing upon advances in single-cell omics, systems biology, and smart biomaterials, we will discuss the potential for reprogramming the spinal cord at multiple levels, from transcriptional programming to biomechanical scaffolds, to change the course from an irreversible degeneration toward a directed regenerative pathway. We will place special emphasis on using AI to improve diagnostic/prognostic and inferred responses, gene and cell therapies enabled by genomic editing, and bioelectronics capable of rehabilitating functional connectivity. Although many of the technologies described below are still in development, they are becoming increasingly disruptive capabilities of what it may mean to recover from an SCI. Instead of prescribing a particular therapeutic fix, we provide a future-looking synthesis of interrelated biological, computational, and bioengineering approaches that conjointly chart a course toward adaptive, personalized neuroregeneration. Our intent is to inspire a paradigm shift to resolve paralysis through precision recovery and to be grounded in a spirit of humility, rigor, and an interdisciplinary approach.},
}
@article {pmid40725041,
year = {2025},
author = {Fizikova, A and Prokhorova, A and Churikova, D and Konstantinov, Z and Ivanov, R and Karabelsky, A and Rybtsov, S},
title = {Hepatocytes as Model for Investigating Natural Senotherapeutic Compounds and Their Effects on Cell Cycle Dynamics and Genome Stability.},
journal = {International journal of molecular sciences},
volume = {26},
number = {14},
pages = {},
doi = {10.3390/ijms26146794},
pmid = {40725041},
issn = {1422-0067},
support = {Agreement No. 18-03 on 10 September 2024//the state program of the "Sirius" Federal Territory "Scientific and technological development of the "Sirius" Federal Territory"/ ; },
mesh = {Humans ; *Genomic Instability/drug effects ; Animals ; *Senotherapeutics/pharmacology ; *Cell Cycle/drug effects ; *Hepatocytes/drug effects/metabolism/cytology ; Cellular Senescence/drug effects ; DNA Damage ; },
abstract = {DNA is inherently unstable and is susceptible to damage from both endogenous sources (such as reactive oxygen species) and exogenous factors (including UV, ionizing radiation, and chemicals). The accumulation of DNA damage manifests as genetic mutations, chromosomal instability, and the stalling of DNA replication and transcription processes. Accumulated DNA damage influences apoptosis and cell cycle checkpoints, serving as one of the key triggers for the manifestation of the senescent phenotype. Both aging and cancer are associated with the accumulation of mutations in somatic cells. Disruption of cell cycle control and uncontrolled proliferation are fundamental characteristics of any cancer cell, with the majority of anticancer drugs acting as inhibitors of cyclin-dependent kinases, thereby inducing a transition of cells into a senescent state. Consequently, disturbances in the dynamics and regulation of inflammatory responses, oxidative stress, cell proliferation, DNA damage repair, and epigenetic anomalies, along with the influence of retroviruses and transposons, lead to the accumulation of senescent cells within the human body, characterized by blocked replication and cell cycle, as well as a distinct secretory phenotype. The age-related or disease-associated accumulation of these senescent cells significantly alters the physiology of tissues and the organism as a whole. Many secondary metabolites of higher plants exhibit senolytic and senomorphic activities, although most of them are not fully characterized. In this review, we will explore the principal signaling pathways in mammalian cells that govern the cell cycle and cellular senescence, with a particular emphasis on how their dynamics, expression, and regulation have been modified through the application of senotherapeutic compounds. The second section of the review will identify key target genes for the metabolic engineering, primarily aimed at enhancing the accumulation of plant secondary metabolites with potential therapeutic benefits. Lastly, we will discuss the rationale for utilizing liver cells as a model system to investigate the effects of senolytic compounds on human physiology and health, as well as how senotherapeutic substances can be leveraged to improve gene therapy approaches based on CRISPR/Cas9 and prime-editing technologies.},
}
@article {pmid40724990,
year = {2025},
author = {Montagna, C and Maiani, E and Pieroni, L and Consalvi, S},
title = {Duchenne Muscular Dystrophy: Integrating Current Clinical Practice with Future Therapeutic and Diagnostic Horizons.},
journal = {International journal of molecular sciences},
volume = {26},
number = {14},
pages = {},
doi = {10.3390/ijms26146742},
pmid = {40724990},
issn = {1422-0067},
abstract = {Duchenne muscular dystrophy (DMD) is a severe X-linked disorder characterized by progressive muscle degeneration due to mutations in the dystrophin gene. Despite major advancements in understanding its pathophysiology, there is still no curative treatment. This review provides an up-to-date overview of current and emerging therapeutic approaches-including antisense oligonucleotides, gene therapy, gene editing, corticosteroids, and histone deacetylases(HDAC) inhibitors-aimed at restoring dystrophin expression or mitigating disease progression. Special emphasis is placed on the importance of early diagnosis, the utility of genetic screening, and the innovations in pre-and post-natal testing. As the field advances toward personalized medicine, the integration of precision therapies with cutting-edge diagnostic technologies promises to improve both prognosis and quality of life for individuals with DMD.},
}
@article {pmid40724964,
year = {2025},
author = {Ruiz-Moreno, HA and Valderrama-Rincon, JD and Cala, MP and Fernández-Niño, M and Valderruten Cajiao, M and Villegas-Torres, MF and González Barrios, AF},
title = {Enhanced Outer Membrane Vesicle Production in Escherichia coli: From Metabolic Network Model to Designed Strain Lipidomic Profile.},
journal = {International journal of molecular sciences},
volume = {26},
number = {14},
pages = {},
doi = {10.3390/ijms26146714},
pmid = {40724964},
issn = {1422-0067},
support = {822-2017//Colciencias/ ; },
mesh = {*Escherichia coli/metabolism/genetics ; *Lipidomics/methods ; *Metabolic Networks and Pathways ; *Bacterial Outer Membrane/metabolism ; Mutation ; Escherichia coli Proteins/genetics/metabolism ; CRISPR-Cas Systems ; Gene Knockout Techniques ; *Extracellular Vesicles/metabolism ; Bacterial Outer Membrane Proteins/metabolism/genetics ; Gene Editing ; },
abstract = {Bacterial structures formed from the outer membrane and the periplasm components carry biomolecules to expel cellular material and interact with other cells. These outer membrane vesicles (OMVs) can encapsulate bioactive content, which confers OMVs with high potential as alternative drug delivery vehicles or as a platform for novel vaccine development. Single-gene mutants derived from Escherichia coli JC8031 were engineered to further enhance OMV production based on metabolic network modelling and in silico gene knockout design (ΔpoxB, ΔsgbE, ΔgmhA, and ΔallD). Mutants were experimentally obtained by genome editing using CRISPR-Cas9 and tested for OMVs recovery observing an enhanced OMV production in all of them. Lipidomic analysis through LC-ESI-QTOF-MS was performed for OMVs obtained from each engineered strain and compared to the wild-type E. coli JC8031 strain. The lipid profile of OMVs from the wild-type E. coli JC8031 did not change significantly confirmed by multivariate statistical analysis when compared to the mutant strains. The obtained results suggest that the vesicle production can be further improved while the obtained vesicles are not altered in their composition, allowing further study for stability and integrity for use in therapeutic settings.},
}
@article {pmid40724866,
year = {2025},
author = {Nidhi, F and Tomatsu, S},
title = {Integrase-Deficient Lentiviral Vector as a Platform for Efficient CRISPR/Cas9-Mediated Gene Editing for Mucopolysaccharidosis IVA.},
journal = {International journal of molecular sciences},
volume = {26},
number = {14},
pages = {},
doi = {10.3390/ijms26146616},
pmid = {40724866},
issn = {1422-0067},
support = {1R01HD102545-01A1/GF/NIH HHS/United States ; },
mesh = {Animals ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; Mice ; *Lentivirus/genetics ; *Mucopolysaccharidosis IV/therapy/genetics ; *Genetic Vectors/genetics ; Humans ; NIH 3T3 Cells ; Genetic Therapy/methods ; *Chondroitinsulfatases/genetics/metabolism ; *Integrases/genetics/deficiency ; Fibroblasts/metabolism ; Disease Models, Animal ; },
abstract = {Mucopolysaccharidosis IVA (MPS IVA) is a lysosomal storage disorder causing systemic skeletal dysplasia due to a deficiency of N-acetyl-galactosamine-6-sulfate sulfatase (GALNS) enzyme activity, leading to the impaired degradation and accumulation of glycosaminoglycans (GAGs), keratan sulfate (KS) and chondroitin-6-sulfate. While treatments such as enzyme replacement therapy (ERT) and hematopoietic stem cell transplantation (HSCT) are available, they have significant limitations regarding efficacy in skeletal tissues and long-term safety, highlighting the need for more effective therapies. We evaluated a novel gene therapy approach using a dual Integrase-deficient lentiviral vector (IDLV) to deliver an expression cassette that includes human GALNS cDNA and Cas9 sgRNA, targeting the upstream region of the mouse Galns initial codon. This approach leverages the endogenous promoter to drive transgene expression. We assessed in vitro transduction, editing, and functional correction in NIH3T3 and MPS IVA mouse fibroblasts. In vivo efficacy was successfully evaluated via the facial vein injection in MPS IVA newborn mice. In vitro, this IDLV platform demonstrated supraphysiological GALNS activity in cell lysate, resulting in the normalization of KS levels. In vivo direct IDLV platform in newborn MPS IVA mice led to sustained plasma GALNS activity, reduced plasma KS, and favorable biodistribution. Partial correction of heart and bone pathology was observed, with no vector toxicity and minimal antibody responses. This dual IDLV-CRISPR/Cas9 approach effectively mediated targeted GALNS knock-in, yielding sustained enzyme activity, reduced KS storage, and partial pathological amelioration in MPS IVA mice. In conclusion, IDLVs represent an efficient, safe platform for delivering the CRISPR/Cas9 gene editing system for MPS IVA.},
}
@article {pmid40722665,
year = {2025},
author = {Zhang, A and Zhang, I and Liu, F},
title = {Applications of CRISPR-Cas-Based Genome Editing Approaches Against Human Cytomegalovirus Infection.},
journal = {Biomedicines},
volume = {13},
number = {7},
pages = {},
doi = {10.3390/biomedicines13071590},
pmid = {40722665},
issn = {2227-9059},
support = {Start-Up Fund 101//University of California, Berkeley/ ; },
abstract = {Human cytomegalovirus (HCMV), a globally ubiquitous herpesvirus with the ability to carry out both lytic productive and lifelong latent infections, is a major cause of congenital infections, often leading to intellectual disabilities and neurological disorders. Moreover, HCMV is an opportunistic pathogen commonly found in immunocompromised individuals such as organ transplant recipients, HIV-positive individuals, and cancer patients, causing severe and life-threatening complications. While effective in inhibiting viral lytic infection, current FDA-approved compounds cannot eliminate the latent viral genome and have little effect on viral latent infection. Developing novel antiviral therapeutic approaches to eliminate HCMV lytic and latent infections is a major public health priority for controlling HCMV infection and preventing viral-associated diseases. The genome-editing technology based on the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated protein (Cas) RNA-guided nuclease system represents a novel and promising antiviral approach through modifying or destroying the genetic material of human viruses. This review summarizes the recently published progress in using the CRISPR-Cas approach to study and inhibit HCMV infections and discusses prospects for developing the CRISPR-based genome-editing technology for therapeutic applications against HCMV infection and associated diseases.},
}
@article {pmid40721002,
year = {2025},
author = {Ahi, EP and Khorshid, M},
title = {Potentials of RNA biosensors in developmental biology.},
journal = {Developmental biology},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.ydbio.2025.07.011},
pmid = {40721002},
issn = {1095-564X},
abstract = {RNA-based/associated biosensors represent a rapidly expanding area of research, providing highly sensitive tools for detecting and monitoring RNA in diverse biological contexts. These sensors offer the ability to track RNA localization, modifications, and interactions in real-time, making them particularly well-suited for developmental biology research. Despite their demonstrated utility in fields such as diagnostics, synthetic biology and environmental science, the application of RNA biosensors in developmental biology has only begun to emerge within the past decade. This gap is notable given the potential of these tools to address key questions about spatiotemporal RNA regulation and cellular signaling during development. This perspective review presents a selection of RNA biosensors, including fluorescent RNA aptamers, CRISPR-Cas-based systems, riboswitches, and catalytic RNA sensors, which have gained attraction in other scientific disciplines. These tools can be used not only to study intrinsic RNA biology, such as RNA expression, splicing, and localization, but also to detect the effects of extrinsic physical and chemical factors, including pH, temperature, redox state, and mechanical stress, on RNA behavior during developmental processes. These examples illustrate how RNA biosensors could be adapted to study developmental mechanisms in model organisms, enabling investigations into RNA dynamics and their role in shaping developmental processes. By revisiting these underutilized tools, this review highlights their relevance for advancing the understanding of molecular mechanisms in developmental biology studies.},
}
@article {pmid40716775,
year = {2025},
author = {Nalefski, EA and Hedley, S and Rajaraman, K and Kooistra, RM and Parikh, I and Sinan, S and Finklestein, IJ and Madan, D},
title = {Unleashing high trans-substrate cleavage kinetics of Cas12a for nucleic acid diagnostics.},
journal = {Nucleic acids research},
volume = {53},
number = {14},
pages = {},
doi = {10.1093/nar/gkaf712},
pmid = {40716775},
issn = {1362-4962},
support = {//College of Natural Sciences Catalyst/ ; //Global Health Labs/ ; F-1808//Welch Foundation/ ; },
mesh = {*CRISPR-Associated Proteins/metabolism/genetics ; *CRISPR-Cas Systems ; Kinetics ; *Endodeoxyribonucleases/metabolism/genetics ; *Bacterial Proteins/metabolism/genetics ; DNA/metabolism/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats ; RNA, Guide, CRISPR-Cas Systems/genetics ; Substrate Specificity ; *Nucleic Acids/genetics/analysis ; Ribonucleoproteins/metabolism ; },
abstract = {CRISPR (clustered regularly interspaced short palindromic repeats)-based nucleic acid diagnostics enable rapid, sensitive pathogen detection. Cas12a is frequently used in these assays because target-activated trans cleavage of a reporter molecule generates an easily detectable signal. However, variable activity across assays suggests that the catalytic potential of Cas12a has been limited via unknown mechanisms. Here, we show that Cas12a trans-nuclease activity is auto-inhibited by long PAM-proximal DNA (>120 bp) following cis-cleavage of targets. Short targets (<100 bp), optimized trans cleavage substrates, and low salt buffers unleash high catalytic efficiency (≈108 M-1 s-1) and turnover (≈1 s-1) across Cas12a orthologs. Pooling multiple Cas12a ribonucleoproteins (RNPs) targeting clustered protospacers overcomes cis-cleavage auto-inhibition, further boosting sensitivity. Optimized CRISPR RNA pools enable sub-femtomolar sensitivity for target detection without any pre-amplification. This mechanistic insight and mitigation strategy broaden the application of CRISPR-Cas enzymes for nucleic acid diagnostics.},
}
@article {pmid40716264,
year = {2025},
author = {Souza-Neves, M and Pórfido, JL and Crispo, M and Menchaca, A},
title = {Electroporation of sheep zygotes as an alternative to microinjection for the generation of CRISPR/Cas genome edited models.},
journal = {Theriogenology},
volume = {248},
number = {},
pages = {117603},
doi = {10.1016/j.theriogenology.2025.117603},
pmid = {40716264},
issn = {1879-3231},
abstract = {Zygote microinjection is considered the most suitable technique to introduce CRISPR/Cas9 reagents for efficient genome editing in livestock. In this study, zygote electroporation was evaluated as an alternative to microinjection for CRISPR/Cas9-mediated genome editing in sheep. Four experiments were conducted on 3548 cumulus-oocyte complexes. Acid Tyrode's solution (AT) was used to partially degrade the zona pellucida (ZP) to improve reagent entry, resulting in ZP thinning with longer AT exposure (P < 0.05). Although early embryo development was impaired by AT exposure (P < 0.05), blastocyst rates were similar across all groups by day 8. Electroporation conditions were optimized by testing pulse length (1 or 3 ms), with the best results from 6 pulses of 20 V for 3 ms with AT during 60 s. Electroporation with 500 ng/μL Cas9 and 300 ng/μL sgRNA with AT during 60 s achieved a 38.5 % mutation rate. When compared with conventional microinjection, electroporation had higher developmental rates but a lower mutation rate (21.4 % vs. 60.0 %; P < 0.05). These findings suggest that electroporation is a viable, cost-effective technique for genome editing in sheep. Nevertheless, further research will be required to fine-tune electroporation conditions and enhance efficiency in terms of mutation rate.},
}
@article {pmid40715925,
year = {2025},
author = {Westarp, P and Keller, T and Brand, J and Horvat, S and Albrecht, K and Beisel, C and Groll, J},
title = {Efficient encapsulation of CRISPR-Cas9 RNP in bioreducible nanogels and release in a cytosol-mimicking environment.},
journal = {Discover nano},
volume = {20},
number = {1},
pages = {119},
pmid = {40715925},
issn = {2731-9229},
abstract = {CRISPR/Cas9-mediated programmable gene editing has disrupted the biotechnology industry since it was first described in 2012. Safe in vivo delivery is a key bottleneck for its therapeutic use. Viral vector-mediated delivery raises concerns due to immunogenicity, long-term expression, and genomic disruption. Delivery of pre-complexed ribonucleoprotein (RNP) reduces off-target effects, and recombinant Cas9 production is more cost-effective than viral vector synthesis. CRISPR-Cas RNPs do not possess intrinsic cell entry mechanisms, and physical delivery methods are confined to ex vivo editing, necessitating non-viral delivery approaches. Nanogels (NG) are biocompatible polymeric nanoparticles capable of entrapping proteins. Here, we report the first proof of principle that NGs from thiol-functionalized polyglycidol can entrap active RNPs with high efficiency (60 ± 2%). We call these particles CRISPR-Gels. A commercially available E. coli lysate for cell-free transcription and translation (TXTL) was used to mimic the intracellular reductive degradation of NGs while providing a real-time fluorescence readout of RNP activity. Degradation and RNP activity were observed within 30-90 min. The described TXTL assay can be utilized to evaluate the release of RNP in a cytosol-mimicking environment from redox-sensitive nanoparticles in a high-throughput and cost-effective way. Further studies are needed to assess the in vitro and in vivo performance of CRISPR-Gels.},
}
@article {pmid40715906,
year = {2025},
author = {Garg, A and Chauhan, P and Kaur, C and Arora, PK and Garg, SK and Singh, VP and Singh, KP and Srivastava, A},
title = {Comprehensive heavy metal remediation mechanisms with insights into CRISPR-Cas9 and biochar innovations.},
journal = {Biodegradation},
volume = {36},
number = {4},
pages = {69},
pmid = {40715906},
issn = {1572-9729},
support = {DST/INSPIRE/03/2021/002510//DST-INSPIRE INDIA/ ; },
mesh = {*Metals, Heavy/metabolism ; *Charcoal/chemistry ; *CRISPR-Cas Systems ; Biodegradation, Environmental ; *Soil Pollutants/metabolism ; Plants/metabolism/genetics ; Gene Editing ; Bacteria/metabolism/genetics ; },
abstract = {Heavy metal contamination of the environment is a serious issue, and more efficient and effective bioremediation techniques are needed. This review introduces current heavy metal bioremediation techniques, with focus on phytoremediation and microbial remediation, and recent developments in biochar and CRISPR-Cas9 technology. Phytoremediation employs the natural process of plants to accumulate and detoxify metals as an eco-friendly and sustainable technique. Microbial remediation by fungi and bacteria provides an additional approach through reduction, sequestration, and transformation of metals. Biochar as a high-carbon value-added pyrolytic biomass product improves soil quality, increases microbial activity, and adsorbs heavy metals, making bioremediation more effective. The discovery of CRISPR-Cas9 revolutionized gene engineering by allowing gene editing of plants and microbes to improve their metal tolerance and degradation. This review outlines recent developments, synergistic uses of biochar and CRISPR-Cas9, and how they might enhance phytoremediation and microbial remediation. By combining such novel technologies, strong, sustainable, and scalable solutions could be built for curbing heavy metal pollution and safeguarding environmental health.},
}
@article {pmid40720830,
year = {2025},
author = {Šimečková, H and Bárdy, P and Kuntová, L and Macháčová, E and Botka, T and Bíňovský, J and Houser, J and Farka, Z and Plevka, P and Pantůček, R and Mašlaňová, I},
title = {CRISPR-Cas10-Assisted Structural Modification of Staphylococcal Kayvirus for Imaging and Biosensing Applications.},
journal = {ACS synthetic biology},
volume = {},
number = {},
pages = {},
doi = {10.1021/acssynbio.5c00387},
pmid = {40720830},
issn = {2161-5063},
abstract = {Recent advances in genome editing techniques based on CRISPR-Cas have opened up new possibilities in bacteriophage engineering and, thus, enabled key developments in medicine, nanotechnology, and synthetic biology. Although staphylococcal phage genomes have already been edited, the modification of their structural proteins has not yet been reported. Here, the structure of Staphylococcus phage 812h1 of the Kayvirus genus was modified by inserting a poly histidine tag into an exposed loop of the tail sheath protein. A two-strain editing strategy was applied, utilizing homologous recombination followed by CRISPR-Cas10-assisted counter-selection of the recombinant phages. The His-tagged phage particles can be recognized by specific antibodies, enabling the modified bacteriophages to be employed in numerous techniques. The attachment of the engineered phage to bacteria was visualized by fluorescence microscopy, and its functionality was confirmed using biolayer interferometry biosensing, enzyme-linked immunosorbent assay, and flow cytometry, demonstrating that the genetic modification did not impair its biological activity.},
}
@article {pmid40720017,
year = {2025},
author = {Maruyama, R and Yokota, T},
title = {Creation of DMD Muscle Cell Model Using CRISPR-Cas9 Genome Editing to Test the Efficacy of Antisense-Mediated Exon Skipping.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2964},
number = {},
pages = {157-162},
pmid = {40720017},
issn = {1940-6029},
mesh = {Humans ; *CRISPR-Cas Systems ; *Muscular Dystrophy, Duchenne/genetics/therapy/pathology ; *Gene Editing/methods ; *Exons/genetics ; *Dystrophin/genetics ; *Oligonucleotides, Antisense/genetics ; Cell Line ; Mutation ; },
abstract = {Duchenne muscular dystrophy (DMD) is a devastating muscle disorder caused by mutations in the DMD gene. Antisense-mediated exon skipping is a promising strategy to treat DMD. The approval of Exondys 51 (eteplirsen) targeting exon 51 was the most noteworthy accomplishment in 2016. To evaluate and optimize the sequence of antisense oligonucleotides (AOs), muscle cell lines with DMD mutations are useful tools. However, there are several immortalized muscle cell lines with DMD mutations available that can be used to test the efficacy of exon skipping in vitro. In addition, an invasive muscle biopsy is required to obtain muscle cells from patients. Furthermore, many DMD mutations are very rare and it is hard to find a patient with a specific mutation for muscle biopsy in many cases. Here, we describe a novel approach to create an immortalized muscle cell line with a DMD deletion mutation using the human rhabdomyosarcoma (RD) cell line and the CRISPR/Cas9 system that can be used to test the efficacy of exon skipping.},
}
@article {pmid40719726,
year = {2025},
author = {Deivarajan, HR and Senthilkumar, K and Sekar, HV and Elamurugan, V and Pandian, J and Venugopal, A and Kuppamuthu, D and Prajna, L and Prajna, VN and Narendran, S},
title = {Rapid one-tube RPA-coupled CRISPR/Cas12a-based RID-MyC assay for the diagnosis of fungal keratitis.},
journal = {Indian journal of ophthalmology},
volume = {73},
number = {8},
pages = {1208-1212},
doi = {10.4103/IJO.IJO_1613_24},
pmid = {40719726},
issn = {1998-3689},
mesh = {Humans ; *Eye Infections, Fungal/diagnosis/microbiology ; Prospective Studies ; Cross-Sectional Studies ; Female ; Male ; *CRISPR-Cas Systems ; Middle Aged ; *Nucleic Acid Amplification Techniques/methods ; *Keratitis/diagnosis/microbiology ; Adult ; *Cornea/microbiology/pathology ; *DNA, Fungal/analysis/genetics ; *Fungi/genetics/isolation &amp; purification ; Aged ; *Recombinases/genetics ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {PURPOSE: This study introduces and evaluates the single-tube rapid identification of mycoses using CRISPR (ST-RID-MyC) assay. This novel diagnostic tool combines recombinase polymerase amplification (RPA) with CRISPR/Cas12a for the rapid and precise diagnosis of fungal keratitis (FK).<br><br>DESIGN: Prospective cross-sectional study.<br><br>METHODS: Corneal scrapings from 61 patients with suspected microbial keratitis were collected at the Cornea Department of a Tertiary Eye Care Center. The study assessed the sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of the ST-RID-MyC assay. Additional measures included concordance rates with traditional diagnostic methods and the time to diagnosis.<br><br>RESULTS: The ST-RID-MyC assay exhibited a sensitivity of 90% and a specificity of 90.48%, with a PPV of 94.74% and an NPV of 82.61%. The ST-RID-MyC showed substantial agreement with culture and microscopy and perfect concordance with conventional RID-MyC. The mean time to diagnosis was significantly reduced (P < 0.001) using the ST-RID-MyC assay, compared to the traditional RID-MyC assay (6 vs. 32 minutes). Visual assessments demonstrated a high level of inter-observer agreement (kappa = 0.832).<br><br>CONCLUSIONS: The ST-RID-MyC assay, combining RPA and CRISPR/Cas12a in a single-tube system, offers a rapid, accurate, and resource-efficient diagnostic method for FK, potentially transforming clinical management of this condition by enabling faster therapeutic decisions.},
}
@article {pmid40715281,
year = {2025},
author = {Lee, S and Kyung, M and Park, M and Park, S and Lee, J and Kim, S and Lee, S and Jo, M and Jung, ST and Lee, HW},
title = {Advanced human FcRn knock-in mice for pharmacokinetic profiling of therapeutic antibodies.},
journal = {Scientific reports},
volume = {15},
number = {1},
pages = {27186},
pmid = {40715281},
issn = {2045-2322},
support = {2020M3F7A1094089//National Research Foundation of Republic of Korea/ ; 2020M3F7A1094089//National Research Foundation of Republic of Korea/ ; 2020M3F7A1094089//National Research Foundation of Republic of Korea/ ; 2020M3F7A1094089//National Research Foundation of Republic of Korea/ ; 2020M3F7A1094089//National Research Foundation of Republic of Korea/ ; 2023-00224201//Ministry of Science and ICT, South Korea/ ; 2023-00224201//Ministry of Science and ICT, South Korea/ ; 2023-00224201//Ministry of Science and ICT, South Korea/ ; 2023-00224201//Ministry of Science and ICT, South Korea/ ; 2023-00224201//Ministry of Science and ICT, South Korea/ ; },
mesh = {Animals ; *Receptors, Fc/genetics/metabolism ; *Histocompatibility Antigens Class I/genetics/metabolism ; Humans ; Mice ; *Gene Knock-In Techniques ; *Immunoglobulin G ; Mice, Transgenic ; CRISPR-Cas Systems ; },
abstract = {IgG-based therapeutic antibodies are increasingly adopted for diverse human diseases, such as cancer and autoimmune disorders displaying remarkable therapeutic performance. A key factor in their success lies in the extended half-life of IgG molecules, which is regulated by the pH-dependent interaction between IgG and neonatal Fc receptor (FcRn). This interaction prevents lysosomal degradation of IgG. Despite the frequent use of humanized rodent models expressing human FcRn (hFcRn) in preclinical studies, these models often fail to accurately replicate human antibody pharmacokinetics (PK) due to the use of non-native promoters that influence FcRn expression. To overcome this limitation, we developed an innovative humanized FcRn knock-in (hiFcRn) mouse model using CRISPR/Cas9 technology. This model integrates hFcRn cDNA into the endogenous locus of the mouse Fcgrt gene, completely replacing native mouse FcRn (mFcRn) expression. The hiFcRn mouse model offers a more human-relevant platform for the preclinical evaluation of therapeutic antibodies and Fc-fusion proteins.},
}
@article {pmid40715076,
year = {2025},
author = {Bell, HW and Feng, R and Shah, M and Yao, Y and Douglas, J and Doerfler, PA and Mayuranathan, T and O'Dea, MF and Li, Y and Wang, YD and Zhang, J and Mackay, JP and Cheng, Y and Quinlan, KGR and Weiss, MJ and Crossley, M},
title = {Removal of promoter CpG methylation by epigenome editing reverses HBG silencing.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {6919},
pmid = {40715076},
issn = {2041-1723},
support = {2020861//Department of Health | National Health and Medical Research Council (NHMRC)/ ; K01DK132453//Foundation for the National Institutes of Health (Foundation for the National Institutes of Health, Inc.)/ ; R01 156647//Foundation for the National Institutes of Health (Foundation for the National Institutes of Health, Inc.)/ ; },
mesh = {Humans ; *Promoter Regions, Genetic/genetics ; *DNA Methylation/genetics ; *CpG Islands/genetics ; *Gene Silencing ; *Gene Editing/methods ; CRISPR-Cas Systems ; Ubiquitin-Protein Ligases/genetics/metabolism ; CCAAT-Enhancer-Binding Proteins/genetics/metabolism ; *gamma-Globins/genetics/metabolism ; *Epigenome/genetics ; Cell Line ; DNA (Cytosine-5-)-Methyltransferase 1/metabolism/genetics ; DNA-Binding Proteins/genetics/metabolism ; Epigenome Editing ; },
abstract = {β-hemoglobinopathies caused by mutations in adult-expressed HBB can be treated by re-activating the adjacent paralogous genes HBG1 and HBG2 (HBG), which are normally silenced perinatally. Although HBG expression is induced by global demethylating drugs, their mechanism is poorly understood, and toxicity limits their use. We identify the DNMT1-associated maintenance methylation protein UHRF1 as a mediator of HBG repression through a CRISPR/Cas9 screen. Loss of UHRF1 in the adult-type erythroid cell line HUDEP2 causes global demethylation and HBG activation that is reversed upon localized promoter re-methylation. Conversely, targeted demethylation of the HBG promoters activates their genes in HUDEP2 or primary CD34[+] cell-derived erythroblasts. Mutation of MBD2, a CpG-methylation reading component of the NuRD co-repressor complex, recapitulates the effects of promoter demethylation. Our findings demonstrate that localized CpGmethylation at the HBG promoters facilitates gene silencing and identify a potential therapeutic approach for β-hemoglobinopathies via epigenomic editing.},
}
@article {pmid40613420,
year = {2025},
author = {Yuan, T and Du, J and Hu, J and Luo, J and Pu, X and Zhu, X and Chen, B and Teng, Y and Li, H and Li, S and Jiang, L and Xiong, E},
title = {CRISPR-Cas13a-based dual-channel AND-logic gated biosensor for the simultaneous assay of APE1 and miRNA-224.},
journal = {Chemical communications (Cambridge, England)},
volume = {61},
number = {62},
pages = {11673-11676},
doi = {10.1039/d5cc03222a},
pmid = {40613420},
issn = {1364-548X},
mesh = {*MicroRNAs/analysis/genetics ; *Biosensing Techniques/methods ; *CRISPR-Cas Systems ; *DNA-(Apurinic or Apyrimidinic Site) Lyase/analysis/genetics ; Humans ; },
abstract = {Simultaneous detection of multiple targets is of great significance for the precise diagnosis of diseases. Herein, we report a dual-channel AND-logic gated biosensing platform based on the CRISPR-Cas13a system for the simultaneous detection of APE1 and miRNA-224, which showed superior specificity, sensitivity and potential for practical applications. Our study not only expands the CRISPR toolbox beyond nucleic acid assay but also establishes a new paradigm for multi-analyte diagnostic systems.},
}
@article {pmid40495486,
year = {2025},
author = {Jeong, YY and Han, JH and Yu, J and Bae, S and Seo, PJ},
title = {Identification of optimal adenine and cytosine base editors for genome editing in Arabidopsis and soybean.},
journal = {BMB reports},
volume = {58},
number = {7},
pages = {288-292},
pmid = {40495486},
issn = {1976-670X},
mesh = {*Gene Editing/methods ; *Arabidopsis/genetics ; *Glycine max/genetics ; *Cytosine/metabolism ; *Adenine/metabolism ; Genome, Plant/genetics ; CRISPR-Cas Systems/genetics ; },
abstract = {Base editors, including adenine base editors (ABEs) and cytosine base editors (CBEs), are widely used in numerous organisms to introduce site-specific sequence modifications in genomic DNA without causing double-strand breaks (DSBs). However, these editors exhibit low editing efficiencies, particularly in dicot plants, thereby limiting their application in dicot plant genome engineering. In this study, we assessed the editing efficiencies of various base editors to identify those optimal for base editing in dicot plants. We discovered that ABE8e, an ABE variant, demonstrated superior A-to-G base editing efficiency within A5-A8 windows, and A3A/Y130F-V04, a CBE variant, exhibited the highest C-to-T base editing efficiency within C4-C15 windows in both Arabidopsis and soybean protoplasts. Overall, we recommend these two base editors as prime choices for efficient genome engineering in a range of crop plants. [BMB Reports 2025; 58(7): 288-292].},
}
@article {pmid40441113,
year = {2025},
author = {Shen, C and Chen, X and Yu, Y and Qin, L and Xu, G and Wei, F and Yang, J and Hu, Q and Cen, Y},
title = {A novel fluorescent sensing platform for miRNA-210 detection based on signal amplification via SDA and self-assembly Cas12a system via RCT.},
journal = {Talanta},
volume = {295},
number = {},
pages = {128382},
doi = {10.1016/j.talanta.2025.128382},
pmid = {40441113},
issn = {1873-3573},
mesh = {*MicroRNAs/analysis/genetics ; Humans ; *Biosensing Techniques/methods ; *Nucleic Acid Amplification Techniques/methods ; *CRISPR-Cas Systems ; *CRISPR-Associated Proteins/metabolism/genetics/chemistry ; *Endodeoxyribonucleases/metabolism/genetics ; Limit of Detection ; Spectrometry, Fluorescence ; *Fluorescent Dyes/chemistry ; Fluorescence ; Bacterial Proteins ; },
abstract = {In this work, a biosensing platform for miR-210 which is a potent biomarker for the early diagnosis of breast cancer was innovatively designed based on CRISPR/Cas12a by integrating strand displacement amplification and rolling circle transcription. The target opened the hairpin through toe-hold, allowing polymerization, incision and extension to occur which involved SDA. This process produced two chains: cycle chain and trigger. The cycle chain then complemented to the loop of the hairpin to open it, repeating the above process to generate additional trigger circularly. The trigger was bound to the notched dumbbell through base complementary pairing and then the dumbbell became intact by T4 DNA ligase. The closed dumbbell strand served as the initiator of transcription and the template for crRNA under the influence of T7 enzyme, and was responsible for the transcription of numerous crRNA sequences in a process called RCT. These sequences subsequently bound to Cas12a proteins, forming a binary complex. In the transcriptional state, the dumbbell was in an unwinding configuration, with the stem portion served as activator binding to the binary complex to facilitate trans-cleavage activity of Cas12a, which resulted in cleavage of the F-Q, generating fluorescent signals. The above platform could sensitively detect miR-210 with a detection limit of 6.67 fM. The platform has the advantages of being easy to use and flexible to sequence according to different target, making it feasible to detect different biomarkers in clinic settings.},
}
@article {pmid40435758,
year = {2025},
author = {Ren, K and Ding, S and Shi, J and Dong, J and Du, F and Tang, Z},
title = {Detection of lead contamination using DNAzyme and split activator-triggered CRISPR/Cas12a.},
journal = {Talanta},
volume = {295},
number = {},
pages = {128385},
doi = {10.1016/j.talanta.2025.128385},
pmid = {40435758},
issn = {1873-3573},
mesh = {*Lead/analysis ; *DNA, Catalytic/metabolism/chemistry ; *CRISPR-Cas Systems ; *Endodeoxyribonucleases/metabolism/chemistry/genetics ; *Biosensing Techniques/methods ; *CRISPR-Associated Proteins/metabolism/chemistry ; *Bacterial Proteins/metabolism/chemistry ; Limit of Detection ; *Water Pollutants, Chemical/analysis ; Humans ; Drinking Water/analysis ; },
abstract = {Widespread Pb[2+] contamination represents a significant global health threat, particularly to children, highlighting the critical need for accurate monitoring and quantification to mitigate its adverse effects. The integration of DNAzymes with the programmable nuclease Cas12a has emerged as a promising approach for achieving specific and ultrasensitive detection of Pb[2+]. However, conventional DNAzyme-Cas12a systems suffer from inevitable background signals caused by dynamic instability between DNAzymes and Cas12a activators, which compromises analytical reliability. Herein, we present a novel DNAzyme-Cas12a assay featuring a split activator-based Cas12a switch. We demonstrate that the split activator containing a flap region effectively prevents Cas12a activation, thereby suppressing background noise induced by "DNA breathing" phenomena. Upon Pb[2+]-dependent activation of the GR-5 DNAzyme, the flap is cleaved, enabling reconstitution of the Cas12a activator and triggering trans-cleavage activity for signal amplification. This strategy achieves a detection limit of 615 pM for Pb[2+] while maintaining high specificity against interfering metal ions. Notably, the assay eliminates requirements for DNA amplification or nanoparticle modification, enabling rapid Pb[2+] detection at ambient temperature. The method demonstrated high accuracy in detecting contaminated tap and drinking water, suggesting its potential as a reliable analytical tool for monitoring Pb2+ contamination in practical samples.},
}
@article {pmid40398044,
year = {2025},
author = {Xu, J and Zhang, Y and Yuan, B and Wang, Y and Wang, J and Yuan, Y},
title = {A novel one-tube RPA/CRISPR melting curve detection sensing system based on unique 3'-toehold nucleic acid aptamer for Bacillus anthracis detection.},
journal = {Talanta},
volume = {295},
number = {},
pages = {128306},
doi = {10.1016/j.talanta.2025.128306},
pmid = {40398044},
issn = {1873-3573},
mesh = {*Bacillus anthracis/isolation &amp; purification/genetics ; *Aptamers, Nucleotide/chemistry/genetics ; *Biosensing Techniques/methods ; *CRISPR-Cas Systems ; *Nucleic Acid Amplification Techniques/methods ; Spores, Bacterial/isolation &amp; purification ; },
abstract = {Developing effective detection methods for Bacillus anthracis is essential for our public health system to accurately detect hidden anthrax outbreaks. Herein, we introduce a unique 3'-toehold nucleic acid aptamer (probes) into Cas12a biosensor, combined with RPA, to establish a rapid (1h), specific, and sensitive (1copy/μL) detection method for B. anthracis. The design behind this approach is that the target sequence is amplified via RPA, and the amplification product triggers the crRNA/Cas12a complex to degrade the 3' toehold probes, which are analyzed using melting curve analysis on a specific instrument, naming a one-tube RPA/CRISPR melting curve detection (ORCMD) sensing system. Furthermore, ORCMD is used to detect the B. anthracis spores-positive or negative soil samples from the location of world War-II site (Harbin, China), B. anthracis was precisely identified as other methods, suggesting its significant practical application potential. This system enriches the CRISPR detection technology toolbox, compared to other CRISPR-based sensing strategies, the concept of the 3' toehold probes offers distinct advantages in the development of CRISPR-based multi-target detection methods.},
}
@article {pmid40185317,
year = {2025},
author = {Lee, EG and Kim, KH},
title = {Transposition of transposable element IS1 in Edwardsiella piscicida mutant generated by CRISPR/Cas9 along with λ-Red recombineering system.},
journal = {Research in microbiology},
volume = {176},
number = {5-6},
pages = {104297},
doi = {10.1016/j.resmic.2025.104297},
pmid = {40185317},
issn = {1769-7123},
mesh = {*CRISPR-Cas Systems ; *DNA Transposable Elements/genetics ; *Gene Editing/methods ; *Edwardsiella/genetics ; Mutation ; Recombination, Genetic ; Genome, Bacterial ; },
abstract = {This study aimed to investigate unintended mutations introduced by the CRISPR/Cas9 genome editing system in Edwardsiella piscicida. Whole-genome sequencing was conducted on the wild-type E. piscicida NH1 and its alanine racemase knockout mutants (E. piscicida Δalr325 NH1 and E. piscicida Δalr50 NH1) generated using CRISPR/Cas9 with a λ-Red recombineering system. Comparative genomic analyses revealed that the insertion sequence 1 (IS1) transpositions occurred in the CRISPR/Cas9-edited mutants, disrupting the type I restriction-modification system subunit M gene, in addition to the targeted gene deletion. Interestingly, no IS1 transpositions were detected in mutants produced via conventional plasmid-based allelic exchange, indicating the potential link between CRISPR/Cas9-mediated editing and transposition events. These results suggest that genome editing via CRISPR/Cas9 could trigger IS1 transposition, potentially due to double-stranded DNA breaks. The lack of sequence similarity between the single guide RNA (sgRNA) and the transposed regions suggests that transpositions are not CRISPR/Cas9 off-target effects. This study provides evidence of interactions between mobile genetic elements and genome editing systems, requiring further investigation into their underlying mechanisms.},
}
@article {pmid40719891,
year = {2025},
author = {Rather, GA and Ayzenshtat, D and Kumar, M and Aisemberg, E and Bocobza, S},
title = {Direct haploid formation in Arabidopsis using transgenic CENH3-based inducers.},
journal = {Plant cell reports},
volume = {44},
number = {8},
pages = {182},
pmid = {40719891},
issn = {1432-203X},
support = {20-01-0245//Chief Scientist - Ministry of Agriculture and Rural Development/ ; },
abstract = {This study introduces a streamlined transgenic method for generating haploid inducers using a single T-DNA construct, combining CENH3 disruption, functional complementation, and a visual marker for efficient haploid screening. The development of doubled haploid lines is crucial for plant breeding programs, but conventional inbreeding methods are laborious and costly. Centromere-mediated genome elimination using modified CENH3 histones offers an efficient single-generation approach to induce haploidy. However, this approach necessitates the generation of haploid inducer lines, which typically involves cumbersome random mutagenesis screens. In this study, we implemented a transgenic strategy to circumvent this and directly create haploid inducers in Arabidopsis. This was achieved by knocking out endogenous AtCENH3 using CRISPR/Cas while complementing it with mutated AtCENH3 variants on the same T-DNA. Four constructs with truncated or full-length AtCENH3 harboring the G83E mutation alone or with the L130F mutation, and one negative control without mutations, were transformed into Arabidopsis. Stable homozygous transgenic lines were obtained and pollinated with a glabra mutant (Atgl1). Progenies lacking RFP fluorescence and exhibiting a glabrate phenotype were recovered, and flow cytometry analyses showed their haploidy, suggesting genome elimination. Comparatively, the G83E variants showed the highest haploid induction rate. This transgenic approach directly generated haploid inducer lines in Arabidopsis while avoiding random mutagenesis. This novel transgenic strategy provides a powerful tool to rapidly establish haploid inducer lines in additional transformable crops.},
}
@article {pmid40719158,
year = {2025},
author = {Torres-Barceló, C and Boyer, C and Garneau, JR and Poussier, S and Robène, I and Moury, B},
title = {A Phylogenetic Host-Range Index Reveals Ecological Constraints in Phage Specialisation and Virulence.},
journal = {Molecular ecology},
volume = {},
number = {},
pages = {e70052},
doi = {10.1111/mec.70052},
pmid = {40719158},
issn = {1365-294X},
abstract = {Phages are typically known for having a limited host range, targeting particular strains within a bacterial species, but accurately measuring their specificity remains challenging. Factors like the genetic diversity or population dynamics of host bacteria are often disregarded despite their potential influence on phage specialisation and virulence. This study focuses on the Ralstonia solanacearum species complex (RSSC), which comprises genetically diverse bacteria responsible for a major plant disease. It uses a diversified collection of RSSC phages to develop new host-range analysis methods and to test ecological and evolutionary hypotheses on phage host range. We introduce a new 'phylogenetic host-range index' that employs an ecological diversity index to account for the genetic diversity of bacterial hosts, allowing systematic classification of phages along a continuum between specialists and generalists. We propose and provide evidence that generalist phages are more likely to be represented in CRISPR-Cas immune system of bacteria than specialist phages. We explore the hypothesis that generalist phages might exhibit lower virulence than specialist ones due to potential evolutionary trade-offs between host-range breadth and virulence. Importantly, contrasted correlations between phage virulence and host range depend on the epidemiological context. A trade-off was confirmed in a context of low bacterial diversity, but not in a context of higher bacterial diversity, where no apparent costs were detected for phages adapted to a wide range of hosts. This study highlights the need for genetic analyses in phage host range and of investigating ecological trade-offs that could improve both fundamental phage knowledge and applications in biocontrol or therapy.},
}
@article {pmid40717612,
year = {2025},
author = {Bagi, M and Jamalzadegan, S and Steksova, A and Wei, Q},
title = {CRISPR-Cas based platforms for RNA detection: fundamentals and applications.},
journal = {Chemical communications (Cambridge, England)},
volume = {},
number = {},
pages = {},
doi = {10.1039/d5cc03257a},
pmid = {40717612},
issn = {1364-548X},
abstract = {The detection of RNA biomarkers is crucial for diagnosing many urgent diseases such as infections and cancer. Conventional RNA detection techniques such as RT-PCR, LAMP, and microarrays are effective, but often face limitations in terms of speed, sensitivity, and equipment demands. In recent years, CRISPR/Cas systems have emerged as versatile platforms for RNA detection, which offer high specificity, programmability, and adaptability across a wide range of diagnostic applications. This review first categorizes different CRISPR-based RNA detection systems according to the CRISPR effectors employed, including Cas13, Cas12, Cas14, Cas9, and newly characterized enzymes such as Cas7-11 and Cas10, detailing their mechanisms of target recognition, cleavage activity, and signal generation. The CRISPR detection platforms are coupled with or without pre-amplification steps to meet the different sensitivity needs. Preamplification-based systems integrate CRISPR with methods like RT-PCR and isothermal amplification to enhance sensitivity. In parallel, preamplification-free strategies, such as split-crRNA or split-activator systems, are gaining attention for their balanced assay performance and simplicity, which are especially attractive for point-of-care (POC) settings. Then, the diagnostic applications of these technologies are explored across two major domains: infectious disease detection and cancer biomarker identification via miRNAs, demonstrating the clinical potential of CRISPR-based RNA detection platforms. In addition, we explore ongoing challenges such as improving sensitivity in amplification-free formats, and developing field-deployable, cost-effective systems. The review concludes by outlining emerging trends and future directions in CRISPR-based RNA diagnostics, emphasizing their transformative potential in clinical settings.},
}
@article {pmid40713745,
year = {2025},
author = {Jiang, M and Chen, X and Song, Y and Wei, M and Cao, J and Lu, W and Lan, F and Bai, Y and Cui, M},
title = {NEXN deficiency leads to dilated cardiomyopathy in human pluripotent stem cell-derived cardiomyocytes.},
journal = {Stem cell research &amp; therapy},
volume = {16},
number = {1},
pages = {402},
pmid = {40713745},
issn = {1757-6512},
support = {7232088//Beijing Natural Science Foundation/ ; BRWEP2024W014090201//Beijing Research Ward Excellence Clinical Study Program/ ; 2024003//Key Clinical Projects of Peking University Third Hospital/ ; 82070272//National Natural Science Foundation of China/ ; },
mesh = {*Myocytes, Cardiac/metabolism/pathology/cytology ; Humans ; *Cardiomyopathy, Dilated/pathology/metabolism/genetics ; *Induced Pluripotent Stem Cells/metabolism/cytology ; Cell Differentiation ; CRISPR-Cas Systems ; Oxidative Stress ; },
abstract = {BACKGROUND: Dilated cardiomyopathy (DCM) constitutes a major cause of heart failure, characterized by high mortality rates and a limited availability of effective therapeutic options. A substantial body of evidence indicates that mutations in the Nexilin (NEXN) gene are significant pathogenic contributors to DCM, but the pathogenic mechanism for dilated cardiomyopathy is unclear.<br><br>METHODS: A human NEXN homozygous knockout cardiomyocyte model was established by combining CRISPR/Cas9 gene editing technology and human induced pluripotent stem cells (hiPSCs)-directed differentiation technology. Cell model phenotypic assays were done to characterize the pathological features of the resulting NEXN-deficient cardiomyocytes.<br><br>RESULTS: NEXN gene knockout did not affect the pluripotency and differentiation efficiency of hiPSCs. NEXN-deficient cardiomyocytes showed disordered junctional membrane complexes, abnormal excitation-contraction coupling, increased oxidative stress and decreased energy metabolism level. Moreover, levo-carnitine and sarcoplasmic reticulum calcium ATPase (SERCA2a) Activator 1 were identified as promising therapeutic agents for the treatment of DCM.<br><br>CONCLUSION: We demonstrated that NEXN was one of the important components in maintaining the structure and function of cardiomyocyte junctional membrane complexes (JMCs), excitation-contraction coupling and energy metabolism of cardiomyocytes, while the loss of its function would lead to DCM. This model represents an important tool to gain insight into the mechanism of DCM, elucidate the gene-phenotype relationship of NEXN deficiency and facilitate drug screening.},
}
@article {pmid40713739,
year = {2025},
author = {Amina, S and Wei, M and Zhang, S and Jiaqi, F and Ma, S and Abbas, MK and Lan, F and Jiang, H},
title = {Metabolism-related gene PDK1 regulates myocardial cell remodeling and its mechanism.},
journal = {Stem cell research &amp; therapy},
volume = {16},
number = {1},
pages = {404},
doi = {10.1186/s13287-025-04518-9},
pmid = {40713739},
issn = {1757-6512},
support = {NSFC 82370440//Beijing Natural Science Foundation/ ; },
mesh = {*Myocytes, Cardiac/metabolism/cytology ; Humans ; *Pyruvate Dehydrogenase Acetyl-Transferring Kinase/genetics/metabolism ; Induced Pluripotent Stem Cells/metabolism/cytology ; Apoptosis ; Cell Differentiation ; Oxidative Stress ; Gene Knockout Techniques ; Cell Hypoxia ; *Myocardium/metabolism ; },
abstract = {BACKGROUND: The progression of ischemic heart disease results from various forms of cardiomyopathies, which begin with cardiac remodelling. Pyruvate Dehydrogenase Kinase 1 (PDK1) is one of the basic kinase family components responsible for oxidative phosphorylation. However, due to the lack of a suitable research model, there is no evidence that remodelling pathogenesis in humans causes death by PDK1 knockout (KO). In the current study, we established a PDK1-deficient human cardiomyocyte (CM) model under conditions imitating the human PDK1-KO model. We determined the role of PDK1 in myocardial apoptosis induced by hypoxia and its implicit mechanism.<br><br>METHODS: A human PDK1-KO CM's model was established by combining CRISPR/Cas-9 gene-editing and human induced pluripotent stem cells (hiPSC) directed differentiation technology. The pathological features of PDK1-KO cardiomyocytes were assessed using a phenotypic cell model under basal and hypoxic conditions.<br><br>RESULTS: We found that pluripotency and differentiation efficiency of hiPSCs after PDK1 knockout remain intact. Cardiomyocytes with a PDK1 gene knockout showed hypoxia-induced myocardial apoptosis by disturbing mitochondrial metabolism, increased oxidative stress levels, and decreased cell energy and viability. In addition, lentivirus transfection significantly improved the metabolism and cell viability in PDK1-deficient cardiomyocytes.<br><br>CONCLUSIONS: Our study established a PDK1 knockout model under hypoxia that exhibits mitochondrial metabolism dysregulation, elevated oxidative stress, and decreased cell viability. This model is an important tool for understanding the mechanism of hypoxia-induced myocardial apoptosis, elucidating the gene-phenotype relationship of PDK1 deficiency, and providing evidence to mitigate the damage against hypoxia.},
}
@article {pmid40711918,
year = {2025},
author = {Roberts, A and Adler, BA and Cress, BF and Doudna, JA and Barrangou, R},
title = {Phage-based delivery of CRISPR-associated transposases for targeted bacterial editing.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {122},
number = {30},
pages = {e2504853122},
doi = {10.1073/pnas.2504853122},
pmid = {40711918},
issn = {1091-6490},
support = {DE-AC02-05CH11231//U.S. Department of Energy (DOE)/ ; },
mesh = {*Gene Editing/methods ; *Transposases/genetics/metabolism ; *CRISPR-Cas Systems/genetics ; *Escherichia coli/genetics/virology ; *Bacteriophage lambda/genetics ; Genome, Bacterial ; },
abstract = {Phage λ, a well-characterized temperate phage, has been recently leveraged for bacterial genome editing by selectively delivering base editors into targeted bacterial species. We extend this concept by engineering phage λ to deliver CRISPR-guided transposases, accomplishing large insertions and targeted gene disruptions. To achieve this, we engineered phage λ using homologous recombination paired with Cas13a-based counterselection for precise phage modifications. Initially, we established the utility of Cas13a in phage λ by conducting minimal recoding edits, deletions, and insertions. Subsequently, we scaled up the engineering to embed the comprehensive DNA-editing CRISPR-Cas transposase (DART) system within the phage genome, creating λ-DART phages. These modified λ-DART phages were then employed to infect Escherichia coli, generating CRISPR RNA-guided transposition events in the host genome. Applying our engineered λ-DART phages to monocultures and a mixed bacterial community comprising three genera led to efficient, precise, and specific gene knockouts and insertions in the targeted E. coli cells, achieving editing efficiencies surpassing 50% of the population. This research enhances phage-mediated genome editing by enabling efficient in situ gene integrations in bacteria, offering an avenue for further application in microbial community contexts. This scalable method enables flexible microbial genome editing in situ to manipulate the function and composition of diverse ecosystems.},
}
@article {pmid40709174,
year = {2025},
author = {Han, B and Liu, W and Wang, W and Li, Z and You, B and Liu, D and Nan, Y and Ding, T and Dai, Z and Zhang, Y and Zhang, W and Liu, Q and Li, X},
title = {CRISPR/Cas9-based discovery of ccRCC therapeutic opportunities through molecular mechanism and immune microenvironment analysis.},
journal = {Frontiers in immunology},
volume = {16},
number = {},
pages = {1619361},
pmid = {40709174},
issn = {1664-3224},
mesh = {Humans ; *Tumor Microenvironment/immunology/genetics ; *Carcinoma, Renal Cell/genetics/immunology/drug therapy/pathology ; *Kidney Neoplasms/genetics/immunology/drug therapy/pathology ; *CRISPR-Cas Systems ; Biomarkers, Tumor/genetics ; Gene Expression Regulation, Neoplastic ; Prognosis ; Mutation ; },
abstract = {INTRODUCTION: Clear cell renal cell carcinoma is a common and aggressive form of renal cell carcinoma. Its incidence continues to rise, and metastatic recurrence leads to poor clinical outcomes. Current prognostic biomarkers lack reliability. We integrated multi-omics data to discover key ccRCC genes and build a prognostic model to improve risk prediction and guide treatment decisions.<br><br>METHODS: Our study integrated genome-wide CRISPR screening data from DepMap and transcriptomic profiles from TCGA to identify key genes associated with ccRCC pathogenesis. Initial screening identified 11 candidate genes through differential expression analysis and CRISPR functional validation. Using LASSO and Cox regression, we selected five key genes (GGT6, HAO2, SLPI, MELK, and EIF4A1) for model construction. The functional role of MELK was tested by knockdown experiments. Additional analyses included tumor mutation burden, immune microenvironment assessment, and drug response prediction.<br><br>RESULTS: The model stratified patients into high-risk and low-risk groups with distinct survival outcomes. High-risk cases showed higher mutation loads, immunosuppressive features, and activated cytokine pathways, whereas low-risk cases displayed metabolic pathway activity. MELK knockdown reduced cancer cell proliferation and migration. High-risk patients exhibited better responses to targeted drugs such as pazopanib and sunitinib.<br><br>DISCUSSION: Our study demonstrates the pivotal role of MELK in ccRCC progression. This multi-omics-driven model elucidates MELK-mediated mechanisms and their interactions with the tumor microenvironment, providing novel strategies for risk stratification and targeted therapy. Future studies will validate these findings in independent cohorts and investigate the regulatory networks of MELK to identify potential therapeutic targets.},
}
@article {pmid40707831,
year = {2025},
author = {Sereika, M and Mussig, AJ and Jiang, C and Knudsen, KS and Jensen, TBN and Petriglieri, F and Yang, Y and Jørgensen, VR and Delogu, F and Sørensen, EA and Nielsen, PH and Singleton, CM and Hugenholtz, P and Albertsen, M},
title = {Genome-resolved long-read sequencing expands known microbial diversity across terrestrial habitats.},
journal = {Nature microbiology},
volume = {},
number = {},
pages = {},
pmid = {40707831},
issn = {2058-5276},
support = {130690//Villum Fonden (Villum Foundation)/ ; 50093//Villum Fonden (Villum Foundation)/ ; },
abstract = {The emergence of high-throughput, long-read DNA sequencing has enabled recovery of microbial genomes from environmental samples at scale. However, expanding the terrestrial microbial genome catalogue has been challenging due to the enormous complexity of these environments. Here we performed deep, long-read Nanopore sequencing of 154 soil and sediment samples collected during the Microflora Danica project, yielding genomes of 15,314 previously undescribed microbial species, recovered using our custom mmlong2 workflow. The recovered microbial genomes span 1,086 previously uncharacterized genera and expand the phylogenetic diversity of the prokaryotic tree of life by 8%. The long-read assemblies also enabled the recovery of thousands of complete ribosomal RNA operons, biosynthetic gene clusters and CRISPR-Cas systems. Furthermore, the incorporation of the recovered genomes into public genomic databases substantially improved species-level classification rates for soil and sediment metagenomic datasets. These findings demonstrate that long-read sequencing allows cost-effective recovery of high-quality microbial genomes from highly complex ecosystems, which remain an untapped source of biodiversity.},
}
@article {pmid40661409,
year = {2025},
author = {Hoffmann, FT and Wiegand, T and Palmieri, AI and Glass-Klaiber, J and Xiao, R and Tang, S and Le, H and Meers, C and Lampe, GD and Chang, L and Sternberg, SH},
title = {Exapted CRISPR-Cas12f homologs drive RNA-guided transcription.},
journal = {bioRxiv : the preprint server for biology},
volume = {},
number = {},
pages = {},
pmid = {40661409},
issn = {2692-8205},
abstract = {Bacterial transcription initiation is a tightly regulated process that canonically relies on sequence-specific promoter recognition by dedicated sigma (σ) factors, leading to functional DNA engagement by RNA polymerase (RNAP) [1] . Although the seven σ factors in E. coli have been extensively characterized [2] , Bacteroidetes species encode dozens of specialized, extracytoplasmic function σ factors (σ [E]) whose precise roles are unknown, pointing to additional layers of regulatory potential [3] . Here we uncover an unprecedented mechanism of RNA-guided gene activation involving the coordinated action of σ [E] factor in complex with nuclease-dead Cas12f (dCas12f). We screened a large set of genetically-linked dCas12f and σ [E] homologs in E. coli using RIP-seq and ChIP-seq experiments, revealing systems that exhibited robust guide RNA enrichment and DNA target binding with a minimal 5'-G target-adjacent motif (TAM). Recruitment of σ [E] was dependent on dCas12f and guide RNA (gRNA), suggesting direct protein-protein interactions, and co-expression experiments demonstrated that the dCas12f-gRNA-σ [E] ternary complex was competent for programmable recruitment of the RNAP holoenzyme. Remarkably, dCas12f-RNA-σ [E] complexes drove potent gene expression in the absence of any requisite promoter motifs, with de novo transcription start sites defined exclusively by the relative distance from the dCas12f-mediated R-loop. Our findings highlight a new paradigm of RNA-guided transcription (RGT) that embodies natural features reminiscent of CRISPRa technology developed by humans [4,5] .},
}
@article {pmid40661357,
year = {2025},
author = {Henriques, WS and Bowman, J and Hall, LN and Gauvin, CC and Wei, H and Kuang, H and Zimanyi, CM and Eng, ET and Santiago-Frangos, A and Wiedenheft, B},
title = {Structures reveal how the Cas1-2/3 integrase captures, delivers, and integrates foreign DNA into CRISPR loci.},
journal = {bioRxiv : the preprint server for biology},
volume = {},
number = {},
pages = {},
pmid = {40661357},
issn = {2692-8205},
abstract = {UNLABELLED: Cas1 and Cas2 are the hallmark proteins of prokaryotic adaptive immunity. However, these two proteins are often fused to other proteins and the functional association of these fusions often remain poorly understood. Here we purify Cas1 and the Cas2/3 fusion protein from Pseudomonas aeruginosa . We determine multiple structures of the Cas1-2/3 complex at distinct stages of CRISPR adaptation. Collectively, these structures reveal a prominent, positively charged channel on one face of the integration complex that captures short fragments of foreign DNA. Foreign DNA binding triggers conformational changes in Cas2/3 that expose new DNA binding surfaces necessary for homing the DNA-bound integrase to specific CRISPR loci. The length of the foreign DNA substrate determines if Cas1-2/3 docks completely onto the CRISPR repeat to successfully catalyze two sequential transesterification reactions required for integration. Taken together, these structures clarify how the Cas1-2/3 proteins orchestrate foreign DNA capture, site-specific delivery, and integration of new DNA into the bacterial genome.<br><br>HIGHLIGHTS: - A positively charged channel on the Cas1-2/3 complex captures fragments of DNA- A loop in the RecA1 domain controls access to the Cas3 nuclease active site- Foreign DNA binding allosterically regulates access to additional DNA binding sites- Distortion of the CRISPR repeat sequence licenses complete foreign DNA integration.},
}
@article {pmid40652511,
year = {2025},
author = {Wei, T and Li, J and Lei, X and Lin, R and Wu, Q and Zhang, Z and Shuai, S and Tian, R},
title = {Multimodal CRISPR screens uncover DDX39B as a global repressor of A-to-I RNA editing.},
journal = {Cell reports},
volume = {44},
number = {7},
pages = {116009},
doi = {10.1016/j.celrep.2025.116009},
pmid = {40652511},
issn = {2211-1247},
mesh = {*RNA Editing/genetics ; *DEAD-box RNA Helicases/metabolism/genetics ; Humans ; *Adenosine/metabolism/genetics ; *CRISPR-Cas Systems/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; HEK293 Cells ; *Inosine/metabolism/genetics ; RNA-Binding Proteins/metabolism/genetics ; },
abstract = {Adenosine-to-inosine (A-to-I) RNA editing is a critical post-transcriptional modification that diversifies the transcriptome and influences various cellular processes, yet its regulatory mechanisms remain largely unknown. Here, we present two complementary CRISPR-based genetic screening platforms: CREDITS (CRISPR-based RNA editing regulator screening), which enables genome-scale identification of editing regulators using an RNA recorder-based reporter system, and scCREDIT-seq (single-cell CRISPR-based RNA editing sequencing), which provides multiplexed single-cell characterization of transcriptome and editome changes for pooled perturbations. By screening 1,350 RNA-binding proteins, we identified a series of A-to-I editing regulators. Mechanistic investigation revealed DDX39B as a global repressor of A-to-I editing, which functions by preventing double-stranded RNA accumulation through its helicase activity. Targeting DDX39B significantly enhances the efficiency of RNA-editing-based tools, such as CellREADR (cell access through RNA sensing by endogenous ADAR) and LEAPER (leveraging endogenous ADAR for programmable editing of RNA), and disrupts hepatitis D virus (HDV) RNA editing homeostasis. These technological advances not only expand our understanding of RNA editing regulation but also provide powerful tools for exploring tissue-specific and context-dependent RNA modification mechanisms, with broad implications for therapeutic development.},
}
@article {pmid40600880,
year = {2025},
author = {Kristof, A and Karunakaran, K and Ferry, Y and Briggs, S and Allen, C and Mizote, P and Jian, Z and Arvanitis, C and Blazeck, J},
title = {Dissection of the MeCP2 repressor protein enables CRISPR platform optimization via localization engineering.},
journal = {Protein engineering, design &amp; selection : PEDS},
volume = {38},
number = {},
pages = {},
doi = {10.1093/protein/gzaf008},
pmid = {40600880},
issn = {1741-0134},
mesh = {*Methyl-CpG-Binding Protein 2/genetics/metabolism/chemistry ; Humans ; *CRISPR-Cas Systems ; HEK293 Cells ; *Protein Engineering/methods ; Nuclear Localization Signals/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Gene Knockdown Techniques ; Animals ; Protein Domains ; },
abstract = {Clustered regularly interspaced short palindromic repeat interference (CRISPRi), the fusion of nuclease-inactive Cas9 with transcriptional repressor domains, is a powerful platform enabling site-specific gene knockdown across diverse biological contexts. Previously described CRISPRi systems typically utilize two distinct domain classes: (1) Krüppel-associated box domains and (2) truncations of the multifunctional protein, MeCP2. Despite widespread adoption of MeCP2 truncations for developing CRISPRi platforms, individual contributions of subdomains within MeCP2's transcriptional repression domain (TRD) toward enhancing gene knockdown remain unclear. Here, we dissect MeCP2's TRD and observe that two subdomains, the expected NcoR/SMRT interaction domain (NID) and an embedded nuclear localization signal (NLS), can separately enhance gold-standard CRISPRi platform performance beyond levels attained with the canonical MeCP2 protein truncation. Incorporating side-by-side analyses of nuclear localization and gene knockdown for over 30 constructs featuring MeCP2 subdomains or virus-derived NLS sequences, we demonstrate that appending C-terminal NLS motifs to dCas9-based transcriptional regulators, both repressors and activators, can significantly improve their effector function across several cell lines. We also observe that NLS placement greatly impacts CRISPRi repressor performance, and that modifying the subdomain configuration natively found within MeCP2 can also enhance gene suppression capabilities in certain contexts. Overall, this work demonstrates the interplay of two complimentary chimeric protein design considerations, transcriptional domain 'dissection' and NLS motif placement, for optimizing CRISPR-mediated transcriptional regulation in mammalian systems.},
}
@article {pmid40466269,
year = {2025},
author = {Jiang, C and Lei, D and Xu, B and Wang, Z and Fang, R and Tang, Y and Wang, H},
title = {High-efficient molecular detection system termed RAA-based CRISPR-Cas13a for novel duck orthoreovirus.},
journal = {Poultry science},
volume = {104},
number = {8},
pages = {105327},
pmid = {40466269},
issn = {1525-3171},
mesh = {Animals ; *Poultry Diseases/diagnosis/virology ; *Ducks ; *Reoviridae Infections/veterinary/diagnosis/virology ; *Orthoreovirus, Avian/isolation &amp; purification/genetics ; *CRISPR-Cas Systems ; *Nucleic Acid Amplification Techniques/veterinary/methods ; Sensitivity and Specificity ; Recombinases/metabolism ; },
abstract = {The novel duck orthoreovirus (NDRV) is an immunosuppressive pathogen that significantly impacts the health of waterfowl breeding. Accurate, efficient, and convenient detection techniques are crucial for the prevention and control of NDRV, particularly in terms of field detection. By employing recombinase aided amplification (RAA) and clustered regularly interspaced short palindromic repeats (CRISPR)-Cas13a, we have developed a highly sensitive enzymatic molecular system that combines Cas13a with T7 in vitro transcription and RAA, enabling efficient and accurate detection of NDRV at a sensitivity level of 10° copies/μL. Furthermore, the integration of portable lateral flow dipstick can effectively reduce the point-of-care testing time to 40 min, while exhibiting no cross-reactivity with duck hepatitis a virus, Tembusu virus and novel duck-origin goose parvovirus. Both this system and the reverse-transcriptase real-time quantitative polymerase chain reaction (RT-qPCR) method demonstrated a consistent 100% accuracy in clinical samples. This study facilitated the development of an optimized assay, which enables specific detection of NDRV through a simplified procedure and significantly reduces the risk of contamination. This highlights the potential applicability of this assay for point-of-care testing.},
}
@article {pmid40449106,
year = {2025},
author = {Sun, D and Zhu, Y and Wang, M and Wang, J and Cheng, W and Li, Z and Deng, Y and Ou, X and Jia, R and Chen, S and Zhu, D and Liu, M and Zhao, X and Yang, Q and Wu, Y and Zhang, S and Huang, J and He, Y and Wu, Z and Cheng, A},
title = {A RT-ERA-CRISPR/Cas12a assay for rapid point-of-care duck hepatitis A virus detection.},
journal = {Poultry science},
volume = {104},
number = {8},
pages = {105316},
pmid = {40449106},
issn = {1525-3171},
mesh = {Animals ; *Ducks ; *Poultry Diseases/diagnosis/virology ; *Hepatitis Virus, Duck/isolation &amp; purification ; Point-of-Care Systems ; *Hepatitis, Viral, Animal/diagnosis/virology ; *Picornaviridae Infections/veterinary/diagnosis/virology ; *Nucleic Acid Amplification Techniques/veterinary/methods ; Sensitivity and Specificity ; CRISPR-Cas Systems ; },
abstract = {Duck hepatitis A virus (DHAV) is a severe pathogen that threatens the duck industry. DHAV is transmitted primarily through the respiratory and gastrointestinal tracts. Therefore, developing accurate and rapid diagnostic technologies is crucial to prevent the spread of this infectious disease. Currently, the most widely used DHAV detection methods in clinical diagnosis include PCR, real-time PCR (RT-PCR) and ELISA, which require specialized equipment or trained professionals. Isothermal amplification technologies are often combined with lateral flow assays or visual readout methods for detecting pathogen nucleic acids in non-laboratory settings. CRISPR nucleases have accelerated the development of nucleic acid detection, increasing the sensitivity to a higher degree. Here, we applied reverse transcription-enzymatic recombinase amplification (RT-ERA) assisted by a Cas12a-fluorescence assay and a Cas12a-lateral flow assay for the detection of DHAV-1. Based on the sequence of DHAV-1, RT-ERA primers and crRNAs were designed, and different concentrations of ssDNA/Cas12a/crRNA were established to optimize the CRISPR reaction. The LoD for the Cas12a-fluorescence assay was 10 copies/μL, and this assay effectively differentiated DHAV-1 from other avian pathogens, exhibiting high sensitivity and specificity. Additionally, the Cas12a-lateral flow assay is user-friendly and can achieve point-of-care detection. Sixty-four clinical samples were tested and compared with quantitative real-time PCR (RT-PCR). This accurate and rapid point-of-care assay has significant potential for detecting DHAV-1 in clinical applications, especially for duck farms in rural areas.},
}
@article {pmid40707593,
year = {2025},
author = {Mahajan, D and Chia, HM and Lu, L},
title = {Knocking out ARL13B completely abolishes primary ciliogenesis in cell lines.},
journal = {Scientific reports},
volume = {15},
number = {1},
pages = {26980},
pmid = {40707593},
issn = {2045-2322},
support = {Tier 1 RG 25/22//Ministry of Education - Singapore/ ; },
mesh = {*Cilia/metabolism/genetics ; *ADP-Ribosylation Factors/genetics/metabolism ; CRISPR-Cas Systems ; Hedgehog Proteins/metabolism ; Signal Transduction ; Cell Line ; Animals ; Humans ; Gene Knockout Techniques ; Mice ; Smoothened Receptor/metabolism ; },
abstract = {Small G protein ARL13B localizes to the cilium and plays essential roles in cilium biogenesis, organization, trafficking, and signaling. Here, we established multiple ARL13B knockout cell lines using the CRISPR/Cas9 system. Surprisingly, all our cell lines lost their cilia completely, in contrast to the reported short cilium and reduced ciliogenesis phenotype. We found that multiple regions of ARL13B are necessary for a complete rescue. Additionally, we found that ARL13B knockout cells also lost their response to SMO-mediated hedgehog stimulation. Our work demonstrates the critical requirement of ARL13B for ciliogenesis and hedgehog signaling, at least in cultured cells, and suggests that ARL13B plays a more crucial role in ciliary function than previously understood.},
}
@article {pmid40707336,
year = {2025},
author = {Li, J and Lau, CH and Zhu, H},
title = {CRISPR/Cas-based personal glucose meters for nucleic acid detection.},
journal = {Trends in biotechnology},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.tibtech.2025.07.006},
pmid = {40707336},
issn = {1879-3096},
abstract = {Through integration with clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) systems, personal glucose meters (PGMs) have been repurposed to detect non-glucose targets. PGMs enable the intuitive readout of electrochemical signals, while CRISPR/Cas offers rapid detection and signal amplification capability. Their integration can realize point-of-care (POC) diagnostics. Herein, we critically discuss their advances, pitfalls, and future perspectives.},
}
@article {pmid40704512,
year = {2025},
author = {Baig, MMFA and Chien, WT and Chair, SY},
title = {Nanotechnological approaches for the targeted delivery of CRISPR-Cas systems for genomic modifications, biomolecular sensing, and precision medicine.},
journal = {Biomaterials science},
volume = {},
number = {},
pages = {},
doi = {10.1039/d5bm00711a},
pmid = {40704512},
issn = {2047-4849},
abstract = {The integration strategies of CRISPR-Cas gene editing systems with nanotechnological approaches have achieved synergistic effects in targeting genes; correcting genetic disorders; and treating, sensing, and diagnosing a variety of cancers and metabolic, immunological, and complex infectious diseases-all having connectivity with distinct genetic cues and mutations. Numerous recent studies have demonstrated the use of the nano-scale properties of nanomaterials to tremendously improve the genomic-editing efficiencies of CRISPR/Cas systems for achieving 50% enhanced bioavailability, improved cell targetability, and gene-level specificity while minimizing immunogenicity, compared with conventional/ordinary delivery techniques. Thus, nano-delivery methods utilizing the unique properties of nanomaterials, molecular interactions, biocompatibility, targeted cellular uptake, and nuclear delivery capability effectively overcame the challenges of inefficient biomolecular delivery, and off-target effects were effectively overcome. Nano -carriers made up of materials such as DNA lattices, lipids, dendrimers, polymers, peptides, and metals (gold, silver, etc.) that were explored for facilitating the precise delivery of CRISPR/Cas components, sensing biomolecules, and diagnostic purposes are discussed in this review report. The ability of DNA scaffold materials to incorporate nano-CRISPR systems, to sense biomolecules, and for targeted cellular delivery of payloads (e.g., Cas9, Cas12, Cas13, and Cas14 proteins and single-guide RNAs (sgRNAs)) maximized gene targeting and improved therapeutic outcomes while achieving up to 90% efficiency compared with common/trivial delivery methods.},
}
@article {pmid40704023,
year = {2025},
author = {Niu, M and Dong, Z and Yu, L and Dong, X and An, J and Han, Y and Yan, Y and Yi, W and Sun, Y and Li, H},
title = {Anti-RNA virus crRNA targets efficient screening platform based on bioinformatics and CRISPR detection.},
journal = {Molecular therapy. Nucleic acids},
volume = {36},
number = {3},
pages = {102619},
pmid = {40704023},
issn = {2162-2531},
abstract = {The mutation and evolution of RNA viruses pose significant challenges in treatment efforts. The CRISPR-Cas system is a promising antiviral tool because of its powerful programmability. However, traditional cell screening methods for CRISPR targets are time-consuming, limiting their application. Here, we developed a rapid and efficient screening platform for crRNA targets by combining the CaSilico-based bioinformatics method with CRISPR in vitro detection technology. Using a bioinformational approach to design and screen crRNAs, the characteristics of crRNAs and the corresponding target sequences can be rapidly determined. CRISPR is used for secondary screening in vitro, enabling swift identification of the target site with optimal cleavage efficiency. This method significantly reduces the screening time for antiviral targets compared with traditional cell screening. We successfully designed and screened effective crRNA targeting SARS-CoV-2 conserved N gene regions and demonstrated its inhibition function in HEK 293T cells. We also designed and screened crRNAs targeting DENV to validate the feasibility of the platform. E-2330 crRNA reduced more than 90% of the DENV RNA load in multiple mammalian cell lines and effectively inhibited the replication of all four DENV serotypes. This study provides a new approach for screening antiviral crRNAs for antivirus research.},
}
@article {pmid40702552,
year = {2025},
author = {Vahabi, M and Xu, G and Sarkisjan, D and Hassouni, BE and Mantini, G and Donati, V and Wang, B and Lencioni, G and Honeywell, RJ and Deng, D and Strano, S and Peters, GJ and Blandino, G and Giovannetti, E},
title = {Unraveling resistance mechanisms to the novel nucleoside analog RX-3117 in lung cancer: insights into DNA repair, cell cycle dysregulation and targeting PKMYT1 for improved therapy.},
journal = {Journal of experimental &amp; clinical cancer research : CR},
volume = {44},
number = {1},
pages = {217},
pmid = {40702552},
issn = {1756-9966},
support = {IG 24444//Fondazione AIRC per la ricerca sul cancro ETS/ ; IG 24444//Fondazione AIRC per la ricerca sul cancro ETS/ ; IG 24444//Fondazione AIRC per la ricerca sul cancro ETS/ ; IG 24444//Fondazione AIRC per la ricerca sul cancro ETS/ ; IG 24444//Fondazione AIRC per la ricerca sul cancro ETS/ ; IG 24444//Fondazione AIRC per la ricerca sul cancro ETS/ ; },
mesh = {Humans ; *Lung Neoplasms/drug therapy/genetics/pathology ; *Drug Resistance, Neoplasm ; *DNA Repair ; Cell Line, Tumor ; Cell Cycle/drug effects ; *Carcinoma, Non-Small-Cell Lung/drug therapy/genetics/pathology ; *Cytidine/analogs &amp; derivatives/pharmacology ; *Nucleoside-Phosphate Kinase/antagonists &amp; inhibitors ; CRISPR-Cas Systems ; *Nucleosides/pharmacology ; },
abstract = {BACKGROUND: Nucleoside analogues are crucial in treating non-small cell lung cancer (NSCLC), but resistance hampers patient outcomes. The cytidine analogue RX-3117 shows promise in gemcitabine-resistant cancers, yet mechanisms underlying acquired resistance to this drug remain unexplored. This study includes a comprehensive investigation into RX-3117 resistance mechanisms by leveraging new preclinical models and cutting-edge genomic tools, including a CRISPR-Cas9 knockout screen and transcriptomics.<br><br>METHODS: NSCLC cell lines A549 and SW1573 were exposed to stepwise increasing concentrations of RX-3117 to establish stable resistant subclones, confirmed by SRB and clonogenic assays. Intracellular RX-3117 nucleotide levels were measured via LC/MS-MS, prompting the evaluation and modulation of the expression of key metabolic enzymes by Western blot and siRNA. A CRISPR-Cas9 screen identified genes whose loss increased RX-3117 sensitivity, while RNA-sequencing with differential expression analyses revealed resistance-related pathways, further investigated through cell cycle distribution, knock-out, and ELISA assays.<br><br>RESULTS: Resistant clones exhibited decreased accumulation of RX-3117 nucleotides, which however, was not associated to reduced expression of activation enzymes (UCK2, UMPK, CMPK, NME1/NDPK, RR1 and RR2). Instead, increased expression was observed in certain DNA repair and deactivation enzymes (NT5C3) but pharmacological inhibition and silencing of the latter did not circumvent resistance. Remarkably, a comprehensive approach with CRISPR-Cas9 screen highlighted DNA-repair and cell cycle determinants as key sensitizing genes. XL-PCR and RNA-sequencing confirmed aberrations in DNA-repair and pathways involved in cell cycle regulation. Knock-out and pharmacological inhibition validated the role of PKMYT1, a protein kinase involved in G2/M transition and genomic stability. RX-3117-resistant A549 cells showed enhanced sensitivity to the PKMYT1 inhibitor lunresertib and its synergism with RX-3117, suggesting further studies, especially in patients with high PKMYT1 expression who have significantly shorter survival rates, as observed in public databases and validated in an internal cohort of NSCLC patients.<br><br>CONCLUSION: By integrating CRISPR-Cas9 with functional assays and transcriptomics, our study established a framework for decoding resistance mechanisms and highlights potential therapeutic strategies to enhance RX-3117 efficacy in NSCLC. We demonstrated for the first time that aberrant DNA repair and cell cycle dysregulation led resistance, identifying PKMYT1 as a promising target.},
}
@article {pmid40702188,
year = {2025},
author = {Baglaenko, Y and Mu, Z and Curtis, M and Mire, HM and Jayanthi, V and Al Suqri, M and Liu, C and Agnew, R and Nathan, A and Mah-Som, AY and Liu, DR and Newby, GA and Raychaudhuri, S},
title = {Precisely defining disease variant effects in CRISPR-edited single cells.},
journal = {Nature},
volume = {},
number = {},
pages = {},
pmid = {40702188},
issn = {1476-4687},
abstract = {Genetic studies have identified thousands of individual disease-associated non-coding alleles, but the identification of the causal alleles and their functions remains a critical bottleneck[1]. CRISPR-Cas editing has enabled targeted modification of DNA to introduce and test disease alleles. However, the combination of inefficient editing, heterogeneous editing outcomes in individual cells and nonspecific transcriptional changes caused by editing and culturing conditions limits the ability to detect the functional consequences of disease alleles[2,3]. To overcome these challenges, we present a multi-omic single-cell sequencing approach that directly identifies genomic DNA edits, assays the transcriptome and measures cell-surface protein expression. We apply this approach to investigate the effects of gene disruption, deletions in regulatory regions, non-coding single-nucleotide polymorphism alleles and multiplexed editing. We identify the effects of individual single-nucleotide polymorphisms, including the state-specific effects of an IL2RA autoimmune variant in primary human T cells. Multimodal functional genomic single-cell assays, including DNA sequencing, enable the identification of causal variation in primary human cells and bridge a crucial gap in our understanding of complex human diseases.},
}
@article {pmid40617066,
year = {2025},
author = {Wang, L and He, X and Tian, T and Cheng, J and Cao, R and Hou, J and Lin, H and Li, Y},
title = {Functional characterization of CEL3C reveals its critical role in regulating cellulase gene expression in Trichoderma reesei Rut C30.},
journal = {Enzyme and microbial technology},
volume = {190},
number = {},
pages = {110706},
doi = {10.1016/j.enzmictec.2025.110706},
pmid = {40617066},
issn = {1879-0909},
mesh = {*Cellulase/genetics/metabolism ; *Gene Expression Regulation, Fungal ; *Fungal Proteins/genetics/metabolism ; *Hypocreales/genetics/enzymology ; *beta-Glucosidase/genetics/metabolism ; CRISPR-Cas Systems ; },
abstract = {The nuclear-localized β-glucosidase CEL3C in Trichoderma reesei plays a pivotal role in cellulase regulation, though its mechanism remains poorly understood. To address this, we disrupted CEL3C in the hypercellulolytic strain T. reesei Rut C30 via CRISPR-Cas9 and evaluated cellulase production under sophorose-rich MGD induction. Deletion of CEL3C significantly enhanced total cellulase activity by 31.28 % (p < 0.05), with β-glucosidase, endoglucanase, and cellobiohydrolase activities increasing by 94.97 %, 19.40 %, and 28.99 %, respectively. These improvements were driven by transcriptional upregulation of core cellulase genes (CEL7A: 2.01-fold; CEL6A: 1.5-fold; CEL12A: 2.0-fold; CEL5A: 1.32-fold) and β-glucosidases (CEL3A: 6.41-fold; CEL3B: 5.02-fold), confirming transcriptional-level control as the dominant regulatory mechanism. Transcriptomic profiling identified 688 differentially expressed genes (399 upregulated, 299 downregulated), with key changes including activation of transcriptional activators XYR1 (59.6 % increase), ACE3 (75.49 % increase), and RXE1 (161.56 % increase), suppression of repressors RCE1 (65.86 % decrease) and RCE2 (65.23 % decrease), and induction of sugar transporters (TrireC30_133589: 13.41-fold) and ER chaperones (BIP1: 1.26-fold; PDI1: 1.55-fold). These alterations collectively enhanced inducer uptake, enzyme maturation, and secretion while alleviating MAPK-mediated repression (TMK2: 110.54 % decrease). Intracellular sugar profiling revealed that gentiobiose and cellobiose were undetectable in the T. reesei ΔCEL3C, whereas glucose and sophorose levels increased by 31.71 % and 13.45 % (p < 0.05), respectively. These results suggest that CEL3C deletion enhances β-glucosidase-mediated hydrolysis of disaccharides into glucose and possibly promotes sophorose formation via transglycosylation. In parallel, the upregulation of disaccharide transporters may facilitate sophorose uptake. Together, these two mechanisms contributed to the intracellular enrichment of sophorose, thereby amplifying cellulase gene induction and enzyme production. Our findings establish CEL3C as a dual-function nuclear regulator that balances cellulase synthesis through transcriptional and enzymatic pathways, providing actionable targets for engineering T. reesei with optimized industrial cellulase yields.},
}
@article {pmid40541708,
year = {2025},
author = {Yoon, DS and Byeon, E and Yun, SC and Jeong, H and Lee, JS and Sun, Y and Won, EJ and Park, HG and Yang, Z and Hagiwara, A and Lee, MC and Lee, JS},
title = {Effects on life parameters and fatty acid expression profiles in response to elevated temperatures in CRISPR/Cas9-mediated gene-targeted mutants of two desaturase (Δ9-3 and Δ5/6) genes in the water flea Daphnia magna.},
journal = {Comparative biochemistry and physiology. Part B, Biochemistry &amp; molecular biology},
volume = {279},
number = {},
pages = {111119},
doi = {10.1016/j.cbpb.2025.111119},
pmid = {40541708},
issn = {1879-1107},
mesh = {Animals ; *Daphnia/genetics/enzymology/metabolism/physiology ; *Fatty Acid Desaturases/genetics/metabolism ; *CRISPR-Cas Systems ; *Fatty Acids/metabolism/genetics ; *Mutation ; *Hot Temperature ; Daphnia magna ; },
abstract = {Fatty-acid desaturation is central to membrane homeostasis and thermal performance in ectotherms. We disrupted the Δ9-3 (MUFA-specific) and Δ5/6 (long-chain PUFA-specific) desaturase genes in Daphnia magna with CRISPR/Cas9 and compared wild-type and knockout lines at 23 °C and 28 °C. Loss of Δ9-3 substantially depleted monounsaturated fatty acids, especially oleic and palmitoleic acids, with a compensatory rise in total polyunsaturates. These lipid shifts coincided with enhanced growth and fecundity under benign temperature but translated into reduced reproductive output when heat stress was imposed, indicating a context-dependent trade-off. In contrast, deletion of Δ5/6 selectively impaired the n-6 pathway at high temperature, leading to pronounced reductions in total PUFA reserves and a severe decline in offspring production; the usual growth benefit of warming was also abolished. Together, the data reveal that Δ9-3 supports MUFA supply needed for baseline metabolism, whereas Δ5/6 maintains PUFA reserves critical for reproduction under thermal challenge. Divergent desaturase functions thus play distinct roles in lipid remodeling and contribute to climate resilience in freshwater zooplankton.},
}
@article {pmid40476445,
year = {2025},
author = {Xu, R and Zhu, S and Zhang, W and Xu, H and Tu, C and Wang, H and Wang, L and He, N and Liu, T and Guo, X and Ren, X and Li, Z},
title = {A Dual Approach with Organoid and CRISPR Screening Reveals ERCC6 as a Determinant of Cisplatin Resistance in Osteosarcoma.},
journal = {Advanced science (Weinheim, Baden-Wurttemberg, Germany)},
volume = {12},
number = {28},
pages = {e2500632},
doi = {10.1002/advs.202500632},
pmid = {40476445},
issn = {2198-3844},
support = {82172500//National Natural Science Foundation of China/ ; 82272664//National Natural Science Foundation of China/ ; 82103228//National Natural Science Foundation of China/ ; CX20230372//Hunan Provincial Innovation Foundation for Postgraduate/ ; },
mesh = {*Cisplatin/pharmacology/therapeutic use ; *Osteosarcoma/genetics/drug therapy/metabolism/pathology ; Humans ; *Drug Resistance, Neoplasm/genetics ; Mice ; *Poly-ADP-Ribose Binding Proteins/genetics/metabolism ; Animals ; *Organoids/metabolism/drug effects ; *Bone Neoplasms/genetics/drug therapy/metabolism ; Cell Line, Tumor ; Antineoplastic Agents/pharmacology ; CRISPR-Cas Systems ; DNA Helicases ; DNA Repair Enzymes ; },
abstract = {Osteosarcoma (OS), the most prevalent primary bone malignancy in adolescents, is typically treated with cisplatin-based chemotherapy. However, the development of cisplatin resistance often leads to relapse or metastasis, significantly impairing therapeutic efficacy. To tackle this issue, patient-derived osteosarcoma organoids (OSOs) is established that accurately reflect the cellular composition and heterogeneity of the original tumors, as validated by single-cell RNA sequencing, bulk RNA sequencing, and histopathology analysis. Cisplatin resistance is successfully induced in these OSOs, creating a clinically relevant model for investigating chemoresistance. Utilizing RNA sequencing in cisplatin-resistance OSOs and CRISPR screening in OS cell line, ERCC6 is identified as a pivotal regulator of cisplatin resistance. Knockdown of ERCC6 markedly enhanced cisplatin sensitivity in vitro and in vivo. Mechanistically, ERCC6 interacts with HNRNPM, influencing the PI3K/AKT signaling pathway and alternative splicing of pre-mRNA for BAX. Notably, the knockdown of ERCC6 and HNRNPM increased expression of full-length BAX and reduced skipping of exon 2, thus promoting apoptosis. This exon skipping in BAX results in a frameshift and introduces a premature stop codon (TGA) within the BH3 domain. These findings underscore the utility of OSOs in elucidating resistance mechanisms and highlight ERCC6 and HNRNPM as potential therapeutic targets.},
}
@article {pmid40701722,
year = {2025},
author = {Long, K and Han, T and Hu, W and Yang, M and Huo, D and Huang, Z and Hou, C},
title = {Double special bodyguard: Selenium-enhanced loop-mediated isothermal amplification combined with CRISPR/Cas12a biosensing strategy.},
journal = {Analytica chimica acta},
volume = {1369},
number = {},
pages = {344353},
doi = {10.1016/j.aca.2025.344353},
pmid = {40701722},
issn = {1873-4324},
mesh = {*Biosensing Techniques/methods ; *CRISPR-Cas Systems ; *Nucleic Acid Amplification Techniques/methods ; *Selenium/chemistry ; Humans ; *DNA, Viral/analysis/genetics ; Limit of Detection ; Papillomaviridae/genetics/isolation &amp; purification ; Molecular Diagnostic Techniques ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {Cervical cancer is closely associated with human papillomavirus (HPV) infection, underscoring the necessity for effective HPV detection. Various biosensors utilizing isothermal amplification techniques have been developed for HPV DNA detection. These methods offer high sensitivity and specificity, making them suitable for point-of-care testing. However, the in vitro DNA polymerases used in these isothermal amplification methods lack a repair system, may leading to polymerization errors. This limitation highlights the need for improved detection methods that can enhance accuracy and reliability in HPV testing. Here, a sensing strategy called selenium nucleic acid-enhanced loop-mediated isothermal amplification (Se-LAMP) combined with CRISPR/Cas12a (SLC) was developed. The SLC has two specific bodyguards, which ensure the accuracy and specificity of biosensors through two aspects. One bodyguard is dNTPαSe, a supplement added to the initial Se-LAMP step, which guarantees the accuracy of DNA polymerase and reduces background signals. The other bodyguard is CRISPR system, integrating three functions of specific verification, signal amplification and signal output. CRISPR activation can only rely on Se-LAMP products successfully rechecked by crRNA. As a readable signal of SLC, the detection limit was as low as 0.38 copies/μL (0.64 fM) within 65 min, which was one order of magnitude lower than that of non-selenium-modified methods and two orders of magnitude lower than that of qPCR. The SLC is the first to use CRISPR system as the output mode of Se-LAMP, proving the CRISPR/Cas12a system has good recognition ability for selenium-modified nucleic acid. The ultrasensitive dual-specificity guard enables SLC, ensuring accuracy in biodetection. This innovation offers novel clinical testing approaches and holds significance for diagnosing and monitoring disease progression.},
}
@article {pmid40699345,
year = {2025},
author = {Schöllkopf, AI and Ehrenreich, A and Liebl, W},
title = {SMC-like Wadjet system prevents plasmid transfer into Clostridium cellulovorans.},
journal = {Applied microbiology and biotechnology},
volume = {109},
number = {1},
pages = {170},
pmid = {40699345},
issn = {1432-0614},
support = {161B0930//German Federal Ministry of Education and Research/ ; 161B0930//German Federal Ministry of Education and Research/ ; 161B0930//German Federal Ministry of Education and Research/ ; },
mesh = {*Plasmids/genetics ; *Conjugation, Genetic ; *Gene Transfer, Horizontal ; *Clostridium cellulovorans/genetics ; *Chromosomes, Bacterial/genetics ; },
abstract = {This study demonstrates the impact of a Structure Maintenance of Chromosome (SMC)-like Wadjet system on the horizontal gene transfer of plasmids by conjugation to a recipient that naturally containing such a system for the first time. A Clostridium cellulovorans mutant with dramatically improved efficiency to receive plasmid DNA by conjugation was isolated and sequenced. Three spontaneous chromosomal deletions included a type II restriction-modification system, a putative CRISPR system, and a cluster of ORFs named jetABCD encoding a putative Wadjet system. Since nearly nothing is known about the role of naturally occurring Wadjet systems in their native host bacteria, markerless chromosomal deletion of jetABCD in the C. cellulovorans wildtype strain 743B was achieved and the effect on conjugative plasmid uptake was studied. The transconjugation frequency of the jetABCD mutant was increased by about five orders of magnitude compared to wildtype C. cellulovorans recipient cells. Bioinformatic analysis of genome sequences of the Bacillota phylum revealed near-complete mutually exclusive possession of either plasmids < 40 kb or jetABCD genes, indicating high efficiency of Wadjet systems in small plasmid prevention in bacteria. Importantly, the implications of this study go beyond the case of C. cellulovorans. Our study demonstrates that the eradication of Wadjet systems can dramatically improve the uptake of recombinant plasmids and thereby enhance genetic engineering of bacterial strains of interest for biotechnological applications. KEY POINTS: • Native Wadjet system inhibits plasmid transfer by conjugation in C. cellulovorans • Deleting jetABCD increased plasmid uptake by about five orders of magnitude • Possession of Wadjet systems efficiently block plasmid maintenance in Bacillota.},
}
@article {pmid40698645,
year = {2025},
author = {Shah, KM and Crozier, AFF and Assaraf, A and Arya, M and Grevitt, P and Mardakheh, F and Plevin, MJ and Sharp, TV},
title = {C-terminal tagging impairs AGO2 function.},
journal = {RNA biology},
volume = {22},
number = {1},
pages = {1-24},
doi = {10.1080/15476286.2025.2534028},
pmid = {40698645},
issn = {1555-8584},
mesh = {*Argonaute Proteins/metabolism/genetics/chemistry ; Humans ; CRISPR-Cas Systems ; A549 Cells ; Protein Binding ; Autoantigens/metabolism/genetics ; DNA-Binding Proteins/metabolism/genetics ; RNA Interference ; MicroRNAs/genetics/metabolism ; Recombinant Fusion Proteins/genetics/metabolism ; Animals ; RNA-Binding Proteins ; },
abstract = {MicroRNA-mediated gene silencing is a conserved mechanism of post-transcriptional gene regulation across metazoans. It depends on base pairing between small RNAs and mRNAs, and on protein complexes including the RNA-induced silencing complex (RISC), where Argonaute 2 (AGO2) plays a central role. A full understanding of RNA silencing requires reliable molecular tools to study AGO2 and RISC. Affinity tagging and antibody-based methods can introduce artefacts, and both the N- and C-terminal domains of AGO2 are critical for its function. While N-terminal tags are frequently used, and a recent study in mice showed altered activity in N-terminal HaloTag-AGO2 fusions, the consequences of C-terminal tagging remain underexplored. CRISPaint, a CRISPR-Cas9-based technique, enables endogenous C-terminal tag fusions without requiring homology arms. Using this system, we generated the first C-terminal HaloTag fusion of AGO2 (AGO2HALO) in human A549 cells. We found that the AGO2HALO fusion protein exhibits reduced binding with TNRC6A, with no effect on cell viability. However, it significantly impairs RNA cleavage, silencing activity, and nuclear localization. We further compared AGO2-EGFP and EGFP-AGO2 using transient transfection. N-terminally tagged AGO2 retained wild-type-like function and localization, while C-terminally tagged AGO2 was impaired in siRNA and miRNA silencing, nuclear import, and P-body localization. These results demonstrate that a C-terminal HaloTag compromises AGO2 functionality and is unsuitable for studying RISC biology. Our findings highlight the importance of validating tagging strategies to avoid misleading conclusions due to tag-induced functional defects. Pre-print, bioRxiv.},
}
@article {pmid40698183,
year = {2025},
author = {Sailer, AL and Wörtz, J and Smith, V and Stachler, AE and Blau, F and Daratha, M and Maier, LK and Allers, T and Marchfelder, A},
title = {CRISPR-Cas induced self-targeting identifies key players in archaeal microhomology-mediated end joining.},
journal = {microLife},
volume = {6},
number = {},
pages = {uqaf015},
pmid = {40698183},
issn = {2633-6693},
abstract = {DNA repair processes are the foundation for genome integrity and survival, especially in extreme environments where DNA damage occurs more frequently and where archaea are found. Nevertheless, first-hand experimental information on repair pathways in archaea is scarce, and assignment of repair proteins is currently largely based on homology. We showed previously that DNA lesions induced by clustered regularly interspaced short palindromic repeats Cas (CRISPR-Cas) self-targeting are repaired by microhomology-mediated end joining (MMEJ). To identify proteins involved in the archaeal MMEJ pathway, we used deletion strains devoid of proteins assigned to the key steps of MMEJ, to examine changes in the repair outcome. In addition, we used aphidicolin to inhibit the activity of the essential PolB1 protein. For the first time, we were thereby able to experimentally identify proteins involved in this repair pathway in the euryarchaeal model organism Haloferax volcanii. This study confirms that Mre11, Rad50, Fen1, PolB1, LigA, and LigN take part in MMEJ, as previously inferred. In addition, we show that Cas1 and Hel308a are also involved in the MMEJ pathway.},
}
@article {pmid40697666,
year = {2025},
author = {Han, H and Sun, X and Guo, X and Wen, J and Zhao, X and Zhou, W},
title = {CRISPR/Cas9 technology in tumor research and drug development application progress and future prospects.},
journal = {Frontiers in pharmacology},
volume = {16},
number = {},
pages = {1552741},
pmid = {40697666},
issn = {1663-9812},
abstract = {The CRISPR/Cas9 system is an acquired immune defense mechanism that has evolved in bacteria and archaea to protect against viral and plasmid attacks. It consists of regularly spaced clusters of short palindromic repeats (CRISPR) and CRISPR-associated proteins (Cas). By adapting the simplest type II CRISPR system to utilize special small guide RNA (sgRNA) and Cas9 nucleic acid endonuclease, precise cuts can be made at specific locations in double-stranded DNA, facilitating gene knockout or knock-in. Due to its efficient gene editing capabilities, CRISPR/Cas9 technology has been widely adopted across various biological and scientific research fields, demonstrating significant potential in tumor research and drug development. This article reviews the progress and future prospects of CRISPR/Cas9 technology in tumor genome editing, drug target screening and validation, and new drug development. It details the fundamental role of this technology in cancer biology research, encompassing various aspects such as gene transcription editors, epigenetic editors, precision genome engineering, and CRISPR-Cas systems targeting RNA. Additionally, the article discusses key applications of CRISPR/Cas9 in anticancer drug discovery, including drug target identification, drug target screening and validation, combinatorial genetic screening, screening of small molecules to overcome resistance to CAR-T therapies, and multimodal functional genomics integration strategies. Finally, although CRISPR/Cas9 has demonstrated great potential for efficient gene editing, precise target discovery, and promotion of personalized therapy and drug screening in oncology research, its application still faces technical bottlenecks such as off-target effects, genomic instability, and low editing efficiency in solid tumors, as well as ethical controversies in gene editing, safety assessment of delivery systems and immune responses in clinical translation, and other ethical and translational challenges.},
}
@article {pmid40695321,
year = {2025},
author = {Ara, PS and Casacuberta, E and Scazzocchio, C and Ruiz-Trillo, I and Najle, SR},
title = {CRISPR-Cas9 genome editing in Corallochytrium limacisporum,a key species for understanding animal origins.},
journal = {Open biology},
volume = {15},
number = {7},
pages = {250066},
doi = {10.1098/rsob.250066},
pmid = {40695321},
issn = {2046-2441},
support = {4973.01//Moore Foundation - MMI Experimental Model Systems/ ; PID2023-153273NB-I00//MICIU/ AEI /10.13039/501100011033/ ; "ERDF A way of making Europe", PID2023-153273NB-I00//MICIU/AEI/10.13039/501100011033/ ; exp. 2021 SGR 00751//Departament de Recerca i Universitats de la Generalitat de Catalunya/ ; },
mesh = {*CRISPR-Cas Systems ; *Gene Editing/methods ; Animals ; Point Mutation ; },
abstract = {Microbial holozoans are the closest unicellular relatives of animals. They share a substantial gene repertoire with animals and exhibit complex life cycles. Studying these organisms is crucial for understanding the evolution of multicellularity, and significant progress has been made in uncovering key aspects of the biology of the four microbial holozoans lineages: choanoflagellates, filastereans, ichthyosporeans and corallochytreans. However, reverse genetic tools are still lacking in corallochytreans, one of the earliest-branching holozoan lineages and the only known group with both coenocytic and binary fission development. Here, we present CRISPR-Cas9-mediated gene inactivation and point mutation methodologies in the corallochytrean Corallochytrium limacisporum. As a proof of concept, we inactivated the fkb12 gene, a component of the mTOR pathway, conferring rapamycin resistance, and introduced a point mutation in sdhB, encoding a subunit of succinate dehydrogenase, conferring carboxin resistance. Our results demonstrate the presence of both non-homologous end-joining and homology-directed repair pathways in C. limacisporum and shows an editing efficiency of approximately 2%. Furthermore, simultaneous gene targeting revealed a co-editing frequency of approximately 20%. Finally, this study establishes unequivocally that C. limacisporum is haploid, making it an ideal model for genetic studies and gene editing applications to unravel the molecular mechanisms involved in animal origins.},
}
@article {pmid40695225,
year = {2025},
author = {Birocchi, F and Maus, MV},
title = {Cas12a knockin mice expand the toolbox for in vivo and ex vivo multiplexed genome editing.},
journal = {Developmental cell},
volume = {60},
number = {14},
pages = {1931-1933},
doi = {10.1016/j.devcel.2025.05.012},
pmid = {40695225},
issn = {1878-1551},
mesh = {Animals ; *Gene Editing/methods ; Mice ; *Gene Knock-In Techniques/methods ; *CRISPR-Cas Systems/genetics ; *CRISPR-Associated Proteins/genetics/metabolism ; *Endodeoxyribonucleases/genetics/metabolism ; Mice, Transgenic ; Bacterial Proteins ; },
abstract = {Multiplex genome editing is critical for advancing transformative gene therapies and unraveling complex genetic interactions. In a recent issue of Nature Biomedical Engineering, Tang et al. introduced Cas12a knockin mice, providing a versatile platform for disease modeling and the development of multiplexed therapeutic strategies.},
}
@article {pmid40694851,
year = {2025},
author = {Song, G and Li, J and Han, J and Gao, X and Tian, C and Zhang, F and Tian, Y},
title = {Identification of regulatory sequences in Aca11 and Aca13 for detection of anti-CRISPR and protein-protein interaction.},
journal = {Nucleic acids research},
volume = {53},
number = {14},
pages = {},
doi = {10.1093/nar/gkaf694},
pmid = {40694851},
issn = {1362-4962},
support = {2022YFF0710700//National Key R&amp;D Program of China/ ; 2024YFF0728700//National Key R&amp;D Program of China/ ; 2021YFF0702800//National Key R&amp;D Program of China/ ; 2020YFA0803501//National Key R&amp;D Program of China/ ; 32270567//National Natural Science Foundation of China/ ; 32070533//National Natural Science Foundation of China/ ; KFJ-BRP-005//Biological Resource Program of Chinese Academy of Sciences/ ; },
mesh = {Humans ; *Bacterial Proteins/genetics/metabolism ; *CRISPR-Cas Systems/genetics ; Promoter Regions, Genetic ; CRISPR-Associated Protein 9/metabolism/genetics ; Operon ; *CRISPR-Associated Proteins/genetics/metabolism ; Streptococcus/genetics ; *Regulatory Sequences, Nucleic Acid ; Protein Binding ; },
abstract = {Anti-CRISPR (Acr) proteins are frequently co-encoded with the anti-CRISPR associated (Aca) proteins, which act as repressors for regulating Acr expression within acr-aca operons. We previously identified three aca genes (aca11-13) from Streptococcus mobile genetic elements, but their regulatory mechanisms remained unclear. Here, we showed that Aca11 and Aca13 mediate bidirectional regulation in acr-aca operons through recognition of their inverted repeat (IR) sequences within the acr promoters. Based on the bioinformatics search using Aca13 with its IR sequences, we discovered a novel type II-A Acr (named AcrIIA35). AcrIIA35 exhibits a potent inhibitory activity against St1Cas9 by interfering with DNA recognition of Cas9 in bacterial and human cells. We also developed a novel Aca-driven protein-protein interaction detection (APID) system by integrating Aca-tagged target proteins with fluorescently labeled IR-DNA probes. The APID system enables efficient detection of protein-protein interaction using proteins or crude cell lysates. Utilizing the APID system, we have further elucidated the mechanism of AcrIIA24, which can interact with the HNH nuclease domain of St3Cas9 to inhibit the DNA cleavage activity of Cas9. Collectively, our work expands the understanding of Aca functions to modulate Acrs and expands the potential for Aca-based applications in CRISPR technologies.},
}
@article {pmid40694850,
year = {2025},
author = {Eleveld, TF and Ylstra, B and Looijenga, LHJ},
title = {Low-amplitude copy number gains shape cancer through known and novel oncogenes with associated therapeutic vulnerabilities.},
journal = {Nucleic acids research},
volume = {53},
number = {14},
pages = {},
doi = {10.1093/nar/gkaf689},
pmid = {40694850},
issn = {1362-4962},
support = {KWF 20161-1-10271//Dutch Cancer Society/ ; //KIKA/ ; },
mesh = {Humans ; *Oncogenes/genetics ; *Neoplasms/genetics/drug therapy ; *DNA Copy Number Variations ; Drug Resistance, Neoplasm/genetics ; *Gene Dosage ; Biomarkers, Tumor/genetics ; CRISPR-Cas Systems ; },
abstract = {Large chromosomal copy number gains are ubiquitous throughout cancer types. However, which genes drive their selective advantage is not well established, and therefore they are hardly utilized in clinical practice. Our analysis of copy number patterns in pan-cancer datasets suggests that the selective advantage of copy number gains is largely driven by known oncogenes. Analysis of CRISPR screening data identifies a list of 101 genes that are likely to mediate the effect of these gains, which is highly enriched in annotated oncogenes but also contains genes that have not been implicated in cancer so far. Moreover, we show that specific gains are associated with drug sensitivity or resistance, with a strong enrichment of gains of oncogenes with increased sensitivity to inhibitors targeting these specific genes. Finally, we provide examples where gains can function as relevant clinical biomarkers for diagnosis and treatment. Thus, large copy number gains exert their selective advantage through known and novel oncogenes, and their systematic analysis could advance precision oncology.},
}
@article {pmid40693713,
year = {2025},
author = {Piccolo, D and Sladen, P and Guarascio, R and Ziaka, K and Cheetham, ME},
title = {Investigation of ABCA4 Missense Variants and Potential Small Molecule Rescue in Retinal Organoids.},
journal = {Investigative ophthalmology &amp; visual science},
volume = {66},
number = {9},
pages = {58},
doi = {10.1167/iovs.66.9.58},
pmid = {40693713},
issn = {1552-5783},
mesh = {Humans ; *Mutation, Missense ; *Organoids/metabolism/drug effects ; *ATP-Binding Cassette Transporters/genetics/metabolism ; Induced Pluripotent Stem Cells/metabolism ; *Retinal Diseases/genetics/metabolism/pathology ; *Retina/metabolism ; CRISPR-Cas Systems ; Cells, Cultured ; Cell Differentiation ; },
abstract = {PURPOSE: ABCA4-related retinopathy is the most common monogenic eye disorder in the world and is currently untreatable. Missense variants in ABCA4 constitute ∼60% of causal ABCA4-related retinopathy variants, often resulting in misfolded or dysfunctional protein products. Despite their prevalence, the molecular mechanisms by which these missense mutations impair ABCA4 function are not fully understood, primarily due to limitations in suitable cellular models. In this study, we investigated the cellular and molecular consequences of ABCA4 missense variants using a human photoreceptor-like model system.<br><br>METHODS: We used CRISPR/Cas9 technology to introduce two ABCA4 missense misfolding variants, T983A and R2077W, which are associated with ABCA4-associated retinopathy, into control induced pluripotent stem cells (iPSCs). The iPSCs were differentiated into retinal organoids, characterized and treated with small molecules.<br><br>RESULTS: The expression level of ABCA4 missense proteins was reduced compared to WT ABCA4 suggesting the variants were degraded in a photoreceptor-like environment. The localization of the missense variants was also altered with negligible ABCA4 detectable in the retinal organoid outer segments compared to the isogenic control. Two small molecule compounds, AICAR and 4-PBA, previously identified as potential ABCA4 folding correctors in vitro, were tested for their ability to enhance ABCA4 traffic to the outer segment. The compounds did not appear to promote ABCA4 folding and traffic in photoreceptors and instead led to a decrease in ABCA4 transcript levels and protein.<br><br>CONCLUSIONS: These data highlight that retinal organoids are an exquisite model to investigate pathogenic variants in ABCA4 and test small compounds for translation to the human retina.},
}
@article {pmid40690290,
year = {2025},
author = {Huang, J and Zhao, X and Zang, X and Jin, Z and Zhang, X and Huang, Y and Zhang, L and Xue, X and Zhang, P},
title = {Identification of ATM1 gene involved in antifungal resistance based on CRISPR/Cas9 technology in Cryptococcus gattii.},
journal = {Medical mycology},
volume = {63},
number = {7},
pages = {},
doi = {10.1093/mmy/myaf061},
pmid = {40690290},
issn = {1460-2709},
support = {2021YFC2302100//National Key Research and Development Program of China/ ; 31900130//National Foundation of Science of China/ ; },
mesh = {*Cryptococcus gattii/genetics/drug effects/growth &amp; development/pathogenicity ; *CRISPR-Cas Systems ; *Drug Resistance, Fungal/genetics ; *Antifungal Agents/pharmacology ; Cryptococcosis/microbiology ; Animals ; *ATP-Binding Cassette Transporters/genetics/metabolism ; Mice ; *Fungal Proteins/genetics/metabolism ; Virulence ; Humans ; },
abstract = {Cryptococcus gattii is a fungal pathogen that poses significant threats to human health, affecting both immunocompromised and immunocompetent individuals. Treatment of C. gattii infections typically involves the use of antifungal agents, such as azoles. However, the increasing emergence of antifungal resistance in C. gattii is a growing concern, highlighting the critical need for novel therapeutic strategies. In our previous study, we identified a mitochondrial ATP-binding cassette (ABC) transporter, Atm1, as potentially involved in antifungal resistance in C. gattii through transcriptome sequencing, but its function remains unclear and requires additional confirmation and investigation. In this study, we developed a "suicide" clustered regularlyinterspaced short palindromic repeats-CRISPR-associated protein 9 system in C. gattii, based on the system used in C. neoformans, and successfully validated its functionality by targeting the ADE2 gene. We subsequently generated C. gattii mutants lacking ATM1 and assessed their growth under various stress conditions. Our data suggest that Atm1 is involved in the iron-sulfur cluster biosynthesis process. Besides, disruption of ATM1 resulted in various growth impairments, including reduced stress tolerance, impaired capsule formation, and diminished virulence. Importantly, we observed compromised antifungal drug resistance in the atm1∆ mutant and performed RNA sequencing-based transcriptome analysis and gene ontology analysis with and without antifungal treatment for further investigation. In conclusion, our findings indicate that ATM1 plays a role in iron homeostasis and is critical for antifungal resistance in C. gattii, offering new insights into potential drug development strategies for the clinical treatment of cryptococcosis.},
}
@article {pmid40674406,
year = {2025},
author = {Dinesh, RK and Wang, C and Qu, Y and Rustagi, A and Cousins, H and Zengel, J and Wang, X and Barnard, TR and Johnson, WA and Xu, G and Zhang, T and Magazine, N and Beck, A and Heilbroner, LM and Peters-Schulze, G and Wilk, AJ and Wang, M and Huang, W and Howitt, BE and Carette, J and Altman, R and Blish, CA and Cong, L},
title = {Membrane-wide screening identifies potential tissue-specific determinants of SARS-CoV-2 tropism.},
journal = {PLoS pathogens},
volume = {21},
number = {7},
pages = {e1013157},
doi = {10.1371/journal.ppat.1013157},
pmid = {40674406},
issn = {1553-7374},
mesh = {Humans ; *SARS-CoV-2/physiology ; *COVID-19/virology/metabolism ; *Virus Internalization ; *Viral Tropism/physiology ; Angiotensin-Converting Enzyme 2/metabolism/genetics ; HEK293 Cells ; Organ Specificity ; CRISPR-Cas Systems ; Antigens, Differentiation ; },
abstract = {While SARS-CoV-2 primarily infects the respiratory tract, clinical evidence indicates that cells from diverse cell types and organs are also susceptible to infection. Using the CRISPR activation (CRISPRa) approach, we systematically targeted human membrane proteins in cells with and without overexpression of ACE2, thus identifying unrecognized host factors that may facilitate viral entry. Validation experiments with replication-competent SARS-CoV-2 confirmed the role of newly identified host factors, particularly the endo-lysosomal protease legumain (LGMN) and the potassium channel KCNA6, upon exogenous overexpression. In orthogonal experiments, we show that disruption of endogenous LGMN or KCNA6 decreases viral infection and that inhibitors of candidate factors can reduce viral entry. Additionally, using clinical data, we find possible associations between expression of either LGMN or KCNA6 and SARS-CoV-2 infection in human tissues. Our results identify potentially druggable host factors involved in SARS-CoV-2 entry, and demonstrate the utility of focused, membrane-wide CRISPRa screens in uncovering tissue-specific entry factors of emerging pathogens.},
}
@article {pmid40639395,
year = {2025},
author = {Ge, Y and Zheng, W and Hou, Z and Zhang, Y and Du, B and Wei, S and Liu, X and Chen, Z and Zhang, H},
title = {Ultra-sensitive detection of microRNA in intraocular fluid using optical fiber sensing technology for central nervous system lymphoma diagnosis.},
journal = {Reports on progress in physics. Physical Society (Great Britain)},
volume = {88},
number = {7},
pages = {},
doi = {10.1088/1361-6633/adee44},
pmid = {40639395},
issn = {1361-6633},
mesh = {*MicroRNAs/analysis/genetics/metabolism ; Humans ; *Lymphoma/diagnosis/genetics ; *Optical Fibers ; *Central Nervous System Neoplasms/diagnosis/genetics ; Limit of Detection ; *Aqueous Humor/metabolism ; *Fiber Optic Technology ; Surface Plasmon Resonance ; *Biosensing Techniques ; CRISPR-Cas Systems ; },
abstract = {MicroRNA (miRNA) in aqueous humor holds significant promise as a non-invasive biomarker of primary central nervous system lymphoma (PCNSL), enabling early diagnosis and prognosis. However, current methods for miRNA detection often suffer from limitations affecting sensitivity, specificity, and clinical applicability. This study introduces a novel black phosphorus (BP)-enhanced fiber-optic surface plasmon resonance sensor integrated with a clustered regularly interspaced short palindromic repeats (CRISPR)-Cas13a system for ultrasensitive and single-base-specific detection of PCNSL-associated miRNA in aqueous humor. The BP nano-interface significantly enhances the surface plasmon resonance signal, while the CRISPR-Cas13a technology enables highly specific detection of miRNA, down to single nucleotide mismatches. This system achieves a detection limit as low as 21 aM without the need for amplification and demonstrates robust performance in analyzing clinical samples. With its unparalleled sensitivity, specificity, label-free operation, and potential for portability, this biosensing platform offers transformative capabilities for early PCNSL diagnosis, prognosis, and treatment monitoring.},
}
@article {pmid40615675,
year = {2025},
author = {Chan, PY and Alexander, D and Mehta, I and Matsuyama, LSAS and Harle, V and Olvera-León, R and Park, JS and Arriaga-González, FG and van der Weyden, L and Cheema, S and Iyer, V and Offord, V and Barneda, D and Hawkins, PT and Stephens, L and Kozik, Z and Woods, M and Wong, K and Balmus, G and Vinceti, A and Thompson, NA and Del Castillo Velasco-Herrera, M and Wessels, L and van de Haar, J and Gonçalves, E and Sinha, S and Vázquez-Cruz, ME and Bisceglia, L and Raimondi, F and Choudhary, J and Patiyal, S and Venkatesh, A and Iorio, F and Ryan, CJ and Adams, DJ},
title = {The synthetic lethal interaction between CDS1 and CDS2 is a vulnerability in uveal melanoma and across multiple tumor types.},
journal = {Nature genetics},
volume = {57},
number = {7},
pages = {1672-1683},
pmid = {40615675},
issn = {1546-1718},
support = {220540/Z/20/A//Wellcome Trust (Wellcome)/ ; 1/CX/CSRD VA/United States ; 1/CX/CSRD VA/United States ; 1/CX/CSRD VA/United States ; 1/CX/CSRD VA/United States ; 1/CX/CSRD VA/United States ; 1/CX/CSRD VA/United States ; },
mesh = {Humans ; *Melanoma/genetics/pathology ; *Uveal Neoplasms/genetics/pathology ; Uveal Melanoma ; Cell Line, Tumor ; CRISPR-Cas Systems/genetics ; Apoptosis/genetics ; Gene Expression Regulation, Neoplastic ; *Synthetic Lethal Mutations/genetics ; Animals ; },
abstract = {Metastatic uveal melanoma is an aggressive disease with limited effective therapeutic options. To comprehensively map monogenic and digenic dependencies, we performed CRISPR-Cas9 screening in ten extensively profiled human uveal melanoma cell line models. Analysis involved genome-wide single-gene and combinatorial paired-gene CRISPR libraries. Among our 76 uveal melanoma-specific essential genes and 105 synthetic lethal gene pairs, we identified and validated the CDP-diacylglycerol synthase 2 gene (CDS2) as a genetic dependency in the context of low CDP-diacylglycerol synthase 1 gene (CDS1) expression. We further demonstrate that CDS1/CDS2 forms a synthetic lethal interaction in vivo and reveal that CDS2 knockout results in the disruption of phosphoinositide synthesis and increased cellular apoptosis and that re-expression of CDS1 rescues this cell fitness defect. We extend our analysis using pan-cancer data, confirming increased CDS2 essentiality in diverse tumor types with low CDS1 expression. Thus, the CDS1/CDS2 axis is a therapeutic target across a range of cancers.},
}
@article {pmid40615674,
year = {2025},
author = {Arnoldus, T and van Vliet, A and Bleijerveld, OB and de Groot, AFH and Piao, Q and Blomberg, N and Schatton, D and Dong, J and van Hal-van Veen, SE and Harkes, R and Grootemaat, AE and Proost, N and Cabukusta, B and Frezza, C and van de Ven, M and van der Wel, NN and Giera, M and Altelaar, M and Peeper, DS},
title = {Cytidine diphosphate diacylglycerol synthase 2 is a synthetic lethal target in mesenchymal-like cancers.},
journal = {Nature genetics},
volume = {57},
number = {7},
pages = {1659-1671},
pmid = {40615674},
issn = {1546-1718},
mesh = {Humans ; *Synthetic Lethal Mutations/genetics ; *Neoplasms/genetics/pathology ; Cell Line, Tumor ; Gene Expression Regulation, Neoplastic ; CRISPR-Cas Systems ; Apoptosis/genetics ; },
abstract = {Synthetic lethal interactions (SLIs) based on genomic alterations in cancer have been therapeutically explored. We investigated the SLI space as a function of differential RNA expression in cancer and normal tissue. Computational analyses of functional genomic and gene expression resources uncovered a cancer-specific SLI between the paralogs cytidine diphosphate diacylglycerol synthase 1 (CDS1) and CDS2. The essentiality of CDS2 for cell survival is observed for mesenchymal-like cancers, which have low or absent CDS1 expression and account for roughly half of all cancers. Mechanistically, the CDS1-2 SLI is accompanied by disruption of lipid homeostasis, including accumulation of cholesterol esters and triglycerides, and apoptosis. Genome-wide CRISPR-Cas9 knockout screens in CDS1-negative cancer cells identify no common escape mechanism of death caused by CDS2 ablation, indicating the robustness of the SLI. Synthetic lethality is driven by CDS2 dosage and depends on catalytic activity. Thus, CDS2 may serve as a pharmacologically tractable target in mesenchymal-like cancers.},
}
@article {pmid40570838,
year = {2025},
author = {Wang, L and Zhou, Y and Yu, Z and Wu, P and Lu, Z and Ma, L},
title = {TAS-seq enables subcellular single-stranded adenosine profiling by signal peptide-assisted adenosine deamination.},
journal = {Cell reports methods},
volume = {5},
number = {7},
pages = {101087},
doi = {10.1016/j.crmeth.2025.101087},
pmid = {40570838},
issn = {2667-2375},
mesh = {*Adenosine/metabolism ; Deamination ; Humans ; *Protein Sorting Signals ; Single-Cell Analysis/methods ; *Sequence Analysis, RNA/methods ; RNA Stability ; Adenosine Deaminase/metabolism ; HEK293 Cells ; CRISPR-Cas Systems ; },
abstract = {RNA structure plays a crucial role in its function and undergoes dynamic changes throughout its life cycle. To study these dynamics, we developed TAS sequencing (TAS-seq), which expresses the deaminase TadA-8e in specific subcellular compartments to modify single-stranded adenosines, particularly within hairpin loops. We applied TAS-seq to the nucleus, cytosol, and endoplasmic reticulum membrane, identifying adenosine structural variations and compartment-specific regulation of RNA stability. Single-cell TAS-seq revealed structural heterogeneity of cytosolic RNAs. Additionally, adenosines labeled by TAS-seq contribute to guide RNA optimization in the CRISPR-Cas13d system. Our method provides insights into compartment-specific RNA structural dynamics, cell-specific heterogeneity, and their functional implications.},
}
@article {pmid40543637,
year = {2025},
author = {Shanaka, KASN and Madushani, KP and Kim, MJ and Jung, S and Lee, J},
title = {Nlrc5 ablation interferes with MHC-I gene expression and immune cell migration.},
journal = {Developmental and comparative immunology},
volume = {169},
number = {},
pages = {105406},
doi = {10.1016/j.dci.2025.105406},
pmid = {40543637},
issn = {1879-0089},
mesh = {Animals ; *Zebrafish/immunology/genetics ; *Zebrafish Proteins/genetics/metabolism ; Cell Movement/genetics ; *Novirhabdovirus/immunology/physiology ; CRISPR-Cas Systems/genetics ; *Neutrophils/immunology ; *Fish Diseases/immunology ; Gene Knockout Techniques ; *Histocompatibility Antigens Class I/genetics ; Gene Expression Regulation ; Animals, Genetically Modified ; *Genes, MHC Class I/genetics ; },
abstract = {Nucleotide-binding domain and leucine-rich repeat-containing receptors (NLRs) have complex and diverse functions. Studies in mammalian models have indicated a clear role of NLRC5 in the regulation of MHC-I gene expression. The nlrc5 KO zebrafish model was generated using CRISPR/Cas9 technology to understand its function with respect to VHSV. The VHSV infection experiment demonstrated higher mortality in nlrc5 KO fish with higher VHSV copy numbers. Virus-infected pathology, such as tissue swelling, hemorrhage, and eye bulging, was significantly higher in the nlrc5 KO fish. Expressional analysis of ifn system genes indicated the significant upregulation of ifnφ1. Gene expression analysis during VHSV infection indicated a significant downregulation of MHC-I genes in the nlrc5 KO model compared to the wild-type (WT). Owing to the impact of MHC-I, the viral copy number may increase with higher mortality under VHSV infections. For neutrophil migration analysis, a nlrc5 KO and a neutrophil-labeled model were developed (nlrc5[-/-] Tg(mpx:mcherry)). Injury was generated in the caudal fin, and the injury site was stimulated with poly I: C. The number of neutrophils were reduced in the nlrc5 KO fish. Levels of critical cytokines responsible for neutrophil migration were significantly reduced in nlrc5 KO fish during VHSV infection. The data from the current study in zebrafish reconfirm the observations in mammalian models, and neutrophil migration analysis suggests that Nlrc5 may be associated with inflammatory activation in the presence of VHSV.},
}
@article {pmid40532693,
year = {2025},
author = {Modell, SM and Smith, JA and Kardia, SLR},
title = {Progress and Criteria in Public Health Applications of Gene Therapy and Gene Editing: Beyond the White Paper.},
journal = {Public health genomics},
volume = {28},
number = {1},
pages = {241-251},
doi = {10.1159/000546850},
pmid = {40532693},
issn = {1662-8063},
mesh = {*Genetic Therapy/methods/trends ; Humans ; *Gene Editing/methods ; *Public Health ; Anemia, Sickle Cell/therapy/genetics ; CRISPR-Cas Systems ; },
abstract = {BACKGROUND: In 2023, the FDA approved two gene therapies for sickle cell disease (SCD), one of which follows a standard gene therapy protocol and the other a gene editing (CRISPR/Cas9) approach. Other gene therapy protocols for conditions relating to public health continue to advance and are being discussed in academic and professional circles. This review examines the pace of public health-related gene therapy and gene editing development since the publication of a key British white paper dealing with the pace of fruition in this field.<br><br>SUMMARY: Gene therapy developments related to public health fit into three overarching baskets: (1) gene therapy and editing for rare, single-gene disorders (e.g., homozygous familial hypercholesterolemia and hereditary amyloidosis polyneuropathy); (2) gene therapy and editing for high prevalence conditions (e.g., SCD); and (3) genetic engineering and gene editing of mosquitoes transmitting tropical disease. While the protocols listed in this purposive inspection largely center around phase III (comparing treatments), with several in phase II (establishing efficacy) and phase I (assessing safety), costs of actual administration can span USD 2.1 to 3.1 million. By comparison, conventional SCD treatment runs between USD 22,500 and USD 200,000 per year for its most severe forms. Expert and public buy-in of gene editing of mosquitoes to reduce tropical disease and for human germline gene editing contain many caveats, with public health serving a useful monitoring and filtering role for how a technology might be deemed permissible.<br><br>KEY MESSAGES: Gene therapy has advanced beyond the stage where possible consequences serve as an automatic barrier to mainstream use, moving it closer to British white paper objectives. Ethical and feasible adoption by public health, taking into account population needs, will most likely happen through a combination Medicaid and Medicare, as opposed to the system governing newborn screening, under arrangements similar to the Centers for Medicare and Medicaid Services' coverage under evidence development program. Vector gene drives to alleviate tropical disease should remain privately financed, with this type of financing also being used for the vast majority of gene therapies entering the market. Though the criteria for germline applications continue to evolve, in the end such applications do not serve public health purposes. Academic public health has a monitoring role to play as relevant gene therapy and gene editing trials evolve; public health practice a referral and field monitoring role in the T3 (implementation) and T4 (population outcomes) translational research phases for the few applications that could justifiably receive public funding and public health support.},
}
@article {pmid40530850,
year = {2025},
author = {Chen, Y and Shi, Y and Zuo, X and Dong, X and Xiao, X and Chen, L and Xiang, Z and Ren, L and Zhou, Z and Wei, W and Lei, X and Wang, J},
title = {UNC0638 inhibits SARS-CoV-2 entry by blocking cathepsin L maturation.},
journal = {Journal of virology},
volume = {99},
number = {7},
pages = {e0074125},
pmid = {40530850},
issn = {1098-5514},
mesh = {*SARS-CoV-2/physiology/drug effects ; Humans ; *Cathepsin L/metabolism/antagonists &amp; inhibitors/genetics ; *Virus Internalization/drug effects ; COVID-19/virology ; *Antiviral Agents/pharmacology ; HEK293 Cells ; CRISPR-Cas Systems ; Vero Cells ; Animals ; Chlorocebus aethiops ; },
abstract = {Since the outbreak of SARS-CoV-2, viral mutations have posed significant challenges in identifying therapeutic targets and developing broad-spectrum antiviral drugs. Post-translational modifications of genes involved in interferon production and signaling pathways play a crucial role in regulating interferon responses. In this study, we employed CRISPR-Cas9 screening based on adenine base editors to investigate functional amino acids in 1,278 innate immune-related genes. This approach, which converts A-T base pairs into G-C base pairs to probe the functional importance of specific amino acids, allowed us to identify 17 vital factors involved in SARS-CoV-2 infection. Among the candidate genes, genetic knockdown of EHMT2 exhibited the strongest antiviral effect. Further analysis revealed that UNC0638, a selective inhibitor of EHMT2, significantly reduced the endosomal entry of SARS-CoV-2 in pseudovirus assays. The observed inhibitory effect was consistently observed across multiple SARS-CoV-2 variants, including Alpha, Beta, Delta, and Omicron. Mechanistically, UNC0638 reduced mature cathepsin L (CTSL) levels, impairing the proteolytic cleavage of SARS-CoV-2 spike protein and subsequent membrane fusion, a critical step for viral entry. Our findings uncover EHMT2 as a host dependency factor and reveal the antiviral mechanism of EHMT2 inhibitors through CTSL maturation blockade. These results advance the understanding of host factors in SARS-CoV-2 infection and provide a strategic framework for developing host-targeted antiviral therapies.IMPORTANCEIn this study, we demonstrated that knockdown or knockout of EHMT2 inhibited SARS-CoV-2 infection, and inhibitors of EHMT2, including UNC0638, UNC0642, and BIX01294 showed similar restrictive effects. Mechanistically, the EHMT2 inhibitor UNC0638 restricts spike-mediated cell entry by inhibiting the maturation of CTSL, a critical protease required for SARS-CoV-2 entry via the endosomal pathway. Importantly, CTSL is not only essential for SARS-CoV-2 but also plays a key role in the entry of other coronaviruses that utilize similar pathways. Therefore, EHMT2 inhibitors could have broader applications as pan-coronavirus therapeutic agents.},
}
@article {pmid40522367,
year = {2025},
author = {Akiyama, K and Momobayashi, A and Okano, M},
title = {Direct detection of CRISPR-Cas9 ribonucleoprotein gene doping using RNA immunoprecipitation and quantitative PCR.},
journal = {Analytical and bioanalytical chemistry},
volume = {417},
number = {19},
pages = {4449-4460},
pmid = {40522367},
issn = {1618-2650},
mesh = {*CRISPR-Cas Systems/genetics ; *Doping in Sports ; *Ribonucleoproteins/genetics ; Animals ; Myostatin/genetics ; Humans ; Erythropoietin/genetics ; *Immunoprecipitation/methods ; Mice ; Gene Editing/methods ; *Real-Time Polymerase Chain Reaction/methods ; Actinin/genetics ; *RNA/genetics ; Limit of Detection ; Male ; RNA, Guide, CRISPR-Cas Systems/genetics ; },
abstract = {Gene doping, using technologies such as CRISPR-Cas9, poses a considerable threat to the integrity of sports. In 2018, the World Anti-Doping Agency implemented a ban on genome editing, which highlighted the need for sensitive and specific detection methods. Detection techniques that are currently available have shown effectiveness in specific contexts, but are limited by low sensitivity and short detection windows. To overcome these limitations, this study presents a new detection method for CRISPR-Cas9 ribonucleoprotein (RNP) complexes, termed RNA immunoprecipitation followed by quantitative PCR (RIP-qPCR). The primary focus of this research was the in vitro development of a detection method targeting genes critical for doping, including myostatin (MSTN), α-actinin 3 (ACTN3), erythropoietin receptor (EPOR), and erythropoietin (EPO), with in vivo proof-of-concept demonstrated using MSTN. The RIP-qPCR method demonstrated sensitive performance, with a limit of quantification of 0.1 ng/mL in plasma. This method successfully detected single guide RNA targeting MSTN, ACTN3, EPOR, and EPO, along with two types of Cas9 proteins in RNP complexes in vitro. Additionally, the detection capabilities of RIP-qPCR were maintained for up to 30 days when plasma samples were stored at 4 °C. In vivo experiments were performed where RNPs were administered via intramuscular and intravenous injections to target the murine Mstn gene. CRISPR-Cas9 RNPs remained detectable for up to 24 h following intramuscular injection and 12 h after intravenous injection. This study underscores the potential of RIP-qPCR as a powerful tool for anti-doping analysis, with future efforts on expanding the target gene panel to enhance the detection of gene editing in sports doping.},
}
@article {pmid40521885,
year = {2025},
author = {Choe, D and Lee, E and Song, Y and Kim, SC and Jeong, KJ and Palsson, B and Cho, B-K and Cho, S},
title = {CRISPRi screening reveals E. coli's anaerobic-like respiratory adaptations to gentamicin: membrane depolarization by CpxR.},
journal = {mSystems},
volume = {10},
number = {7},
pages = {e0035325},
pmid = {40521885},
issn = {2379-5077},
support = {2021R1A2C1012589//National Research Foundation of Korea/ ; 2018M3A9H024759//National Research Foundation of Korea/ ; RS-2023-00246928//National Research Foundation of Korea/ ; NNF16CC0021858//Novo Nordisk Fonden/ ; },
mesh = {*Escherichia coli/drug effects/genetics/metabolism/physiology ; *Gentamicins/pharmacology ; *Escherichia coli Proteins/genetics/metabolism ; *Anti-Bacterial Agents/pharmacology ; Anaerobiosis ; Gene Expression Regulation, Bacterial/drug effects ; Adaptation, Physiological/drug effects ; Membrane Potentials/drug effects ; Drug Resistance, Bacterial/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats ; CRISPR-Cas Systems ; },
abstract = {Bacterial genes serve diverse cellular functions and many affect fitness in response to environmental challenges. We employed CRISPR interference screening to investigate the fitness effect of each gene in Escherichia coli exposed to gentamicin, aiming to understand the cellular defense mechanisms. Our findings revealed that ribosomal proteins, ribosome-associated proteins, toxin-antitoxin systems, and outer membrane proteins strongly influence the fitness of E. coli in gentamicin. Notably, gentamicin-induced fitness changes resembled those under anaerobic conditions, where resistance to gentamicin was observed. Specifically, genes related to the biosynthesis of cofactors and electron carriers, crucial for the respiratory system, showed reduced essentiality under both gentamicin and anaerobic conditions, suggesting a disruption in membrane potential leading to limited gentamicin uptake. Transcriptomic and genome-wide binding analyses identified the two-component system CpxR as a key regulator of respiratory systems in response to gentamicin. Our study provides insights into cellular defense mechanisms, offering potential strategies for combating antibiotic resistance.IMPORTANCEBacteria can adapt to a variety of stressful environments, including antibiotic exposure. The mechanisms underlying antibiotic resistance remain an active area of investigation. Clustered regularly interspaced short palindromic repeats (CRISPR) interference enables specific silencing of gene expression, allowing researchers to assess the fitness effects of gene knockdowns under given conditions. Using genome-wide CRISPR interference screening on Escherichia coli exposed to gentamicin, we identified anaerobic-like fitness effects of genes involved in respiration and the maintenance of membrane potential-key processes that facilitate gentamicin entrance into the cell. Transcriptomic analysis and immunoprecipitation assays further indicated that the two-component system CpxR modulates respiratory adaptations in response to gentamicin challenge. These findings shed light on the development of antibiotic resistance in bacteria and may offer new insight into strategies for treating gentamicin-resistant pathogens.},
}
@article {pmid40505881,
year = {2025},
author = {Shihan, MTA and Hyodo, T and Fujino, T and Rahman, ML and Hasan, MN and Biswas, M and Vu, LQ and Jahan, N and Mihara, Y and Karnan, S and Ota, A and Tsuzuki, S and Toyoda, A and Hosokawa, Y and Kasahara, M and Konishi, H},
title = {Characterization of targeted knock-in achieved via tandem paired nicking mediated by CRISPR/Cas9 nickases.},
journal = {Methods (San Diego, Calif.)},
volume = {241},
number = {},
pages = {184-195},
doi = {10.1016/j.ymeth.2025.06.004},
pmid = {40505881},
issn = {1095-9130},
mesh = {*CRISPR-Cas Systems/genetics ; *Gene Knock-In Techniques/methods ; *Gene Editing/methods ; Humans ; *Deoxyribonuclease I/genetics/metabolism ; RNA, Guide, CRISPR-Cas Systems/genetics ; },
abstract = {Targeted knock-in of specific DNA sequences using CRISPR/Cas9 is an advanced technology that enables programmed genome alterations including insertions, deletions, and base substitutions exactly as designed. Despite its utility in life sciences and promise for medical and industrial applications, it remains critical to establish a methodology for highly precise and efficient targeted knock-in to facilitate the practical use of this technology. Tandem paired nicking (TPN) is a genome editing methodology leveraging nicking variants of CRISPR/Cas9 nucleases (Cas9 nickases) to create site-specific nicks within the homologous region of the genome and donor DNA. Such nicking configuration promotes precise and efficient targeted knock-in while repressing the formation of unintended insertions and deletions and p53-mediated DNA damage response. In this study, we conducted a detailed characterization of TPN-based targeted knock-in by performing genome editing assays with various nicking configurations modified from TPN. Our results demonstrated that genomic nicks remarkably contribute to TPN-based targeted knock-in, whereas donor nicks play a less critical role. The introduction of additional nicks beyond the standard two-nick configuration did not further improve the efficiency of TPN-based targeted knock-in. Comparison with other Cas9 nickase-based methodologies for targeted knock-in demonstrated largely equivalent knock-in efficiencies achieved by these methodologies. High-throughput long-read sequencing confirmed a lower incidence of undesired insertions and deletions of various lengths by TPN, in comparison with a conventional Cas9 nuclease-based approach. These findings underscore TPN as a methodology for precise and efficient targeted knock-in, and highlight the broad potential of Cas9 nickase-based targeted knock-in for clinical and industrial applications.},
}
@article {pmid40503884,
year = {2025},
author = {Liang, Z and Huang, C and Li, Y and Yang, C and Wang, N and Ma, X and Huo, Y-X},
title = {A recombineering-based platform for high-throughput genomic editing in Escherichia coli.},
journal = {Applied and environmental microbiology},
volume = {91},
number = {7},
pages = {e0019325},
doi = {10.1128/aem.00193-25},
pmid = {40503884},
issn = {1098-5336},
support = {11116022401//The foundation of Muyuan laboratory/ ; C2024105020//Natural Science Foundation of Hebei Province/ ; },
mesh = {*Escherichia coli/genetics ; *Gene Editing/methods ; *Genome, Bacterial ; CRISPR-Cas Systems ; Recombination, Genetic ; },
abstract = {UNLABELLED: Functional analysis of bacterial genes or genomic fragments in vivo primarily relies on the analysis of knockout strains. Although various methods have successfully generated bacterial knockout mutants, the parallel operation of multiple sites, especially in biofoundries, remains challenging. New technological refinements of existing methods are necessary for high-throughput genomic editing in bacteria. In this study, to modify numerous sites in parallel, we optimized the linear donor DNA by adding modification at the different positions and achieved high-efficiency recombination with chemical transformation. Then, by combining with the CRISPR system, we established a guide sequence-independent and donor DNA-mediated genomic editing (GIDGE) method, enabling efficient and scarless engineering of common E. coli strains as well as wild-type strains such as E. coli MG1655, with particularly marked advantages demonstrated in E. coli Nissle 1917. This method allows for high-throughput genomic engineering in a 96-well format and is useful for sequence deletion with a wide range of lengths, sequence insertion, sequence replacement, and point mutation. As a proof-of-concept study, we constructed 96 single-gene knockout mutants and a genomic large-fragment deletion library in E. coli K-12 MG1655 using the GIDGE method. This high-throughput and easy-to-use method has great potential for automation and can be adapted for use in biofoundries.<br><br>IMPORTANCE: With the increasing demand in the microbiology field and the expansion of its application scope, the urgency for genome editing techniques that are not only efficient and versatile but also capable of high-throughput processing and even automation has become increasingly critical. In this study, we enhanced the efficiency of recombination engineering by incorporating modifications and integrated it with the CRISPR system to develop an advanced gene editing method. This method allows for various gene editing events such as insertion, replacement, and long fragment knockout without the need for plasmid construction. It not only demonstrated high efficiency in common E. coli strains but also exhibited marked advantages in the probiotic strain E. coli Nissle 1917. This method is a versatile, efficient approach capable of high-throughput parallel gene editing. Using this method, we successfully constructed a large-scale strain library, significantly accelerating the process of microbial engineering.},
}
@article {pmid40373837,
year = {2025},
author = {Vandendriessche, B and Huyghebaert, J and Rossem, KV and Cremers, TC and Man, K and Sieliwonczyk, E and Boen, H and Akdeniz, D and Rabaut, L and Schippers, J and Ponsaerts, P and Kooy, RF and Loeys, B and Schepers, D and Alaerts, M},
title = {An NGS-based approach for precise and footprint-free CRISPR-based gene editing in human stem cells.},
journal = {Methods (San Diego, Calif.)},
volume = {241},
number = {},
pages = {33-42},
doi = {10.1016/j.ymeth.2025.05.004},
pmid = {40373837},
issn = {1095-9130},
mesh = {Humans ; *Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; *High-Throughput Nucleotide Sequencing/methods ; *Induced Pluripotent Stem Cells/metabolism/cytology ; *Human Embryonic Stem Cells/metabolism/cytology ; Transfection/methods ; },
abstract = {Precise gene editing with conventional CRISPR/Cas9 is often constrained by low knock-in (KI) efficiencies (≈ 2-20 %) in human induced pluripotent stem cells (hiPSCs) and human embryonic stem cells (hESCs). This limitation typically necessitates labour-intensive manual isolation and genotyping of hundreds of colonies to identify correctly edited cells. Fluorescence- or antibiotic-based enrichment methods facilitate the identification process but can compromise cell viability and genomic integrity. Here, we present a footprint-free editing strategy that combines low-density seeding with next-generation sequencing (NGS) to rapidly identify cell populations containing precisely modified clones. By optimising the transfection workflow and adhering to CRISPR/Cas9 KI design principles, we achieved high average editing efficiencies of 64 % in hiPSCs (introducing a Brugada syndrome-associated variant) and 51 % in hESCs (introducing a neurodevelopmental disorder (NDD)-associated variant). Furthermore, under suboptimal CRISPR design conditions, this approach successfully identified hESC clones carrying a second NDD-associated variant, despite average KI efficiencies below 1 %. Importantly, genomic integrity was preserved throughout subcloning rounds, as confirmed by Sanger sequencing and single nucleotide polymorphism (SNP) array analysis. Hence, this NGS-based enrichment strategy reliably identifies desired KI clones under both optimal and challenging conditions, reducing the need for extensive colony screening and offering an effective alternative to fluorescence- and antibiotic-based selection methods.},
}
@article {pmid40324704,
year = {2025},
author = {Quintanilla, I and Azeroglu, B and Sagar, MAK and Stracker, TH and Denchi, EL and Pegoraro, G},
title = {Optical pooled screening for the discovery of regulators of the alternative lengthening of telomeres pathway.},
journal = {Methods (San Diego, Calif.)},
volume = {241},
number = {},
pages = {1-12},
doi = {10.1016/j.ymeth.2025.05.001},
pmid = {40324704},
issn = {1095-9130},
mesh = {Humans ; *Telomere Homeostasis/genetics ; *Telomere/genetics/metabolism ; *In Situ Hybridization, Fluorescence/methods ; CRISPR-Cas Systems ; Telomerase/genetics/metabolism ; },
abstract = {Telomere elongation is essential for the proliferation of cancer cells. Telomere length control is achieved either by the activation of the telomerase enzyme, or by the recombination-based Alternative Lengthening of Telomeres (ALT) pathway. ALT is active in about 10-15% of human cancers, but its molecular underpinnings remain poorly understood, preventing the discovery of potential novel therapeutic targets. Pooled CRISPR-based functional genomic screens enable the unbiased discovery of molecular factors involved in cancer biology. Recently, Optical Pooled Screens (OPS) have significantly extended the capabilities of pooled functional genomics screens to enable sensitive imaging-based readouts at the single cell level and large scale. To gain a better understanding of the ALT pathway, we developed a novel OPS assay that employs telomeric native DNA FISH (nFISH) as an optical quantitative readout to measure ALT activity. The assay uses standard OPS protocols for library preparation and sequencing. As a critical element, an optimized nFISH protocol is performed before in situ sequencing to maximize the assay performance. We show that the modified nFISH protocol faithfully detects changes in ALT activity upon CRISPR knock-out (KO) of the FANCM and BLM genes, which were previously implicated in ALT. Overall, the OPS-nFISH assay is a reliable method that can provide deep insights into the ALT pathway in a high-throughput format.},
}
@article {pmid40143710,
year = {2025},
author = {Ji, C and Han, Y and Li, J and Wei, J and Yang, W and Cai, X and Tian, S and Chen, C and Wang, Y and Zhao, P and Cao, S and Zhang, W and Xu, J and Gu, B and Li, F and Liu, P and Pu, J},
title = {DNA Nanoflower-Powered CRISPR/Cas12a Biosensing Platform for Ultrasensitive Protein Detection in Clinical Samples.},
journal = {Small methods},
volume = {9},
number = {7},
pages = {e2402130},
doi = {10.1002/smtd.202402130},
pmid = {40143710},
issn = {2366-9608},
support = {32371468//National Natural Science Foundation of China/ ; 22204104//National Natural Science Foundation of China/ ; 22474077//National Natural Science Foundation of China/ ; 23ZR1461400 22ZR1459600//Shanghai Municipal Natural Science Foundation/ ; YG2023ZD07 YG2021QN23//Medical-Engineering Joint Funds from the Shanghai Jiao Tong University/ ; 20234Y0201//Foundation of Shanghai Municipal Health Commission/ ; 2022JC002//Foundation of Shanghai Municipal Health Commission/ ; TMSK-2024-203//National Key Scientific Infrastructure for Translational Medicine (Shanghai)/ ; },
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Biosensing Techniques/methods ; *DNA/chemistry ; *Lipocalin-2/urine/blood ; Biomarkers/blood/urine ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {Protein markers secreted by various human cells provide crucial insights for the early diagnosis and prognostic assessment of clinical diseases. However, restricted by efficient protein marker signal amplification in real clinical samples with complex compositions, accurate, sensitive, and rapid detection of protein markers remains largely challenging. Herein, a DNA nanoflower (DNF)-powered CRISPR/Cas12a biosensing platform (DNF-CRISPR) is presented that employs the DNF in upstream to amplify input signals for protein markers, while utilizing the CRISPR system in downstream to amplify output signals by trans cleavage. This upstream and downstream cascade amplification sensing platform exhibits high sensitivity (500 fg mL[-1]), rapid (≤2 h), and a broad dynamic range (2.5 pg mL[-1] to 25 ng mL[-1]). As a proof of concept, DNF-CRISPR biosensing platform enables the quantitative detection of neutrophil gelatinase-associated lipocalin (NGAL) biomarkers in blood and urine samples from kidney injury patients with 91% accuracy. This study provides a powerful and versatile approach for the accurate diagnosis of protein markers in clinical settings, facilitating the application of CRISPR/Cas12a-based sensing platforms for non-nucleic acid markers.},
}
@article {pmid40140561,
year = {2025},
author = {Kufrin, V and Seiler, A and Brilloff, S and Rothfuß, H and Küchler, S and Schäfer, S and Rahimian, E and Baumgarten, J and Ding, L and Buchholz, F and Ball, CR and Bornhäuser, M and Glimm, H and Bill, M and Wurm, AA},
title = {The histone modifier KAT2A presents a selective target in a subset of well-differentiated microsatellite-stable colorectal cancers.},
journal = {Cell death and differentiation},
volume = {32},
number = {7},
pages = {1259-1272},
pmid = {40140561},
issn = {1476-5403},
support = {WU977/2-1//Deutsche Forschungsgemeinschaft (German Research Foundation)/ ; 70114086//Deutsche Krebshilfe (German Cancer Aid)/ ; },
mesh = {Humans ; *Colorectal Neoplasms/genetics/pathology/metabolism ; Animals ; Mice ; Cell Line, Tumor ; *Histone Acetyltransferases/metabolism/genetics ; Cell Differentiation/genetics ; *Microsatellite Instability ; Cell Proliferation ; Histones/metabolism ; Gene Expression Regulation, Neoplastic ; CRISPR-Cas Systems ; },
abstract = {Lysine acetyltransferase 2 A (KAT2A) plays a pivotal role in epigenetic gene regulation across various types of cancer. In colorectal cancer (CRC), increased KAT2A expression is associated with a more aggressive phenotype. Our study aims to elucidate the molecular underpinnings of KAT2A dependency in CRC and assess the consequences of KAT2A depletion. We conducted a comprehensive analysis by integrating CRISPR-Cas9 screening data with genomics, transcriptomics, and global acetylation patterns in CRC cell lines to pinpoint molecular markers indicative of KAT2A dependency. Additionally, we characterized the phenotypic effect of a CRISPR-interference-mediated KAT2A knockdown in CRC cell lines and patient-derived 3D spheroid cultures. Moreover, we assessed the effect of KAT2A depletion within a patient-derived xenograft mouse model in vivo. Our findings reveal that KAT2A dependency is closely associated with microsatellite stability, lower mutational burden, and increased molecular differentiation signatures in CRC, independent of the KAT2A expression levels. KAT2A-dependent CRC cells display higher gene expression levels and enriched H3K27ac marks at gene loci linked to enterocytic differentiation. Furthermore, loss of KAT2A leads to decreased cell growth and viability in vitro and in vivo, downregulation of proliferation- and stem cell-associated genes, and induction of differentiation markers. Altogether, our data show that a specific subset of CRCs with a more differentiated phenotype relies on KAT2A. For these CRC cases, KAT2A might represent a promising novel therapeutic target.},
}
@article {pmid40691444,
year = {2025},
author = {Tian, T and Xiao, H and Guo, X and Chen, Y and Qiu, Z and Zhang, T and Chen, M and Qi, W and Cai, P and Cheng, M and Zhou, X},
title = {Identification of a key nucleotide influencing Cas12a crRNA activity for universal photo-controlled CRISPR diagnostics.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {6694},
pmid = {40691444},
issn = {2041-1723},
support = {32150019//National Natural Science Foundation of China (National Science Foundation of China)/ ; 2023M741238//China Postdoctoral Science Foundation/ ; },
mesh = {*CRISPR-Associated Proteins/genetics/metabolism ; *CRISPR-Cas Systems/genetics ; *Endodeoxyribonucleases/genetics/metabolism ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; *Bacterial Proteins/genetics/metabolism ; Mutation ; Humans ; *Nucleotides/genetics/metabolism ; },
abstract = {Developing a one-pot assay is a critical strategy for enhancing the applicability of CRISPR-based molecular diagnostics; however, it is hindered by CRISPR cleavage interfering with nucleic acid amplification templates. Photo-regulation strategies provide an ideal solution to suppress undesired CRISPR cleavage while maintaining detection efficiency. However, existing photo-controlled CRISPR diagnostic methods face limitations in universality, cost, and detection efficiency. In this study, we systematically examine the impact of mutations in the repeat recognition sequence (RRS), a four-nucleotide segment within the Cas12a crRNA direct repeat (DR) region, on cleavage activity. We observe that mutations at positions 3 or 4 nearly abolished crRNA activity. Based on this discovery, we introduce 6-nitropiperonyloxymethyl (NPOM) photo-caging modifications at positions 3 and 4. Photo-caging at position 4 demonstrates the most effective suppression of enzymatic activity and optimal light-mediated activation. We leverage this finding to develop a photo-controlled CRISPR diagnostic method, enabling a universally adaptable one-pot detection strategy. Furthermore, by incorporating a crRNA splinting strategy, this pre-preparable reagent can be adapted for the detection of virtually any target gene.},
}
@article {pmid40688512,
year = {2025},
author = {Kanazhevskaya, LY and Zhdanova, PV and Chernonosov, AA and Koval, VV},
title = {Off-target interactions in the CRISPR-Cas9 Machinery: mechanisms and outcomes.},
journal = {Biochemistry and biophysics reports},
volume = {43},
number = {},
pages = {102134},
pmid = {40688512},
issn = {2405-5808},
abstract = {The in vivo editing of genetic information necessitates tools of unprecedented accuracy. CRISPR-Cas-based systems have emerged as leading technologies for precisely targeting the genome. The Cas9 endonuclease derived from Streptococcus pyogenes is the most commonly used instrument for targeted DNA cleavage. The development of engineered and chimeric Cas9 variants with enhanced activity and specificity has enabled not only the simple knockout of target genes but also the sophisticated engineering of the epigenome. This advancement has broadened the potential applications of CRISPR-Cas9 technology for the treatment of various disorders characterized by a combination of mutations, deletions, and duplications in the coding and non-coding regions of the genome. The inherent simplicity and predictability of the CRISPR-Cas9 targeting mechanism have led to an explosive growth in the development of prototype gene-editing drugs. However, their therapeutic application is still challenged by potential off-target effects. The erroneous editing of tumor suppressors and oncogenes could lead to adverse outcomes that mitigate the benefits of CRISPR therapy. The evolution of DNA-targeting technologies requires a comprehensive understanding of the mechanisms underlying CRISPR-Cas9 off-target binding and cleavage. The use of massive libraries of DNA targets and guide RNAs, coupled with high-throughput sequencing, contributes significantly to the analysis of mismatch tolerance. Nevertheless, the detection of ultra-low levels of off-target activity is hindered by the sensitivity limitations of current technologies. This review focuses on the mechanisms responsible for off-target interactions during CRISPR-Cas9-mediated gene editing. We discuss the influence of various factors, including nucleotide context, enzyme concentration, guide RNA structure, and the energetics of the RNA-DNA hybrid on mismatch tolerance in vitro and in vivo. Recent advances in the development of technologies for predicting off-target effects are briefly summarized. Particular emphasis is placed on the role of the Cas9 protein structure in the allosteric regulation of the specific and non-specific activity of the Cas9-sgRNA complex.},
}
@article {pmid40687858,
year = {2025},
author = {Bi, Q and Liu, M and Yan, L and Cheng, J and Sun, Q and Dai, Y and Zou, L},
title = {Progress in the application of isothermal amplification technology in the diagnosis of infectious diseases.},
journal = {Frontiers in microbiology},
volume = {16},
number = {},
pages = {1601644},
pmid = {40687858},
issn = {1664-302X},
abstract = {Rapid detection of infectious diseases is critical for global public health prevention and control. However, the use of traditional molecular diagnostic methods, including PCR, has been limited because of their cumbersome procedures, complex equipment requirements, operation at different temperatures, and the level of expertise required for operation. Isothermal amplification technology (IAT) provides a rapid, sensitive, specific, simple and less costly method for diagnosing infectious diseases, which has led to revolutionary breakthroughs in molecular diagnostics. This paper summarizes recent progress in IAT technology, which focuses on the principles and applications of core technologies such as NASBA, LAMP, RPA, and RAA. In addition, the combination of IATs with the CRISPR/Cas system, which further revolutionizes nucleic acid detection technology, is explored in this review.},
}
@article {pmid40617444,
year = {2025},
author = {Zhu, Y and Kuang, S and Yao, Z and Sun, Y and Gao, H and Gao, Q and Ding, W and He, H and Li, Y and Qiu, L},
title = {Doublesex knockout via CRISPR/Cas9 disrupts fertility and sexual dimorphism of wings in the rice stem borer, Chilo suppressalis.},
journal = {Insect biochemistry and molecular biology},
volume = {182},
number = {},
pages = {104356},
doi = {10.1016/j.ibmb.2025.104356},
pmid = {40617444},
issn = {1879-0240},
mesh = {Animals ; *Moths/genetics/growth &amp; development/physiology ; Female ; Male ; *Insect Proteins/genetics/metabolism ; CRISPR-Cas Systems ; Fertility/genetics ; Sex Characteristics ; Wings, Animal/growth &amp; development ; Gene Knockout Techniques ; Phylogeny ; Larva/growth &amp; development/genetics ; Amino Acid Sequence ; },
abstract = {The doublesex (dsx) gene has a conserved role in sex determination in insects, controlling sexual development and mating behavior. Although dsx is known to participate in these critical functions, its role in insect sex determination remains not fully elucidated. Given the economic importance of the rice pest Chilo suppressalis, we employed this species as a model to investigate the function of its dsx homolog. We cloned and characterized the Csdsx gene, which is 1123 bp in length and encodes four sex-specific proteins: three female-specific isoforms of 252, 258, and 254 amino acids, respectively, and one male-specific isoform of 290 amino acids. Phylogenetic analysis revealed that Csdsx is highly conserved within Lepidoptera, containing two domains: DM DNA binding domain and DSX dimer domain. Analysis of transcripts produced from a mini-dsx construct transfected into human HEK293T cells indicates that the female-splicing pattern is the default mode. Quantitative real-time PCR showed that among various developmental stages, Csdsx expression peaked at the first instar larval stage and showed tissue-specific, stage-dependent patterns, with notably high levels in the larval midgut, pupal fat body, and adult thorax in both sexes. In comparison to wild-type (WT) adults, Csdsx-knockout individuals exhibited malformations in their external genitalia, and female wing patterns became masculinized. Dissections revealed that knockout females had a reduced number of mature oocytes, while knockout males displayed a smaller testis area. Furthermore, when Csdsx-knockout females were paired with wild-type males, their mating behavior was significantly impaired. RNA-seq revealed that Csdsx disruption led to sex-biased gene expression shifts, including upregulation of male-associated genes (e.g., OBPs, PBPs, trypsin) and downregulation of female-specific genes (vitellogenin, FAS), indicating partial masculinization at the transcriptional level. These findings underscore the critical role of the dsx gene in reproductive development and sexual dimorphism in C. suppressalis.},
}
@article {pmid40607956,
year = {2025},
author = {Shi, K and Zhang, J and Yuan, R and Xiang, Y},
title = {Target-induced recycling and self-folding hairpin primer-mediated LAMP activation of CRISPR/Cas12a for highly sensitive aptamer-based therapeutic antibody assay.},
journal = {The Analyst},
volume = {150},
number = {15},
pages = {3475-3480},
doi = {10.1039/d5an00458f},
pmid = {40607956},
issn = {1364-5528},
mesh = {*Aptamers, Nucleotide/chemistry/genetics ; *Trastuzumab/analysis/blood ; Humans ; *CRISPR-Cas Systems ; *Nucleic Acid Amplification Techniques/methods ; Limit of Detection ; *CRISPR-Associated Proteins/genetics/metabolism ; DNA Primers/chemistry/genetics ; Bacterial Proteins ; Endodeoxyribonucleases ; Molecular Diagnostic Techniques ; },
abstract = {Owing to their high affinity and specificity for antigen target molecules, therapeutic monoclonal antibodies (mAbs) have been increasingly used for the treatment of different diseases. The sensitive and accurate detection of mAbs is crucial for the evaluation of their efficacy and safety. With a new design of a thiophosphate-modified and self-folding hairpin primer, herein, we described the establishment of an aptamer-based, highly sensitive and simple fluorescent trastuzumab mAb assay method via target-induced recycling and low-temperature LAMP activation of CRISPR/Cas12a signal amplifications. Target trastuzumab molecules bound with and changed the conformation of the hairpin aptamer probes to trigger Bst polymerase-mediated recycling and LAMP reactions with the assistance of hairpin primers to form long dsDNAs containing many protospacer-adjacent motif (PAM) segments. Cas12a/crRNA subsequently associated with these PAMs to exhibit trans-cleavage property for cyclically cutting ssDNA reporter molecules and yielding considerably magnified fluorescence recovery for trastuzumab detection. Owing to target-recycling, LAMP and Cas12a/crRNA-integrated signal amplifications, a low picomolar detection limit (4.17 pM) for trastuzumab was achieved. This assay could also be applied to trace trastuzumab in diluted human serums. With the distinct advantages of low-temperature LAMP activation with minimal primer involvement and the integration of an amplification cascade, this sensing methodology could be employed as a robust signal enhancement methodology for detecting various molecular biomarkers for diverse biomedical and biological applications.},
}
@article {pmid40568758,
year = {2025},
author = {Konde, MV and Inchanalkar, S and Sherkhane, TM and Deshpande, N and Virmani, M and Singh, K and Balasubramanian, N},
title = {CRISPR-Cas9 mediated RALA knockout and reconstitution: insights into the detection and role of RALA S194 phosphorylation in Ras-dependent and Ras-independent cancers.},
journal = {Biology open},
volume = {14},
number = {7},
pages = {},
doi = {10.1242/bio.061884},
pmid = {40568758},
issn = {2046-6390},
support = {35/03/2019-NANO/BMS//Indian Council of Medical Research/ ; 35/03/2019-NANO/BMS//Indian Council of Medical Research (ICMR)/ ; //IISER Pune: Indian Institute of Science Education Research Pune./ ; },
mesh = {Phosphorylation ; Humans ; *CRISPR-Cas Systems ; *Neoplasms/metabolism/genetics/pathology ; Cell Line, Tumor ; Gene Knockout Techniques ; *ras Proteins/metabolism/genetics ; Animals ; },
abstract = {Downstream of oncogenic RAS, RALA is critical for cancer tumorigenesis, possibly regulated by phosphorylation of its Serine194 residue. We made CRISPR-Cas9 RALA knockout (RALA KO) in three RAS-dependent and two RAS-independent cancer cells. Detection of RALA S194 phosphorylation using the commercial anti-phospho-RALA antibody lacks specificity in all three RAS-dependent cancers. siRNA knockdown of RALA and AURKA inhibition by MLN8237 (VMLN) also did not affect pS194RALA detection in these cancers. RALA KO MiaPaCa2 (RAS-dependent) and MCF7 (RAS-independent) cells, stably reconstituted with WT-RALA and S194A-RALA mutants, showed no effect on RALA activation. Tumor growth was, however, restored partly by WT-RALA, but not S194A-RALA mutant. Thus, RALA S194 phosphorylation is needed for tumor formation, not affecting its activation, but possibly through its localization.},
}
@article {pmid40523854,
year = {2025},
author = {Munusamy, S and Zheng, H and Chen, J and Zhou, S and Kong, J and Jahani, R and Zhao, Y and Guan, X},
title = {Gold Nanoparticle-Assisted CRISPR-Cas12a-Based Activity Assay for Highly Sensitive Detection of Trypsin.},
journal = {ACS applied bio materials},
volume = {8},
number = {7},
pages = {6379-6387},
doi = {10.1021/acsabm.5c00854},
pmid = {40523854},
issn = {2576-6422},
mesh = {*Trypsin/analysis/metabolism ; *Gold/chemistry ; *Metal Nanoparticles/chemistry ; *CRISPR-Cas Systems ; Humans ; *Biocompatible Materials/chemistry/chemical synthesis ; Particle Size ; Materials Testing ; Cattle ; Surface Properties ; Biosensing Techniques/methods ; Animals ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {Proteases play important roles in diverse physiological processes, and their malfunction has been implicated in various conditions and diseases. Therefore, development of sensitive methods for protease detection in clinical samples is highly desired for disease diagnosis. Herein, we report an ultrasensitive and selective CRISPR-Cas12a based fluorescent assay for trypsin activity measurement. By taking advantage of the signal amplification brought by a unique magnetic bead-gold nanoparticle assembly, which carries the peptide substrate and tens to hundreds of DNA molecules per peptide molecule, trypsin can be detected with a limit of detection reaching as low as 0.13 ng/mL. Furthermore, the sensor selectivity study was performed by examining several biomolecules commonly present in biological samples, including bovine serum albumin (BSA), human serum albumin (HSA), DNase, RNase, chymotrypsin, elastase, and thrombin. Moreover, trypsin inhibition and serum sample analysis were successfully carried out. Given the ultrahigh sensitivity, the CRISPR-based trypsin activity assay developed in this work can be used as a generic platform for developing sensors for other proteases, offering the potential as a noninvasive/minimally invasive tool for clinical diagnosis.},
}
@article {pmid40398570,
year = {2025},
author = {Zou, Y and Ye, A and Dong, M and Zhou, Y and Wu, W and Tang, Y and Hu, H and Dai, F and Tong, X},
title = {Silkworm mutagenesis using a ribonucleoprotein-based CRISPR/Cas12a system.},
journal = {Insect biochemistry and molecular biology},
volume = {182},
number = {},
pages = {104329},
doi = {10.1016/j.ibmb.2025.104329},
pmid = {40398570},
issn = {1879-0240},
mesh = {*Bombyx/genetics/metabolism ; Animals ; *CRISPR-Cas Systems ; *Gene Editing/methods ; *Ribonucleoproteins/genetics/metabolism ; *Mutagenesis ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {The development of highly efficient genome editing tools has revolutionized developmental biology and genetic studies in silkworm. Although methods based on CRISPR/Cas9 are currently popular, the Cas12a system has emerged as a promising option. However, it has not yet been applied to target the silkworm genome in vivo, and its activity in silkworm has not yet been characterized. In this study, we established a ribonucleoprotein-based CRISPR/Cas12a system, and compared it to the CRISPR/Cas9 system using 19 crRNA and 17 sgRNAs to target three different genes in vivo. Although Cas12a generates mutants less efficiently than Cas9, we used it successfully to generate transmissible indels, and demonstrated its application by targeting the FibH and mp genes to produce mutants with the expected phenotypes. We also assessed the influence of temperature (37 °C vs. 25 °C) on Cas12a activity, and demonstrated that the effects are target dependent. In summary, we have established a ribonucleoprotein-based CRISPR/Cas12a system in silkworm that offers a practical alternative to CRISPR/Cas9 and extends the genome editing tool box available for silkworm research.},
}
@article {pmid40320697,
year = {2025},
author = {Batzel, G and Wang, Y and Bock, A and Chen, E and Neal, S and Lopez-Anido, RN and Lee, Y and Tjeerdema, E and Ignatoff, E and Patil, T and Ramirez, G and Lesoway, MP and Hamdoun, A and Lyons, DC},
title = {CRISPR/Cas9 Knockout of Shell Matrix Protein 1 in the Slipper-Snail Crepidula atrasolea.},
journal = {Journal of experimental zoology. Part B, Molecular and developmental evolution},
volume = {344},
number = {5},
pages = {266-283},
doi = {10.1002/jez.b.23293},
pmid = {40320697},
issn = {1552-5015},
support = {R35 GM133673/GM/NIGMS NIH HHS/United States ; //This study was supported by grants to D.C.L. from the National Science Foundation Faculty Early Career Development (CAREER) Award (1943606) and the National Institute of General Medical Sciences Maximizing Investigators' Research Award (MIRA) (R35GM133673). Additional funding from the National Institutes of Health (OD034075) to A.H./ ; },
mesh = {Animals ; *CRISPR-Cas Systems ; *Snails/genetics/metabolism ; Animal Shells/metabolism ; Gene Editing ; Gene Knockout Techniques ; Gastropoda/genetics ; },
abstract = {Over the course of hundreds of millions of years, biomineralization has evolved independently many times across all kingdoms of life. Among animals, the phylum Mollusca displays a remarkable diversity in biomineral structures, particularly the molluscan shell, which varies greatly in shape, size, pigmentation, and patterning. Shell matrix proteins (SMPs) are key components of these shells, and are thought to drive the precipitation of calcium carbonate minerals and influence shell morphology. However, this structure-function relationship has rarely been studied directly because tools for knocking out genes did not exist in molluscs until recently. In this study, we report the first successful use of CRISPR/Cas9 gene editing to target an SMP in gastropod molluscs. Using the emerging model gastropod Crepidula atrasolea, we generated knockouts of the SMP1 gene. Successful gene editing was confirmed by Sanger and MiSeq sequencing, and loss of SMP1 expression was validated through high-content imaging of crispant embryos. This study establishes C. atrasolea as a valuable model for investigating the genetic basis of shell formation and provides a framework for applying CRISPR/Cas9 technology in other molluscan species. Our approach will enable future studies to thoroughly test the role of SMPs in shaping the diverse array of molluscan shell structures.},
}
@article {pmid40072468,
year = {2025},
author = {Chen, Q and Su, C and Li, S and Zhang, Z and Yang, Y and Yang, Y and Tao, D and Xie, S and Gong, P and Feng, Y},
title = {A sensitive and rapid visual method of chicken sexing based on LAMP-CRISPR/Cas12a system.},
journal = {British poultry science},
volume = {66},
number = {4},
pages = {531-538},
doi = {10.1080/00071668.2025.2454963},
pmid = {40072468},
issn = {1466-1799},
mesh = {Animals ; *Chickens/genetics ; *Sex Determination Analysis/veterinary/methods ; *Nucleic Acid Amplification Techniques/veterinary/methods ; *CRISPR-Cas Systems ; Female ; Male ; Chick Embryo ; Sensitivity and Specificity ; *Molecular Diagnostic Techniques/veterinary ; CRISPR-Associated Proteins/genetics ; Bacterial Proteins ; Endodeoxyribonucleases ; },
abstract = {1. Accurate sex identification of one-day-old chicks is crucial in layer poultry production. Establishing an early sexing method during the chicken embryonic period is essential for animal welfare. However, PCR-based sexing has limitations in terms of specialised equipment and is time-consuming.2. This study presents a rapid, simple and fluorescent visual technique for chicken sex identification based on Loop-mediated isothermal amplification (LAMP)-clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 12a (Cas12a). It targets the chicken Z chromosome gene DMRT1 and W chromosome-specific fragment EE0.6 using designed primers and sgRNA. The LAMP amplicon is cleaved by Cas12a, producing a fluorescent product detectable by a portable light apparatus.3. The method has high sensitivity, capable of detecting as few as two copies per microlitre of the EE0.6 template and 20 copies per microlitre of the DMRT1 template. This has significant potential for distinguishing chicken embryo gender very early in embryonic development.},
}
@article {pmid39702861,
year = {2025},
author = {Su, P and Miao, YL},
title = {A Genome-Wide CRISPR/Cas9 Screen Identifies Regulatory Genes for Stem Cell Aging.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2960},
number = {},
pages = {159-170},
pmid = {39702861},
issn = {1940-6029},
mesh = {*CRISPR-Cas Systems/genetics ; *Cellular Senescence/genetics ; Animals ; Cell Differentiation/genetics ; Mice ; Humans ; Gene Editing/methods ; Mesenchymal Stem Cells/cytology/metabolism ; *Embryonic Stem Cells/cytology/metabolism ; },
abstract = {Aging is a ubiquitous biological phenomenon, characterized by a gradual decline in physiological functions and an increased risk of various diseases. Although it is known that aging involves extensive changes in gene expression and disruptions in cellular metabolism, the molecular mechanisms underlying these processes remain incompletely understood. The CRISPR/Cas9 technology provides an efficient method for gene editing. In recent years, this technique has been successfully applied in various cellular and animal models to identify key genes involved in biological processes such as cancer and genetic diseases, which makes it possible to screen genes that affect cell senescence in the whole genome. Here, we describe a method that involves differentiating embryonic stem cells into mesenchymal progenitor cells and employing CRISPR/Cas9 for genome-wide functional screening to identify genes that regulate aging. Further analysis of the functions and regulatory mechanisms of these genes may provide new targets and strategies for anti-aging research and stem cell therapy.},
}
@article {pmid40685330,
year = {2025},
author = {Far, BF and Akbari, M and Habibi, MA and Katavand, M and Nasseri, S},
title = {CRISPR Technology in Disease Management: An Updated Review of Clinical Translation and Therapeutic Potential.},
journal = {Cell proliferation},
volume = {},
number = {},
pages = {e70099},
doi = {10.1111/cpr.70099},
pmid = {40685330},
issn = {1365-2184},
abstract = {CRISPR-Cas9 technology has rapidly advanced as a transformative genome-editing platform, facilitating precise genetic modifications and expanding therapeutic opportunities across various diseases. This review explores recent developments and clinical translations of CRISPR applications in oncology, genetic and neurological disorders, infectious diseases, immunotherapy, diagnostics, and epigenome editing. CRISPR has notably progressed in oncology, where it enables the identification of novel cancer drivers, elucidation of resistance mechanisms, and improvement of immunotherapies through engineered T cells, including PD-1 knockout CAR-T cells. Clinical trials employing CRISPR-edited cells are demonstrating promising results in hematologic malignancies and solid tumours. In genetic disorders, such as hemoglobinopathies and muscular dystrophies, CRISPR-Cas9 alongside advanced editors like base and prime editors show significant potential for correcting pathogenic mutations. This potential was affirmed with the FDA's first approval of a CRISPR-based therapy, Casgevy, for sickle cell disease in 2023. Neurological disorders, including Alzheimer's, ALS, and Huntington's disease, are increasingly targeted by CRISPR approaches for disease modelling and potential therapeutic intervention. In infectious diseases, CRISPR-based diagnostics such as SHERLOCK and DETECTR provide rapid, sensitive nucleic acid detection, particularly valuable in pathogen outbreaks like SARS-CoV-2. Therapeutically, CRISPR systems target viral and bacterial genomes, offering novel treatment modalities. Additionally, CRISPR-mediated epigenome editing enables precise regulation of gene expression, expanding therapeutic possibilities. Despite these advances, significant challenges remain, including off-target effects, delivery methodologies, immune responses, and long-term genomic safety concerns. Future improvements in editor precision, innovative delivery platforms, and enhanced safety assessments will be essential to fully integrate CRISPR-based interventions into standard clinical practice, significantly advancing personalised medicine.},
}
@article {pmid40684030,
year = {2025},
author = {Lam, HT and Nhi, NHH and Lan, VTH and Van Hau, N and Nghia, NH},
title = {A single-vector CRISPR/Cas9 system for genome editing and heterologous enzyme secretion in Saccharomyces cerevisiae: a case study on pectate lyase for coffee mucilage removal.},
journal = {Biotechnology letters},
volume = {47},
number = {4},
pages = {78},
pmid = {40684030},
issn = {1573-6776},
support = {36-2024-18-02//Viet Nam National University Ho Chi Minh City/ ; },
mesh = {*Polysaccharide-Lyases/genetics/metabolism ; *Saccharomyces cerevisiae/genetics/metabolism ; *CRISPR-Cas Systems ; *Gene Editing/methods ; *Coffee/chemistry ; Pectins/metabolism ; Bacillus subtilis/enzymology/genetics ; Genetic Vectors/genetics ; },
abstract = {The CRISPR/Cas9 system facilitates precise genome editing in various organisms. In this study, a single-vector CRISPR/Cas9 system was developed for Saccharomyces cerevisiae, employing a type II Cas9 enzyme from Streptococcus pyogenes and a single-guide RNA cassette targeting CAN1.Y locus on chromosome V. This system is broadly applicable across yeast strains, as it utilizes G418 selection, eliminating the need for auxotrophic markers. The efficiency of the CRISPR/Cas9 system was demonstrated, with editing efficiencies ranging from 70 to 100%. This system was utilized to integrate a cassette encoding secretory pectate lyase (PL) from Bacillus subtilis 168 into the yeast genome. The engineered S. cerevisiae strain secreted active PL, which exhibited pectin-degrading activity characterized by significant reductions in residual pectin and increased production of reducing sugars. Since pectin constitutes a major component of coffee mucilage, the secreted PL was applied to coffee beans for mucilage removal. The treated beans presented noticeably reduced residual mucilage, a purer green color, and decreased viscosity. These findings suggest the potential of the engineered S. cerevisiae strain for applications in coffee processing, particularly in efficient mucilage removal.},
}
@article {pmid40683967,
year = {2025},
author = {Chang, W and Zhang, B and Yang, S and Zhang, X and Zhang, L and Zhang, S and Lu, J and Wang, W and Shang, P and Yue, Z},
title = {Genome-wide CRISPR/Cas9 screening identifies PTGR2 as a potential therapeutic target for sunitinib resistance in clear cell renal cell carcinoma.},
journal = {Scientific reports},
volume = {15},
number = {1},
pages = {26263},
pmid = {40683967},
issn = {2045-2322},
support = {PR5124025//the Special Fund Project for Doctoral Training Program of Lanzhou University Second Hospital/ ; CY2021-MS-A11//Cuiying Science and Technology Innovation Plan Project of Lanzhou University Second Hospital/ ; 22JR5RA942//Gansu Provincial Science Fund for Distinguished Young Scholars/ ; 22JR5RA1009//Gansu Provincial Natural Science Foundation/ ; lzuyxcx-2022-105//Medical Innovation and Development Project of Lanzhou University/ ; },
mesh = {Humans ; *Sunitinib/pharmacology/therapeutic use ; *Carcinoma, Renal Cell/genetics/drug therapy/pathology/metabolism ; *Drug Resistance, Neoplasm/genetics ; *Kidney Neoplasms/genetics/drug therapy/pathology/metabolism ; *CRISPR-Cas Systems ; Histone Demethylases/metabolism/genetics ; Animals ; Cell Line, Tumor ; Mice ; Xenograft Model Antitumor Assays ; Gene Expression Regulation, Neoplastic/drug effects ; Antineoplastic Agents/pharmacology ; },
abstract = {Acquired and intrinsic resistance to sunitinib is a major obstacle to improving the therapeutic efficacy of treatment for clear cell renal cell carcinoma (ccRCC). This study aimed to identify novel therapeutic targets and the potential molecular mechanisms to overcome sunitinib resistance in ccRCC. Utilizing genome-wide CRISPR/Cas9 screening and resistant transcriptomics, we identified that prostaglandin reductase 2 (PTGR2) is a novel therapeutic target to overcome sunitinib resistance in ccRCC. The silencing of PTGR2 enhanced the cytotoxic effects of sunitinib in ccRCC cells, as measured by cell viability assays, and suppressed tumor growth in xenograft models. Mechanistically, PTGR2 physically interacts with lysine specific demethylase 6A (KDM6A) via endogenous/exogenous co-immunoprecipitation. PTGR2 knockdown reduced KDM6A protein expression, while KDM6A overexpression partially reversed the sensitization effect of PTGR2 silencing, suggesting KDM6A is a major downstream effector. Our findings establish the PTGR2-KDM6A axis as a potential target for overcoming sunitinib resistance in ccRCC. Pharmacological inhibition of PTGR2 or targeted modulation of KDM6A activity represents a promising combination strategy to overcome sunitinib resistance and improve patient outcomes.},
}
@article {pmid40683956,
year = {2025},
author = {Zhang, C and Li, H and Poluektova, LY and Gendelman, HE and Dash, PK},
title = {Unique molecular signatures in rebound viruses from antiretroviral drug and CRISPR-treated HIV-1-infected humanized mice.},
journal = {Communications biology},
volume = {8},
number = {1},
pages = {1077},
pmid = {40683956},
issn = {2399-3642},
support = {R01MH115860//U.S. Department of Health &amp; Human Services | NIH | National Institute of Mental Health (NIMH)/ ; R01MH121402//U.S. Department of Health &amp; Human Services | NIH | National Institute of Mental Health (NIMH)/ ; R24OD018546//U.S. Department of Health &amp; Human Services | NIH | NIH Office of the Director (OD)/ ; },
mesh = {Animals ; *HIV-1/genetics/drug effects ; *HIV Infections/virology/drug therapy/genetics ; Mice ; Humans ; *CRISPR-Cas Systems ; *Anti-HIV Agents/pharmacology/therapeutic use ; Mutation ; *Anti-Retroviral Agents/pharmacology/therapeutic use ; Pyridones ; Heterocyclic Compounds, 3-Ring ; Oxazines ; Piperazines ; },
abstract = {HIV-1 elimination from a subset of virus-infected humanized mice (hu-mice) is reported following sequential dual treatment with long-acting (LA) antiretroviral (ART) and CRISPR-Cas9 therapies. However, viral rebound is observed in > 50% of the dual-treated animals. The molecular signatures of the rebound virus, recovered from plasma, have now been determined. The LA-ART treatment contains nanoformulated dolutegravir, lamivudine, abacavir, and rilpivirine combinations, and the HIV-1 excision treatment is CRISPR-Cas9 targeting the HIV-1-LTR-gag. One-step reverse transcriptase polymerase chain reaction, which avoids spontaneous preparatory mutations, is performed on plasma-derived RNA. Sanger and Next-Generation Sequencing are employed targeting the HIV-1-gag, pol, and env genes. HIV-1 env shows the most divergence. LA-ART, with or without CRISPR, is responsible for the new mutations. The primary and accessory mutations are detected by deep sequencing. Viral evolution reflects changes in the virus as reported by ART-treated and HIV-1-infected patients. No major CRISPR-specific mutations are observed. The molecular viral signatures demonstrate an accelerated HIV-1 drug resistance escape from ART rather than from the generation of CRISPR mutants. These define viral rebound in the dual-treated hu-mice. The data underscores the limited role of CRISPR excision in generating these rebound HIV-1 mutants from dual-treated hu-mice.},
}
@article {pmid40683724,
year = {2025},
author = {Chen, J and Lu, Y and Meng, X and Chen, D and Wu, C and Song, D},
title = {Visual and self-contained diagnosis of Salmonella using RPA-CRISPR on a centrifugal force driven microfluidic system.},
journal = {Food microbiology},
volume = {132},
number = {},
pages = {104844},
doi = {10.1016/j.fm.2025.104844},
pmid = {40683724},
issn = {1095-9998},
mesh = {*Salmonella/genetics/isolation &amp; purification ; *Nucleic Acid Amplification Techniques/methods/instrumentation ; *CRISPR-Cas Systems ; Food Microbiology/methods ; Recombinases/genetics/metabolism ; Food Contamination/analysis ; Lab-On-A-Chip Devices ; Bacterial Proteins/genetics ; DNA, Bacterial/genetics ; },
abstract = {Rapid and sensitive nucleic acid detection is very important for the detection of Salmonella in food. Convenient detection methods show great potential in food safety. CRISPR-Cas12a system has been widely used for nucleic acid detection. In one-pot reaction, Cas12a can perform cis- and trans-cleavage of amplicons and primers, which reduces the sensitivity of the reaction and has certain limitations. In this study, we integrated the Cas12a cutting system with recombinase polymerase amplification (RPA) into one microfluidic chip to avoid cap opening pollution. A portable observation device was integrated to enable visual detection of signals with the naked eye. The detection technology could specifically detect 1 × 10° CFU/mL Salmonella in 50 min at 37 °C. In the field test, there was no need for professional equipment or high-tech means.},
}
@article {pmid40682826,
year = {2025},
author = {Zheng, H and Mao, C and Chen, S and Hou, S and Sun, D},
title = {A quorum sensing-controlled type I CRISPRi toolkit for dynamically regulating metabolic flux.},
journal = {Nucleic acids research},
volume = {53},
number = {14},
pages = {},
doi = {10.1093/nar/gkaf693},
pmid = {40682826},
issn = {1362-4962},
support = {32170083//National Natural Science Foundation of China/ ; 31670084//National Natural Science Foundation of China/ ; 2020C02031//Research and Development Program of Zhejiang Province/ ; 32170083//National Natural Science Foundation of China/ ; 31670084//National Natural Science Foundation of China/ ; },
mesh = {*Quorum Sensing/genetics ; *Bacillus subtilis/genetics/metabolism ; *CRISPR-Cas Systems ; *Metabolic Engineering/methods ; Gene Expression Regulation, Bacterial ; Riboflavin/biosynthesis ; Synthetic Biology/methods ; Bacterial Proteins/genetics/metabolism ; },
abstract = {Synthetic biology advances have enabled dynamic metabolic regulation via quorum sensing (QS) and CRISPR systems. However, the integration of QS with CRISPR-based systems for dynamic control remains largely underexplored. Here, we developed a QS-controlled type I CRISPR interference (QICi) toolkit that modulates target gene expression in response to cell density. By streamlining CRISPR RNA (crRNA) vector construction and optimizing key QS components PhrQ and RapQ, we achieved a twofold enhancement in QICi efficacy. We subsequently implemented the optimized QICi to reprogram Bacillus subtilis for d-pantothenic acid (DPA) and riboflavin (RF) biosynthesis. Dynamic regulation of the citrate synthase gene citZ by QICi, coupled with pantoate pathway engineering, cofactor supply enhancement, and suppression of sporulation, elevated DPA titers to 14.97 g/l in 5-l fed-batch fermentations without precursor supplementation. QICi-mediated metabolic rewiring of key nodes boosted RF production by 2.49-fold. Together, our work provides a robust tool for reprogramming microbial metabolism and advancing sustainable biomanufacturing.},
}
@article {pmid40490244,
year = {2025},
author = {Wang, T and Liu, Y and Wu, X and Wang, X and Shi, S and Song, X and Ma, Y and Zhang, Z and Gao, J and Sun, R and Song, G},
title = {Multi-omics reveals miR-181a-5p regulates PPAR-driven lipid metabolism in Oral squamous cell carcinoma: Insights from CRISPR/Cas9 knockout models.},
journal = {Journal of proteomics},
volume = {319},
number = {},
pages = {105480},
doi = {10.1016/j.jprot.2025.105480},
pmid = {40490244},
issn = {1876-7737},
mesh = {*MicroRNAs/genetics/metabolism ; Animals ; Mice ; *Mouth Neoplasms/metabolism/genetics/pathology ; *Lipid Metabolism/genetics ; Mice, Knockout ; *CRISPR-Cas Systems ; *Carcinoma, Squamous Cell/metabolism/genetics/pathology ; *Peroxisome Proliferator-Activated Receptors/metabolism/genetics ; Gene Expression Regulation, Neoplastic ; Proteomics/methods ; Humans ; 4-Nitroquinoline-1-oxide ; *Squamous Cell Carcinoma of Head and Neck/genetics/metabolism/pathology ; Multiomics ; },
abstract = {Oral squamous cell carcinoma (OSCC) remains a therapeutic challenge due to its complex molecular landscape and metabolic adaptability. This study integrates proteomic and transcriptomic analyses to investigate the role of miR-181a-5p in OSCC pathogenesis using CRISPR/Cas9-generated whole-body knockout (KO) mice. By inducing OSCC with the chemical carcinogen 4-nitroquinoline 1-oxide (4NQO), we identified significant dysregulation of lipid metabolism-associated proteins and tumor regulators in miR-181a-5p-KO tumors compared to wild-type controls. Quantitative proteomics revealed enrichment of the PPAR signaling pathway, with 12 key genes upregulated in KO mice, mechanistically linking miR-181a-5p deficiency to enhanced lipid droplet biogenesis and immunosuppressive microenvironments. Serum biomarker validation demonstrated elevated Cyfra21-1, SCC-Ag, and ISG20 levels in KO mice, correlating with tumor aggressiveness and radioresistance. Multi-omics integration further identified a diagnostic-prognostic protein signature with 89 % specificity for miR-181a-5p-deficient OSCC subtypes. These findings establish miR-181a-5p as a master regulator of PPAR-mediated metabolic reprogramming and immune evasion, offering novel proteome-driven insights into therapeutic targeting of lipid metabolism and biomarker discovery in OSCC. SIGNIFICANCE: This study integrates transcriptomic and proteomic analyses to elucidate the critical role of miR-181a-5p in regulating lipid metabolism via the PPAR signaling pathway during oral squamous cell carcinoma (OSCC) pathogenesis. Loss of miR-181a-5p enhances lipid metabolism, promoting membrane biosynthesis and metastasis. Multi-omics profiling identified a specific diagnostic-prognostic protein signature, highlighting CES3 and ISG20 as potential biomarkers for early diagnosis and therapeutic targeting in miR-181a-5p-deficient OSCC. The research establishes a foundation for miRNA-based liquid biopsy and PPAR-targeted nanotherapy. Mouse knockout models recapitulating human OSCC spatial biology validated miR-181a-5p's role in tumor initiation.},
}
@article {pmid40425868,
year = {2025},
author = {Anderson, RK and Nugen, SR},
title = {Genetic engineering of bacteriophage S16 for Salmonella separation, concentration, and detection.},
journal = {Analytical and bioanalytical chemistry},
volume = {417},
number = {18},
pages = {4069-4081},
pmid = {40425868},
issn = {1618-2650},
support = {2021-67021-33997//National Institute of Food and Agriculture/ ; R01EB027895/EB/NIBIB NIH HHS/United States ; R01EB027895/EB/NIBIB NIH HHS/United States ; },
mesh = {*Biosensing Techniques/methods ; *Genetic Engineering/methods ; *Salmonella Phages/genetics ; *Salmonella/isolation &amp; purification/virology ; Limit of Detection ; Water Microbiology ; CRISPR-Cas Systems ; *Bacteriophages/genetics ; Food Microbiology ; *Salmonella typhimurium/isolation &amp; purification/virology ; },
abstract = {Salmonella contamination in food and water poses a major global health risk, creating an urgent need for rapid, reliable, and cost-effective detection methods. Conventional approaches are often expensive, labor-intensive, and time-consuming, and they frequently yield inconclusive or presumptive positive results. A significant bottleneck to rapid detection is the need to separate the target bacteria into smaller, clean, and concentrated samples. Bacteriophages can recognize, bind, and infect specific bacterial hosts. This study presents a genetically engineered S16 Salmonella-specific bacteriophage as a biosensor, optimized for enhanced sensitivity and efficiency in detecting Salmonella. Using CRISPR-Cas12a, the phages were engineered to include a gene for a NanoLuc luciferase reporter and a monomeric streptavidin (mSA) affinity tag fused to the gene for the capsid protein Soc. This design enabled conjugation of the phages to magnetic nanoparticles, facilitating the capture, concentration, and detection of Salmonella from 10 mL water samples. The modified S16 phage exhibited a detection limit of fewer than 10 CFU of Salmonella in 10 mL of water within a typical work shift. This innovative phage-based detection method offers a promising tool for enhancing food and water safety by providing a faster, more sensitive, and cost-effective approach to pathogen monitoring of Salmonella enterica subsp. enterica serovar Typhimurium.},
}
@article {pmid40052530,
year = {2025},
author = {Li, M and Hu, Z and Huang, Y and Han, Y and Liang, C and Liu, Y and Wu, R and Lu, X and Deng, K and Liu, S and Ou, X and Li, Y and Liu, C and Li, X and Liang, J and Fu, Y and Xu, A},
title = {BiFC and FACS-based CRISPR screening revealed that QKI promotes PABPN1 LLPS in colorectal cancer cells.},
journal = {Protein &amp; cell},
volume = {16},
number = {7},
pages = {557-574},
pmid = {40052530},
issn = {1674-8018},
support = {32470586//National Natural Science Foundation of China/ ; 31971332//National Natural Science Foundation of China/ ; 91942301//National Natural Science Foundation of China/ ; 81430099//National Natural Science Foundation of China/ ; 2022YFA1103900//National Key Research and Development Program of China/ ; 2023B1212060028//Guangdong Science and Technology Department/ ; },
mesh = {Humans ; *Colorectal Neoplasms/metabolism/pathology/genetics ; *RNA-Binding Proteins/metabolism/genetics ; *Poly(A)-Binding Protein I/metabolism/genetics ; *CRISPR-Cas Systems ; Flow Cytometry ; Cell Proliferation ; Cell Line, Tumor ; Cell Movement ; },
abstract = {Protein liquid-liquid phase separation (LLPS), a pivotal phenomenon intricately linked to cellular processes, is regulated by various other proteins. However, there is still a lack of high-throughput methods for screening protein regulators of LLPS in target proteins. Here, we developed a CRISPR/Cas9-based screening method to identify protein phase separation regulators by integrating bimolecular fluorescence complementation (BiFC) and fluorescence-activated cell sorting (FACS). Using this newly developed method, we screened the RNA-binding proteins that regulate PABPN1 phase separation and identified the tumor suppressor QKI as a promoter of PABPN1 phase separation. Furthermore, QKI exhibits decreased expression levels and diminished nuclear localization in colorectal cancer cells, resulting in reduced PABPN1 phase separation, which, in turn, promotes alternative polyadenylation (APA), cell proliferation, and migration in colorectal cancer.},
}
@article {pmid40682401,
year = {2025},
author = {Moso, MA and Roche, M and Cevaal, PM and Lewin, SR},
title = {CRISPR/Cas9 for achieving postintervention HIV control.},
journal = {Current opinion in HIV and AIDS},
volume = {},
number = {},
pages = {},
doi = {10.1097/COH.0000000000000963},
pmid = {40682401},
issn = {1746-6318},
abstract = {PURPOSE OF REVIEW: Recent advances in gene therapy have led to the first clinically approved CRISPR/Cas9 therapy for β-thalassaemia and sickle cell disease. Gene therapy could play an important role in targeting HIV persistence and achieving postintervention HIV control. Here, we review recent updates in CRISPR/Cas9-based HIV gene therapy approaches, including CCR5-editing (protect), proviral targeting (excise or modify), and immune cell engineering (attack).<br><br>RECENT FINDINGS: Recent studies provide additional safety data for use of CRISPR/Cas9-based gene therapies, however low in vivo editing efficiency highlights the need for improved delivery methods. This is particularly relevant for strategies requiring transfection of all HIV-infected cells containing intact proviruses, such as proviral excision. For ex vivo editing approaches, poor engraftment and durability of edited cells present additional challenges. Newer methods such as lipid nanoparticle delivery could provide a mechanism to overcome current limitations with ex vivo and in vivo delivery. Several studies have demonstrated proof-of-concept of combination gene therapy approaches, including gene editing strategies to generate HIV-resistant cells with immune effector functions, providing novel approaches to control and durably suppress viral replication.<br><br>SUMMARY: Several studies have demonstrated feasibility of gene therapy approaches in achieving postintervention HIV control. Improvements in both ex vivo and in vivo delivery methods are required to progress current gene therapy approaches to the clinic.},
}
@article {pmid40682144,
year = {2025},
author = {Motosugi, N and Hasegawa, K and Kurosaki, N and Kawaguchi, E and Izumi, K and Iida, Y and Higashiseto, M and Yokoyama, K and Sasaki, A and Nakabayashi, K and Fukuda, A},
title = {Highly efficient XIST reactivation in female hPSC by transient dual inhibition of TP53 and DNA methylation during Cas9 mediated genome editing.},
journal = {Stem cell research &amp; therapy},
volume = {16},
number = {1},
pages = {389},
pmid = {40682144},
issn = {1757-6512},
support = {22bm0804030h0002//Japan Agency for Medical Research and Development/ ; },
mesh = {Humans ; *RNA, Long Noncoding/genetics/metabolism ; Female ; *Gene Editing/methods ; *DNA Methylation/genetics ; *Tumor Suppressor Protein p53/metabolism/genetics/antagonists &amp; inhibitors ; *CRISPR-Cas Systems/genetics ; X Chromosome Inactivation/genetics ; *Pluripotent Stem Cells/metabolism/cytology ; },
abstract = {The irreversible erosion of X-chromosome inactivation (XCI) due to repression of the long non-coding RNA XIST presents a major challenge for disease modeling and raises safety concerns for the clinical application of female human pluripotent stem cells (hPSCs) due to the aberrant overexpression of X-linked genes. While Cas9-mediated non-homologous end joining (NHEJ) targeting the XIST promoter can induce DNA demethylation and restore XCI by reactivating XIST, its efficiency remains low. Here, we introduce a highly efficient strategy for XIST reactivation by combining TP53 inhibition with suppression of DNA methylation maintenance during Cas9-mediated NHEJ. This dual-inhibition approach increased the proportion of XIST-positive hPSCs from ~ 5 to ~ 43.7%, providing a robust method for stabilizing XCI in female hPSCs for diverse applications.},
}
@article {pmid40681749,
year = {2025},
author = {de Lima Balico, L and Gaucher, EA},
title = {Genomic insertion of ancestral uricase into human liver cells to determine metabolic consequences of pseudogenization.},
journal = {Scientific reports},
volume = {15},
number = {1},
pages = {26093},
pmid = {40681749},
issn = {2045-2322},
mesh = {Humans ; *Urate Oxidase/genetics/metabolism ; Uric Acid/metabolism ; *Hepatocytes/metabolism ; Animals ; Triglycerides/metabolism ; Fructose/metabolism ; *Liver/metabolism/cytology ; CRISPR-Cas Systems ; },
abstract = {The biological role of urate (uric acid) during primate evolution has been unclear ever since it was discovered over 100 years ago that humans have unusually high levels of the small molecule compared to most other mammals. Humans (including all apes) are uncharacteristically susceptible to the build-up of urate because we no longer have a functional uricase enzyme capable of oxidizing this highly insoluble molecule. We have now utilized CRISPR technology to insert functional ancestral uricase into the genome of human liver cells to address recent metabolic hypotheses that our ancestral primates inactivated uricase as a mechanism to increase triglyceride production in response to fructose and/or starvation. Uricase expression is confirmed in both hepatocyte monolayer and spheroid tissue cultures, and its expression reduces intracellular urate levels. The presence of uricase is also shown to prevent an increase in triglyceride production upon cellular uptake of fructose in both culture conditions. Our results make progress that further describes a potential advantageous biological role of urate during primate evolution.},
}
@article {pmid40681400,
year = {2025},
author = {Huang, HC and Wu, LF and Liu, K and Ma, BG},
title = {Opto-CRISPR: new prospects for gene editing and regulation.},
journal = {Trends in biotechnology},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.tibtech.2025.06.018},
pmid = {40681400},
issn = {1879-3096},
abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR) technology represents a landmark advance in the field of gene editing. However, conventional CRISPR/Cas systems are limited by inadequate temporal and spatial control. In recent years, the development of optically controlled CRISPR (Opto-CRISPR) technology has offered a novel solution to this issue. As a combination of optogenetics and the CRISPR technology, the Opto-CRISPR technology enables dynamic space-time-specific gene editing and regulation in cells and organisms. In this review, we concisely introduce the basic principles of Opto-CRISPR, summarize its operational mechanisms, and discuss its applications and recent advances across various research fields. In addition, this review analyzes the limitations of Opto-CRISPR, aiming to provide a reference for the development of this emerging field.},
}
@article {pmid40680677,
year = {2025},
author = {Li, X and Liu, J and Wang, R and Fu, H and Kim, M and Li, X and Peng, W and Man, S and Gao, Z and Ma, L},
title = {CRISPR-based one-pot detection: A game-changer in nucleic acid analysis.},
journal = {Biosensors &amp; bioelectronics},
volume = {288},
number = {},
pages = {117786},
doi = {10.1016/j.bios.2025.117786},
pmid = {40680677},
issn = {1873-4235},
abstract = {The CRISPR/Cas system, originally developed as a gene-editing tool, has rapidly emerged as a powerful platform for nucleic acid detection due to its remarkable specificity, programmability, and robust trans-cleavage activity. While conventional CRISPR-based assays typically require separate amplification and detection steps, introducing complexity, manual intervention, and contamination risks. Recent innovations have led to the development of one-pot detection strategies that integrate target amplification with Cas-mediated signal generation within a single reaction vessel. These streamlined systems not only reduce handling steps and turnaround time but also enhance sensitivity and enable multiplexed detection of low-abundance nucleic acid targets. As such, one-pot CRISPR diagnostics are poised to transform point-of-care testing, pathogen surveillance, and clinical diagnostics. In this review, we summarize the latest advances in one-pot CRISPR-based detection technologies, evaluate their current limitations, and discuss strategies for optimization. Future efforts should focus on refining reaction kinetics, improving reagent stability, reducing costs, and expanding the repertoire of detectable targets. Furthermore, integration with portable platforms, AI-assisted signal interpretation, and the discovery of novel Cas effectors will be pivotal in realizing the full potential of CRISPR-based one-pot systems in diverse real-world applications.},
}
@article {pmid40480571,
year = {2025},
author = {Dong, X and Zhang, T and Liu, L and Chen, S and Li, J and Zhang, Y and Chen, J and Yang, C and Huang, J and Huang, T and Wei, T and Mo, M},
title = {Rapid field visual detection of avian metapneumovirus by integrating MIRA pre-amplification with CRISPR-Cas13a to enhance sensitivity and specificity: Innovative technologies well-suited for real-time large-scale epidemiological surveillance.},
journal = {International journal of biological macromolecules},
volume = {318},
number = {Pt 1},
pages = {144966},
doi = {10.1016/j.ijbiomac.2025.144966},
pmid = {40480571},
issn = {1879-0003},
mesh = {Animals ; *CRISPR-Cas Systems/genetics ; *Nucleic Acid Amplification Techniques/methods ; *Metapneumovirus/genetics/isolation &amp; purification ; *Poultry Diseases/virology/diagnosis/epidemiology ; Sensitivity and Specificity ; Epidemiological Monitoring ; *Paramyxoviridae Infections/diagnosis/veterinary/virology/epidemiology ; Reproducibility of Results ; Molecular Diagnostic Techniques/methods ; },
abstract = {Avian metapnemovirus (aMPV) is associated with swollen head syndrome in poultry and causes significant economic losses due to respiratory disease and reduced egg production, hence the urgent need for rapid and reliable detection methods. To address this challenge, we developed two novel CRISPR-Cas13a combined multiplex isothermal recombinase amplification (MIRA) assays: a fluorescence-based method (Fluorescent-MIRA-Cas13a) and a lateral flow strip-based platform (LF-MIRA-Cas13a), representing the first application of CRISPR-Cas13a for aMPV detection. Through systematic screening of 21 primer pairs and 4 crRNAs, optimal combinations were identified, achieving excellent specificity with no cross-reactivity against 12 common poultry pathogens. The assays demonstrated high sensitivities of 2 copies/reaction (Fluorescent-MIRA-Cas13a) and 5 copies/reaction (LF-MIRA-Cas13a), outperforming qPCR (20 copies/reaction), while maintaining reproducibility across inter- and intra-assay tests. Clinical evaluation using 200 suspected samples revealed 21 % (Fluorescent-MIRA-Cas13a) and 17.5 % (LF-MIRA-Cas13a) positivity rates, aligning closely with qPCR (19 %) and showing strong diagnostic concordance (κ > 0.93). Notably, both methods enabled detection within 1 h, without requiring specialized plug-in instruments (only a handheld UV emitter or lateral flow strips), significantly reducing operational complexity compared to conventional techniques. These field-deployable assays provide a cost-effective solution for rapid on-site diagnosis and large-scale epidemiological surveillance, particularly in resource-limited settings.},
}
@article {pmid40680043,
year = {2025},
author = {Kurniawan, C and Itoh, T},
title = {Off-target sequence variations driven by the intrinsic properties of the Cas-sgRNA-DNA complex in genome editing.},
journal = {PloS one},
volume = {20},
number = {7},
pages = {e0328905},
doi = {10.1371/journal.pone.0328905},
pmid = {40680043},
issn = {1932-6203},
mesh = {*Gene Editing/methods ; *DNA/genetics/metabolism ; Humans ; *CRISPR-Cas Systems/genetics ; *RNA, Guide, CRISPR-Cas Systems/genetics/metabolism ; Mutation ; },
abstract = {Genome-editing technologies hold significant potential across various biotechnological fields, yet concerns about possible risks, including off-target mutations, remain. To ensure safe and effective application, these unintended mutations must be rigorously examined and minimized. Computational approaches are anticipated to streamline the detection of off-target mutations; however, the performance of current prediction tools is limited, likely owing to insufficient knowledge of off-target mutation characteristics. In this study, we collected experimentally validated off-target mutation data and conducted a large-scale analysis of 177 nonredundant datasets obtained from six studies. We developed a method to assess the statistical significance of sequence pattern similarity and diversity between off-target sites. This method is based on a comparison of ordered relative entropy values for aligned target sequences, and it was compared with two other methods on the basis of Euclidean distance and the Pearson correlation coefficient. The three methods demonstrated clear correlations, indicating their validity. These methods were applied to 238 dataset pairs for the same target site, and it was revealed that off-target sequence patterns were quite similar across different experimental conditions, such as varying cell lines and independent experiments, suggesting that the intrinsic properties of the Cas-sgRNA-DNA complex play a key role in determining cleavage sites. However, newly engineered enzymes and those from different bacterial sources occasionally display unique off-target patterns, indicating the need for comprehensive evaluation of each new enzyme to develop reliable prediction tools. The insights gained from this study are expected to contribute to a better understanding of off-target mutation characteristics and support the development of more accurate computational prediction methods.},
}
@article {pmid40678258,
year = {2025},
author = {Zhang, J and Li, J and Zhou, J and Zhong, J and Xu, Y and Mao, X and Xu, M and Luo, S and Yang, Y and Hu, R and Liu, DA and Chen, S and Qiu, Y and Chen, K and Yuan, J and Zhang, X and Li, X},
title = {A Critical Review of the CRISPR-Cas Technology in the Detection of SARS-CoV-2 Variants.},
journal = {The Canadian journal of infectious diseases &amp; medical microbiology = Journal canadien des maladies infectieuses et de la microbiologie medicale},
volume = {2025},
number = {},
pages = {9107724},
pmid = {40678258},
issn = {1712-9532},
abstract = {Coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is still going on, and as the epidemic situation continues, the genome of SARS-CoV-2 is also mutating and evolving, resulting in more and more SARS-CoV-2 mutant strains, which have brought serious pressure on the prevention and control of COVID-19. Given that the COVID-19 is still spreading, it is extremely important to rapidly identify SARS-CoV-2 variants by nucleic acid assays. Thus, developing highly sensitive and specific assays that are suitable for field testing, high-throughput, and automation, as well as other diagnostic applications for SARS-CoV-2 variants, is urgently needed. This paper reviews the research progress of novel CRISPR-based diagnostic methods for SARS-CoV-2 variants.},
}
@article {pmid40632103,
year = {2025},
author = {Tharu, RJ and Hanson, E and Yigit, MV},
title = {Target-Responsive DNA Hydrogels with Encapsulation and Release Properties Using Programmable CRISPR-Cas12a.},
journal = {ACS chemical biology},
volume = {20},
number = {7},
pages = {1805-1812},
doi = {10.1021/acschembio.5c00355},
pmid = {40632103},
issn = {1554-8937},
mesh = {*Hydrogels/chemistry/metabolism ; *CRISPR-Cas Systems ; *DNA/chemistry/metabolism ; Salmonella typhimurium/genetics ; *CRISPR-Associated Proteins/metabolism ; *Endodeoxyribonucleases/metabolism ; Bacterial Proteins ; },
abstract = {We report the development of a DNA hydrogel that disassembles and releases its payload in response to a target of interest. The DNA hydrogel is assembled from Y-shaped DNA motifs with polyA domains and cross-linked via the small molecule cyanuric acid through hydrogen bonding. The hydrogel's structural integrity was rapidly assessed using a simple, instrumentation-free capillary migration assay that provides results within seconds. To evaluate its responsiveness to enzymatic degradation, the hydrogel was exposed to nonspecific nuclease activity using DNase I, resulting in increased mobility and decrease in fluorescence. Later, CRISPR-Cas12a was incorporated to enable programmable, target-specific hydrogel disassembly using a conserved genomic region from Salmonella typhimurium. Guided by crRNA sequences, the target sequences activated Cas12a to selectively degrade hydrogels. This process enabled the controlled release of various payloads, including a small-molecule drug, a fluorescent dye, a nanoparticle-based MRI contrast agent conjugated to a chemotherapeutic agent, and a model protein. To evaluate whether the hydrogel disassembly can be selectively programmed to an intended target, we tested its responsiveness against two serotypes of Salmonella, i.e., conserved genomic regions from Salmonella enteritidis and S. typhimurium. To test the disassembly of this novel DNA hydrogel in the presence of a full genome, we tested the hydrogel with the S. typhimurium genome. The target genome induced an increase in the hydrogel's mobility and loss in fluorescence with as few as 50 copies of full genome. The results demonstrate the potential of these CRISPR-responsive DNA hydrogels as intelligent platforms for target-induced imaging and therapeutic agent release, and biosensing applications.},
}
@article {pmid40629255,
year = {2025},
author = {Thornbury, M and Knoops, A and Summerby-Murray, I and Dhaliwal, J and Johnson, S and Utomo, JC and Joshi, J and Narcross, L and Remondetto, G and Pouliot, M and Whiteway, M and Martin, VJJ},
title = {Sequencing of a Dairy Isolate Unlocks Kluyveromyces marxianus as a Host for Lactose Valorization.},
journal = {ACS synthetic biology},
volume = {14},
number = {7},
pages = {2667-2680},
doi = {10.1021/acssynbio.5c00096},
pmid = {40629255},
issn = {2161-5063},
mesh = {*Kluyveromyces/genetics/metabolism ; *Lactose/metabolism ; Promoter Regions, Genetic/genetics ; CRISPR-Cas Systems/genetics ; Gene Editing/methods ; Polymorphism, Single Nucleotide ; Genome, Fungal/genetics ; Metabolic Engineering/methods ; },
abstract = {The use of genetically modified nonconventional yeast provides significant potential for the bioeconomy by diversifying the tools available for the development of sustainable and novel products. In this study, we sequenced and annotated the genome of Kluyveromyces marxianus Y-1190 to establish it as a platform for lactose valorization. The strain was chosen for rapid growth on lactose-rich dairy permeate, high transformation efficiency, and ease of culturing in bioreactors. Genomic sequencing revealed that K. marxianus Y-1190 possesses single nucleotide polymorphisms associated with efficient lactose metabolism. The strain is diploid with notable genomic heterogeneity, which appears to be critical for its robust growth and acid tolerance. To further exploit this platform strain, we developed protocols for gene and chromosome manipulation using CRISPR editing, constructed and validated a series of promoters compatible with MoClo vectors, and designed synthetically inducible promoters for K. marxianus. These tools enable precise control over gene expression, allowing for the tailored optimization of metabolic pathways and production processes. The synthetic promoters provide flexibility for dynamic expression tuning, while the CRISPR-based editing protocols facilitate targeted genetic modifications with high efficiency. Together, these advancements significantly enhance the genetic toolbox for K. marxianus, positioning it as a versatile platform for industrial biotechnology. These tools open new opportunities for the sustainable production of biobased chemicals, fuels, and high-value products, leveraging lactose-rich feedstocks to contribute to a circular economy.},
}
@article {pmid40562764,
year = {2025},
author = {Wang, T and Ding, S and Xu, J and Cai, G and Zhang, Y and Qi, Y and Jiang, Y and Zhang, P and Wang, T and Xin, F and Shen, T and Liu, G},
title = {Modular Design of a Copper Ion Biosensor Based on the MAPK Signaling Pathway in Saccharomyces cerevisiae.},
journal = {ACS synthetic biology},
volume = {14},
number = {7},
pages = {2854-2864},
doi = {10.1021/acssynbio.5c00276},
pmid = {40562764},
issn = {2161-5063},
mesh = {*Copper/analysis/metabolism ; *Biosensing Techniques/methods ; *Saccharomyces cerevisiae/metabolism/genetics ; Saccharomyces cerevisiae Proteins/genetics/metabolism ; Promoter Regions, Genetic/genetics ; *MAP Kinase Signaling System/genetics ; Pheromones/metabolism/genetics ; Quorum Sensing/genetics ; CRISPR-Cas Systems/genetics ; Green Fluorescent Proteins/genetics/metabolism ; },
abstract = {Copper ion poses serious threats to both the environment and human health. To develop a yeast biosensor with reduced background noise and enhanced detection sensitivity, we constructed a quorum-sensing module with amplified positive feedback. This biosensor employs a copper ion-pheromone communication system, which allows haploid a-type yeast (MATa) to express the α-pheromone gene (mfα2) under the control of the copper ion-inducible promoter pCUP1. The α-pheromone activates the mitogen-activated protein kinase (MAPK) signaling pathway, which in turn induces the expression of the green fluorescent protein (GFP) gene via the pheromone-inducible promoter pprm1. To improve the performance of the biosensor, we optimized the prm1 promoter and constructed the Ste5ΔN-CTM chassis. Specifically, the promoter intensity was improved by converting the three nonconsensus Pheromone Response Elements (PRE) in pprm1 into consensus PRE sequences, resulting in the prm1 Pro promoter. The Ste5ΔN-CTM strain continuously activates the MAPK signaling pathway. Next, to offset the loss of sensitivity and dynamic response range caused by endogenous pheromone degradation, we knocked out the pheromone degradation gene bar1 using CRISPR-Cas9 gene editing technology. Additionally, we established a functional model relating the copper ion concentration to the GFP signal output. In conclusion, this study designed a modular copper ion biosensor system by integrating sensing, amplification, and signal-reporting components, laying a foundation for the development of biosensors for other heavy metals.},
}
@article {pmid40555107,
year = {2025},
author = {Tian, W and Liu, T and Niu, X and Li, P and Li, Z and Yu, S and Zhang, P},
title = {CRISPR/Cas12a-mediated DNAzyme/split-aptamer cascade for label-free detection of site-specific DNA methylation.},
journal = {Biosensors &amp; bioelectronics},
volume = {287},
number = {},
pages = {117720},
doi = {10.1016/j.bios.2025.117720},
pmid = {40555107},
issn = {1873-4235},
mesh = {*DNA, Catalytic/chemistry/genetics ; *DNA Methylation/genetics ; *Biosensing Techniques/methods ; Humans ; *Septins/genetics ; *CRISPR-Cas Systems/genetics ; *Aptamers, Nucleotide/chemistry/genetics ; Limit of Detection ; Colorectal Neoplasms/genetics/diagnosis ; Endodeoxyribonucleases/chemistry/genetics ; DNA/genetics ; CRISPR-Associated Proteins/chemistry/genetics ; Bacterial Proteins ; },
abstract = {DNA methylation serves as a critical epigenetic biomarker for early disease screening and clinical diagnostics, exemplified by the FDA-approved detection of methylated Septin9 gene in peripheral blood for colorectal cancer (CRC) screening. Conventional CRISPR/Cas12a-based detection systems rely on fluorophore-quencher-labeled ssDNA probes to transduce trans-cleavage activity into fluorescence signal, which yet suffers from limitations including complex probe synthesis, background noise induced by incomplete quenching and high cost. In this study, we developed a cascaded and label-free CRISPR/Cas12a system for Septin9 methylation detection using DNAzyme and split DAP-10. The method employed methylation-sensitive AciI digestion to selectively hydrolyze non-methylated DNA, leaving methylated Septin9 DNA fragment intact to activate CRISPR/Cas12a system. Activated Cas12a triggered trans-cleavage of DNAzyme, a bifunctional molecule that acted as both trans-cleavage substrate of Cas12a and hydrolysis regulator of linker DNA that facilitated split DAP-10 reassembly to light-up Auramine O (AO) fluorescence proportionally to methylation levels. The assay achieved a linear range from 2 to 200 nM with a detection limit of 1.74 nM and robust performance in accuracy, precision, and selectivity. The proposed method was successfully applied in methylated Septin9 detection in cell lines, clinical tissues and blood samples, which indicated application potential for CRC screening.},
}
@article {pmid40544649,
year = {2025},
author = {Zhu, X and Jiao, J and Ni, Q and Yao, L and Zhang, Y and Liu, K and Huang, L and Bo, Q and Qin, P},
title = {Development of a universal one-pot CRISPR assay based on multifunctional tagged primer eliminating unstable crRNA input and PAM dependency for point-of-care detection of bacterial infections.},
journal = {Biosensors &amp; bioelectronics},
volume = {287},
number = {},
pages = {117718},
doi = {10.1016/j.bios.2025.117718},
pmid = {40544649},
issn = {1873-4235},
mesh = {*CRISPR-Cas Systems/genetics ; *Biosensing Techniques/methods ; Point-of-Care Systems ; Humans ; *Bacterial Infections/diagnosis/microbiology ; Nucleic Acid Amplification Techniques/methods ; DNA-Directed RNA Polymerases/chemistry ; Pseudomonas aeruginosa/isolation &amp; purification/genetics ; Staphylococcus aureus/isolation &amp; purification/genetics ; Escherichia coli/isolation &amp; purification/genetics ; Bacterial Proteins ; Endodeoxyribonucleases ; Viral Proteins ; CRISPR-Associated Proteins ; },
abstract = {The integration of recombinase polymerase amplification (RPA) with CRISPR/Cas12a holds great promise for point-of-care assay of bacterial infections, but still confronts challenges such as the input of unstable RNA, dependence on PAM, and multi-step operations. To address these challenges, we here present a tagged primer-based one-pot CRISPR/Cas12a assay, called TOP-CRISPR. Strategically, TOP-CRISPR utilized target gene-induced amplicons to generate crRNA sequences with the help of T7 RNA polymerase, thus eliminating the need for additional crRNA. The resulting Cas12a/crRNA complex were activated by the tag sequences at the end of specific primers rather than by the target amplicons, avoiding dependence on the PAM and disruption of the target amplicon by CRISPR/Cas12a. The ingenious design of tagged primer enables perfect compatibility between RPA and CRISPR/Cas12a system in a closed single tube, which is expected to overcome aerosol contamination caused by multi-step and open-cap operations. Under optimal conditions, the method was able to detect target bacteria down to ∼1 CFU/mL and the entire process took only less than 50 min, which is significantly better than the traditional two-step or one-step methods. By grafting the tag sequence onto different specific primers, bacteria such as Staphylococcus aureus, Escherichia coli O157:H7 and Pseudomonas aeruginosa has been detected, respectively. The practicability and robustness of the method was further validated by real samples. We hope that the method can be developed into a point-of-care detection tool for bacterial infections.},
}
@article {pmid40517662,
year = {2025},
author = {Zhang, X and Zhu, L and Yang, L and Zhou, Y and Li, H and Gao, Y and Xiong, X and Zhang, T and Zhu, L},
title = {DNA triangular prism nanostructure and CRISPR/Cas12a empowered electrochemical biosensor for dual detection of alpha-fetoprotein and microRNA 122.},
journal = {Biosensors &amp; bioelectronics},
volume = {287},
number = {},
pages = {117697},
doi = {10.1016/j.bios.2025.117697},
pmid = {40517662},
issn = {1873-4235},
mesh = {*Biosensing Techniques/methods ; *MicroRNAs/blood/isolation &amp; purification/genetics ; *alpha-Fetoproteins/analysis/isolation &amp; purification ; Humans ; Electrochemical Techniques/methods ; CRISPR-Cas Systems/genetics ; *Liver Neoplasms/diagnosis/blood/genetics ; *Carcinoma, Hepatocellular/blood/diagnosis/genetics ; DNA, Catalytic/chemistry ; Nanostructures/chemistry/ultrastructure ; Limit of Detection ; G-Quadruplexes ; Nucleic Acid Hybridization ; },
abstract = {Developing sensitive multi-target-combined detection method is of great significance for the early and accurate diagnosis and prognosis of hepatocellular carcinoma (HCC). Herein, an electrochemical biosensor was proposed for detecting HCC biomarkers miRNA 122 and alpha-fetoprotein (AFP), employing a DNA triangular prism (DTP) nanostructure as an efficient supporting platform integrated with a DNAzyme-hybridization chain reaction (HCR)-CRISPR/Cas12a triple signal amplification strategy. The ssDNA portion at the top of the DTP hybridized with ssDNA (S6) rich in guanine, thereby capturing hemin and forming G-quadruplex/hemin complexes, generating a strong initial electrochemical signal. Target recognition process released Mg[2+]-dependent DNAzyme, which cleaved the substrate hairpin DNA and produced trigger for HCR. The product of HCR activated CRISPR/Cas12a, causing non-specific cleavage of S6 and the ssDNA portion of DTP, which hindered the formation of G-quadruplex/hemin and led to a significant decrease in current signal. The rigid, stable, and size-controllable DTP nano-framework enhanced nucleic acid hybridization and signal molecule binding while minimizing nonspecific adsorption, eliminating masking agent requirements, with its ordered assembly facilitating CRISPR/Cas12a access to ssDNA for improved cleavage efficiency and detection sensitivity. Additionally, the programmable biosensing platform enabled sensitive detection of miRNA 122 and AFP through minimal sequence modifications in target recognition, while maintaining the identical cascade amplification system and DTP framework, demonstrating excellent versatility. Meaningfully, the biosensor sensitively detected miRNA 122 and AFP in real serum samples, and effectively distinguished healthy individuals from HCC patients, indicating its enormous potential for clinical diagnosis.},
}
@article {pmid40499459,
year = {2025},
author = {Zhou, B and Fan, T and Chen, H and Huang, Y and Lai, X and Feng, S and Ding, Z and Chen, Y and Jiang, Y},
title = {Development of a multi-miRNA detection platform for enhanced diagnostics of colorectal cancer.},
journal = {Biosensors &amp; bioelectronics},
volume = {287},
number = {},
pages = {117639},
doi = {10.1016/j.bios.2025.117639},
pmid = {40499459},
issn = {1873-4235},
mesh = {Humans ; *Colorectal Neoplasms/diagnosis/genetics/blood ; *MicroRNAs/genetics/blood/isolation &amp; purification ; *Biosensing Techniques/methods ; *Biomarkers, Tumor/genetics/blood ; Nucleic Acid Amplification Techniques/methods ; CRISPR-Cas Systems ; },
abstract = {The identification of specific miRNA biomarkers has considerably advanced colorectal cancer (CRC) diagnostics. Our prior research identified four key miRNA signatures-miR-10b-5p, miR-130a-3p, miR-561-5p, and miR-4684-5p-as potential diagnostic markers for CRC. To enhance the clinical utility of these biomarkers, we developed a novel multi-miRNA detection platform, RCA-SDA-CRISPR (RS-CRISPR), which integrates rolling circle amplification (RCA), strand displacement amplification (SDA), and CRISPR/Cas12a technologies. This platform employs RCA products as SDA templates, maximizes miRNA binding sites, and utilizes the spatial arrangement of miRNAs to bypass limitations of traditional enzymatic cleavage, enabling rapid, simultaneous detection of multiple miRNAs. RS-CRISPR achieved a detection sensitivity of 57.8fM for target miRNAs. In clinical validation, this method successfully distinguished tumor tissue from adjacent non-cancerous tissue in five patients with CRC. In addition, during the serum analysis of 12 normal controls (NC) and 12 CRC patients, we found that the levels of four specific miRNAs were significantly higher in CRC patients compared to NC (p = 0.00646), underscoring its diagnostic potential. These findings establish RS-CRISPR as a promising, precise, and efficient diagnostic tool for clinical CRC management through multi-miRNA detection.},
}
@article {pmid40480143,
year = {2025},
author = {Khoshbin, Z and Sameiyan, E and Alizadeh, F and Ramezani, M and Alibolandi, M and Abnous, K and Taghdisi, SM},
title = {A CRISPR/Cas12a-powered liquid crystal aptasensor for point-of-care detection of prostate-specific antigen for early-stage cancer diagnosis.},
journal = {Biosensors &amp; bioelectronics},
volume = {287},
number = {},
pages = {117651},
doi = {10.1016/j.bios.2025.117651},
pmid = {40480143},
issn = {1873-4235},
mesh = {*Prostate-Specific Antigen/blood/isolation &amp; purification ; Humans ; *Biosensing Techniques/methods ; *Aptamers, Nucleotide/chemistry/genetics ; CRISPR-Cas Systems/genetics ; *Liquid Crystals/chemistry ; *Prostatic Neoplasms/diagnosis/blood ; Male ; Early Detection of Cancer/methods ; Limit of Detection ; Point-of-Care Systems ; DNA, Single-Stranded/chemistry ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {Herein, an ultrasensitive aptasensor was developed for monitoring prostate-specific antigen (PSA) levels for the first time by incorporating the catalytic activity of CRISPR/Cas12a assembly and directional orientation of liquid crystal (LC) molecules. In the absence of PSA, the specific aptamer was hybridized with crRNA segment of the CRISPR/Cas12a assembly, which activated it for cleaving the ssDNA strand on the sensing substrate. This resulted in a dark background of the sensing substrate. While the assembly was unable to cleave the ssDNA sequence in the presence of PSA, due to forming the PSA-aptamer hybrid and disabling the enzymatic assembly, visualizing the colorful polarized sensing substrate. By changing the polarized background of the LC aptasensor from murky to multicolored, PSA could be detected ranging from 1 ag mL[-1]-1 pg mL[-1] and 1 pg mL[-1]-1 ng mL[-1] with a detection limit of 0.28 ag mL[-1]. With the potency to determine PSA in the spiked human serum samples with the recovery amounts of 97.87-99.57 %, the CRISPR/Cas12a-powered LC aptasensor is a reliable test kit for cancer screening. This was also confirmed by comparing the results obtained from the LC aptasensor with those of ELISA analysis for human serum samples of some patients. Being ultrasensitive, cost-effective, label-free, and user-friendly, the aptasensor can be an efficient future portable gadget for early-stage cancer monitoring.},
}
@article {pmid40347517,
year = {2025},
author = {Celauro, E and Saleh, AF and Mahalingaiah, PK and Mohamet, L and David, R and Nitsch, R},
title = {Exploring the synergy of CRISPR and microphysiological systems.},
journal = {ALTEX},
volume = {42},
number = {3},
pages = {468-478},
doi = {10.14573/altex.2403251},
pmid = {40347517},
issn = {1868-8551},
mesh = {*CRISPR-Cas Systems ; Animals ; *Gene Editing/methods ; Humans ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Genetic Therapy/methods ; Microphysiological Systems ; },
abstract = {Since its discovery as an innate bacterial immune system, the clustered regularly interspaced short palindromic repeats (CRISPR) associated nuclease 9 (CRISPR-Cas9) system has quickly landed on mammalian genomes to become the first-in-class editing technique. CRISPR-Cas9 offered an inval­uable approach to correct pathogenic mutations, thus becoming a promising cure for diseases with highly unmet medical needs. To date, several attempts have been made to understand, categorize and predict the outcome of genetic manipulation with different degrees of success. The lack of an appropriate and translatable model to test CRISPR/Cas9 effects, both wanted and unwanted, has limited its applications to advance gene therapies. Herein we describe the potential of microphysi­ological systems (MPS) as an alternative to the classical models used in CRISPR safety studies, such as immortalized cell lines or small mammals (e.g., rodents), to facilitate the progress of new CRISPR medicines to the clinics.},
}
@article {pmid40676076,
year = {2025},
author = {Borrelli, MJ and Buensuceso, A and Ramos Valdes, Y and Johnston, TPA and Haagsma, J and Shepherd, TG},
title = {CAMKKβ supports growth and viability of epithelial ovarian cancer in vitro and in vivo.},
journal = {Scientific reports},
volume = {15},
number = {1},
pages = {25913},
pmid = {40676076},
issn = {2045-2322},
support = {PGSD3 - 560096 - 2021//Natural Sciences and Engineering Research Council of Canada/ ; 451405/CAPMC/CIHR/Canada ; },
mesh = {Female ; Humans ; *Calcium-Calmodulin-Dependent Protein Kinase Kinase/metabolism/genetics ; Animals ; *Carcinoma, Ovarian Epithelial/pathology/genetics/metabolism ; Cell Line, Tumor ; *Ovarian Neoplasms/pathology/genetics/metabolism ; Mice ; Cell Survival ; Spheroids, Cellular/metabolism/pathology ; Cell Proliferation ; AMP-Activated Protein Kinases/metabolism ; Autophagy ; Gene Knockout Techniques ; CRISPR-Cas Systems ; Xenograft Model Antitumor Assays ; },
abstract = {Epithelial ovarian cancer (EOC) metastasizes predominantly through multicellular aggregates known as spheroids, which disseminate within the peritoneal cavity and initiate secondary disease upon reattachment at distant sites. EOC spheroids resist detachment-induced cell death by upregulating stress responses including AMP-activated protein kinase (AMPK) signaling and AMPK-dependent macroautophagy (autophagy), highlighting these pathways as potential therapeutic targets. Previously, we used a pharmacological approach to putatively identify Ca[2+]/calmodulin-dependent protein kinase kinase 2 (CAMKKβ, encoded by CAMKK2) as the primary activator of AMPK in EOC spheroids. Herein we have generated CAMKK2 knockout EOC cell lines via CRISPR-Cas9 genome editing to confirm this function of CAMKKβ and explore the impacts of its loss using in vitro and in vivo models of metastatic EOC. CAMKK2 knockout spheroids exhibited decreased AMPK activation, autophagic flux, cell viability, and metastatic potential relative to parental spheroids, and intraperitoneal xenograft tumours lacking CAMKKβ grew slower than their CAMKKβ-intact counterparts. Effect magnitudes varied between cell line models, suggesting context-dependent roles for CAMKKβ in EOC and rationalizing further studies to characterize the underlying mechanisms. Altogether, our findings highlight CAMKKβ as an important contributor to metabolic reprogramming in EOC spheroids and as a potential therapeutic target in the setting of advanced disease.},
}
@article {pmid40674699,
year = {2025},
author = {Xin, C and Lu, Y and Gul, SL and Sun, W and Cao, Z and Kong, X and Fan, K and Li, S and Liu, X and Wang, XC and Chen, QJ},
title = {Never-Germinating Arabidopsis Seeds with LbCas12a-Induced Mutations in Six Clade A Type 2C Protein Phosphatase Genes.},
journal = {Plant physiology},
volume = {},
number = {},
pages = {},
doi = {10.1093/plphys/kiaf315},
pmid = {40674699},
issn = {1532-2548},
abstract = {Cas12 nucleases, such as Cas12a, Cas12i, and Cas12f, are genome-editing tools that possess several unique attributes. However, the potential of various Cas12 variants for multiplex genome editing in Arabidopsis (Arabidopsis thaliana) remains insufficiently characterized. In this report, we systematically evaluated 18 additional targets and demonstrated that the LbCas12a variant carrying D156R and E795L mutations exhibits minimal target bias. Furthermore, we achieved an editing efficiency of at least 73.8% (45/61) in generating T1 homozygous sextuple mutants, with more than half of these mutants exhibiting a complete seed germination arrest phenotype. Comparative analysis of seven LbCas12a variants revealed that the optimization of nuclear localization sequences, rather than codon usage, is fundamental for improved editing efficiency, and that the E795L mutation had synergistic effects with other mutations in highly efficient LbCas12a variants. Further investigation into one Cas12i3 and two AsCas12f variants showed that the Cas12i3 variant also exhibits sufficiently high editing efficiency in Arabidopsis, although additional refinements were required to mitigate its target bias. Collectively, in this study, we developed the most efficient CRISPR/Cas tool for multiplex genome editing in Arabidopsis, as demonstrated by the highly efficient generation of never-germinating seeds harboring mutations in six clade A type 2C protein phosphatase genes.},
}
@article {pmid40674424,
year = {2025},
author = {Kaur, K and Zaheer, J and Lang, F and Ribeiro, DL and Zhang, M and Song, H and Zhang, W and Chari, R and Alkaissi, H and Yang, C},
title = {mtKO: A dedicated guide RNA library for mitochondria research.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {122},
number = {29},
pages = {e2502285122},
doi = {10.1073/pnas.2502285122},
pmid = {40674424},
issn = {1091-6490},
mesh = {*Mitochondria/genetics/metabolism ; Humans ; *Superoxide Dismutase/genetics/metabolism ; Isocitrate Dehydrogenase/genetics/metabolism ; *RNA, Guide, CRISPR-Cas Systems/genetics ; Gene Knockout Techniques/methods ; CRISPR-Cas Systems ; Mutation ; Gene Library ; Cell Line, Tumor ; Neoplasms/genetics/metabolism/pathology ; Animals ; },
abstract = {Mitochondria are multifunctional organelles central to both physiological and pathological processes. In malignant cancer cells, mitochondrial reprogramming establishes the metabolic foundation to meet cellular demands, which is particularly important in tumor cells with existing metabolic perturbations. To identify key mitochondrial pathways supporting cancer development, we developed mitochondria Knockout (mtKO), a robust and unbiased CRISPR screening platform to pinpoint critical mitochondria-associated pathways. The mtKO screen revealed that the mitochondrial antioxidant enzyme SOD2 is essential for cells harboring IDH1 mutations. Mechanistically, SOD2 activity determines the disease manifestation of IDH1-mutated cancers, through maintaining redox homeostasis and mitochondrial fitness. This study introduces a powerful functional genomic tool to identify mitochondrial-centered pathways and reveals the selective mitochondrial vulnerability in Krebs cycle-deficient cancers for future therapeutic intervention.},
}
@article {pmid40673874,
year = {2025},
author = {Zhang, XM and Qiu, MY and Liao, L and Han, B and Deng, YD and Zhang, N and Hai, LT and Liang, L and Liu, MJ},
title = {Insights Into Coat Color Variation: Unraveling the Role of ASIP by Gene Editing in Fine-Wool Sheep.},
journal = {FASEB journal : official publication of the Federation of American Societies for Experimental Biology},
volume = {39},
number = {14},
pages = {e70807},
doi = {10.1096/fj.202500355R},
pmid = {40673874},
issn = {1530-6860},
support = {32160777//the National Natural Science Foundation of China/ ; Ky202471//the Public Welfare Research Institutes' Basic Research Fund Project in Autonomous Region/ ; 2023TSYCTD0007//the Scientific and Technological Innovation Team Project of Xinjiang Uygur Autonomous Region/ ; },
mesh = {Animals ; *Agouti Signaling Protein/genetics/metabolism ; Sheep/genetics ; *Gene Editing/methods ; *Hair Color/genetics ; *Pigmentation/genetics ; Female ; Phenotype ; CRISPR-Cas Systems ; Male ; },
abstract = {Sheep coat color is an important phenotypic trait that influences consumer preferences and breeding. Although the Agouti Signaling Protein (ASIP) gene has been linked to pigmentation, the mechanisms underlying its function remain unclear. In a previous study, we generated ASIP gene-edited fine-wool sheep (F0) with diverse coat colors using CRISPR/Cas9. Building on this foundation, the present study investigates coat color variation in 33 F1 offspring produced by mating F0 gene-edited rams with either F0 gene-edited ewes or wild-type white ewes. Comprehensive genotyping identified two inherited ASIP mutations-a 4-bp deletion and a 27-bp deletion with a 1-bp insertion-alongside natural variants (D5, D9) and copy number variation. These genetic differences were associated with a wide range of coat color phenotypes in the F1 population. To explore the underlying mechanisms, we conducted histological analyses (H&amp;E staining), immunohistochemistry, melanin content quantification, qRT-PCR, and Western blotting. Results showed that dark-coated individuals exhibited reduced ASIP expression and elevated levels of TYR and TYRP1, consistent with increased eumelanin synthesis. In contrast, white-coated sheep showed higher ASIP expression and lower levels of melanogenic enzymes. Our findings provide functional validation that ASIP is a central regulator of pigmentation in sheep and reveal how the interplay between gene editing, natural mutations, and gene dosage contributes to diverse coat color outcomes. This work establishes a valuable model for dissecting coat color genetics and offers a molecular framework for future breeding strategies aimed at wool color diversification and economic trait improvement in fine-wool sheep.},
}
@article {pmid40673793,
year = {2025},
author = {Pan, Y and Yang, X and Zeng, Z and Liu, F and Luo, J and Shen, M and Zhou, W and Li, J and Jiang, G and Sun, L and Huang, H and Pan, R},
title = {[124]I-labelled BMSC-Derived Extracellular Vesicles Deliver CRISPR/Cas9 Ribonucleoproteins With a GFP-Reporter System to Inhibit Osteosarcoma Proliferation and Metastasis.},
journal = {Journal of extracellular vesicles},
volume = {14},
number = {7},
pages = {e70130},
doi = {10.1002/jev2.70130},
pmid = {40673793},
issn = {2001-3078},
support = {82060491//the National Natural Science Foundation of China/ ; 82160568//the National Natural Science Foundation of China/ ; [2024]168//the Guizhou Provincial Science and Technology Projects/ ; [2021]395//the Guizhou Provincial Science and Technology Projects/ ; [2024]481//the Guizhou Provincial Science and Technology Projects/ ; },
mesh = {*Osteosarcoma/pathology/therapy/metabolism/genetics ; *CRISPR-Cas Systems ; Humans ; *Extracellular Vesicles/metabolism ; Cell Proliferation ; Animals ; *Mesenchymal Stem Cells/metabolism ; Mice ; Cell Line, Tumor ; *Ribonucleoproteins/metabolism/genetics ; *Bone Neoplasms/pathology/therapy/metabolism/genetics ; Iodine Radioisotopes ; Gene Editing ; Green Fluorescent Proteins/metabolism/genetics ; Mice, Nude ; Neoplasm Metastasis ; },
abstract = {Metastasis constitutes the principal factor leading to the unfavourable prognosis of osteosarcoma patients. Hypoxia, as the inherent microenvironment of osteosarcoma, can upregulate HIF-1α via multiple pathways, thereby facilitating osteosarcoma proliferation and metastasis. Our previous research indicated that the inwardly rectifying potassium channel subfamily J member 2 (KCNJ2) inhibits the degradation of HIF-1α in osteosarcoma. Concurrently, HIF-1α upregulates the expression of KCNJ2 through a positive feedback regulatory mechanism. This positive regulatory mechanism significantly promotes the proliferation and metastasis of osteosarcoma. Therefore, the development of a KCNJ2-targeted therapeutic strategy capable of disrupting this reciprocal regulatory loop represents a crucial intervention for impeding osteosarcoma progression. The CRISPR/Cas9 targeted gene editing technology has garnered extensive attention in the field of tumour treatment due to its high efficiency and low off-target rate. Nevertheless, the relative lag of the delivery systems has restricted its application. The extracellular vesicles (EVs) secreted by bone marrow mesenchymal stem cells (BMSCs) have a natural targeting specificity for osteosarcoma and possess superior biocompatibility, making them ideal carriers for in vivo delivery. However, it is essential to confirm whether the CRISPR/Cas9 system mediated by EVs can accurately function intracellularly. Hence, we developed a fluorescence-based Cas9 editing efficiency reporter system. When CRISPR/Cas9 system induces double-strand breaks at specific target sites and results in frameshift mutations, osteosarcoma cells will stably express GFP. This system enables the transformation of gene editing events into quantifiable fluorescence signals. Furthermore, we engineered radiolabelled EVs derived from BMSCs to deliver the CRISPR/Cas9 system targeting KCNJ2. Using this reporter system, we confirmed their efficient gene-editing capabilities in vitro. Additionally, leveraging their radiolabelling properties, we validated their targeted distribution in vivo. Subsequent investigations revealed that our constructed [124]I@EVs-Cas9 effectively suppresses the proliferation and metastasis of osteosarcoma by targeting the inhibition of KCNJ2 expression and promoting HIF-1α ubiquitin-dependent degradation (as depicted in Graphical Abstract).},
}
@article {pmid40672203,
year = {2025},
author = {Hodge, CA and Donegan, NP and Armstrong, DA and Hayden, MS and Howell, AL},
title = {ENHANCED CLEAVAGE OF GENOMIC CCR5 USING CASX2 [Max].},
journal = {bioRxiv : the preprint server for biology},
volume = {},
number = {},
pages = {},
doi = {10.1101/2025.07.08.663680},
pmid = {40672203},
issn = {2692-8205},
abstract = {Development of novel CRISPR/Cas systems enhances opportunities for gene editing to treat infectious diseases, cancer, and genetic disorders. We evaluated CasX2 (Plm Cas12e), a class II CRISPR system derived from Planctomycetes , a non-pathogenic bacterium present in aquatic and terrestrial soils. CasX2 offers several advantages over Streptococcus pyogenes Cas9 (Sp Cas9) and Staphylococcus aureus Cas9 (Sa Cas9), including its smaller size, distinct protospacer adjacent motif (PAM) requirements, staggered cleavage cuts that promote homology-directed repair, and no known pre-existing immunity in humans. A recent study reported that a three amino acid substitution in CasX2 significantly enhanced cleavage activity (1). Therefore, we compared cleavage efficiency and double-stranded break repair characteristics between the native CasX2 and the variant, CasX2 [Max] , for cleavage of CCR5 , a gene that encodes the CCR5 receptor important for HIV-1 infection. Two CasX2 single guide RNAs (sgRNAs) were designed that flanked the 32 bases deleted in the natural CCR5 Δ32 mutation. Nanopore sequencing demonstrated that CasX2 using sgRNAs with spacers of 17 nucleotides (nt), 20 nt or 23 nt in length were ineffective at cleaving genomic CCR5. In contrast, CasX2 [Max] using sgRNAs with 20 nt and 23 nt spacer lengths, enabled robust genomic cleavage of CCR5 . Structural modeling indicated that two of the CasX2 [Max] substitutions enhanced sgRNA-DNA duplex stability, while the third improved DNA strand alignment within the catalytic site. These structural changes likely underlie the increased activity of CasX2 [Max] in cellular gene excision. In sum, CasX2 [Max] consistently outperformed native CasX2 across all assays and represents a superior gene-editing platform for therapeutic applications.},
}
@article {pmid40672171,
year = {2025},
author = {Sinnott, RW and Solanki, A and Govind, AP and Green, WN and Dickinson, BC},
title = {Engineering a human-based translational activator for targeted protein expression restoration.},
journal = {bioRxiv : the preprint server for biology},
volume = {},
number = {},
pages = {},
doi = {10.1101/2025.07.09.663984},
pmid = {40672171},
issn = {2692-8205},
abstract = {Therapeutic modalities to programmably increase protein production are in critical need to address diseases caused by deficient gene expression via haploinsufficiency. Restoring physiological protein levels by increasing translation of their cognate mRNA would be an advantageous approach to correct gene expression, but has not been evaluated in an in vivo disease model. Here, we investigated if a translational activator could improve phenotype in a Dravet syndrome mouse model, a severe developmental and epileptic encephalopathy caused by SCN1a haploinsufficiency, by increasing translation of the SCN1a mRNA. We identifiy and engineere human proteins capable of increasing mRNA translation using the CRISPR-Cas Inspired RNA-targeting System (CIRTS) platform to enable programmable, guide RNA (gRNA)-directed translational activation with entirely engineered human proteins. We identify a compact (601 amino acid) CIRTS translational activator (CIRTS-4GT3), that can drive targeted, sustained translation increases up to 100% from three endogenous transcripts relevant to epilepsy and neurodevelopmental disorders. AAV-delivery of CIRTS-4GT3 targeting SCN1a mRNA to a Dravet syndrome mouse model led to increased SCN1a translation and improved survivability and seizure threshold - key phenotypic indicators of Dravet syndrome. This work validates a new strategy to address SCN1a haploinsufficiency and emphasizes the preclinical potential translational activation has to address neurological haploinsufficiency.},
}
@article {pmid40671523,
year = {2025},
author = {Ma, E and Chen, K and Shi, H and Wasko, KM and Esain-Garcia, I and Trinidad, MI and Zhou, K and Ye, J and Doudna, JA},
title = {Directed evolution expands CRISPR-Cas12a genome-editing capacity.},
journal = {Nucleic acids research},
volume = {53},
number = {13},
pages = {},
doi = {10.1093/nar/gkaf649},
pmid = {40671523},
issn = {1362-4962},
support = {//Life Sciences Research Foundation/ ; //Rett Syndrome Research Trust/ ; //The Jane Coffin Childs Fund for Medical Research/ ; /HHMI/Howard Hughes Medical Institute/United States ; U01AI142817/GF/NIH HHS/United States ; U19AI171110/AI/NIAID NIH HHS/United States ; U54AI170792/AI/NIAID NIH HHS/United States ; U19AI135990/AI/NIAID NIH HHS/United States ; UH3AI150552/AI/NIAID NIH HHS/United States ; U19NS132303/AI/NIAID NIH HHS/United States ; R21HL173710/HB/NHLBI NIH HHS/United States ; 2334028//NSF/ ; DE-AC02-05CH11231//DOE/ ; 2553571//DOE/ ; B656358//DOE/ ; 24180//Lawrence Livermore National Laboratory/ ; //CB-Hampton University Summer Program/ ; //Mr Li Ka Shing/ ; //Koret-Berkeley-TAU/ ; //Emerson Collective/ ; //Innovative Genomics Institute/ ; },
mesh = {*CRISPR-Cas Systems ; *Gene Editing/methods ; Humans ; *CRISPR-Associated Proteins/genetics/metabolism ; *Directed Molecular Evolution/methods ; *Endodeoxyribonucleases/genetics/metabolism ; *Bacterial Proteins/genetics/metabolism ; Genome, Human ; },
abstract = {CRISPR-Cas12a enzymes are versatile RNA-guided genome-editing tools with applications encompassing viral diagnosis, agriculture, and human therapeutics. However, their dependence on a 5'-TTTV-3' protospacer adjacent motif (PAM) next to DNA target sequences restricts Cas12a's gene targeting capability to only ∼1% of a typical genome. To mitigate this constraint, we used a bacterial-based directed evolution assay combined with rational engineering to identify variants of Lachnospiraceae bacterium Cas12a with expanded PAM recognition. The resulting Cas12a variants use a range of noncanonical PAMs while retaining recognition of the canonical 5'-TTTV-3' PAM. In particular, biochemical and cell-based assays show that the variant Flex-Cas12a utilizes 5'-NYHV-3' PAMs that expand DNA recognition sites to ∼25% of the human genome. With enhanced targeting versatility, Flex-Cas12a unlocks access to previously inaccessible genomic loci, providing new opportunities for both therapeutic and agricultural genome engineering.},
}
@article {pmid40670899,
year = {2025},
author = {Alam, T},
title = {Advances in Tissue Culture-Free Genetic Engineering and Genome Editing of Peanut.},
journal = {Molecular biotechnology},
volume = {},
number = {},
pages = {},
pmid = {40670899},
issn = {1559-0305},
abstract = {Plant transformation and genome editing are pivotal in advancing peanut biotechnology, yet traditional tissue culture-dependent methods are hindered by lengthy protocols, genotype dependency, and somaclonal variation. CRISPR/Cas technologies have revolutionized breeding by enabling precise, multiplex genome editing to improve traits such as disease resistance, allergen reduction, seed quality, and yield. However, variable transformation efficiencies and chimerism remain challenges. This review examines emerging tissue culture-independent techniques such as nanoparticle-based delivery, viral vectors, pollen magnetofection, pollen tube injection, node injection, and vacuum infiltration that offer rapid, cost-effective gene transfer. It also highlights the integration of high-throughput screening, robust selection markers, and automation, including robotics and advanced imaging, to refine transformation pipelines. These methodological breakthroughs promise to overcome current limitations and accelerate the development of improved peanut cultivars for sustainable agriculture.},
}
@article {pmid40670803,
year = {2025},
author = {Admas, T and Shu, J and Shalmani, A and Pan, R and Zhang, W},
title = {Salt stress-responsive transcription factors provide insights to enhance barley improvement: a review.},
journal = {Planta},
volume = {262},
number = {3},
pages = {56},
pmid = {40670803},
issn = {1432-2048},
support = {32372052//Innovative Research Group Project of the National Natural Science Foundation of China/ ; },
mesh = {*Hordeum/genetics/physiology ; *Salt Stress/genetics ; *Transcription Factors/genetics/metabolism ; *Plant Proteins/genetics/metabolism ; Gene Expression Regulation, Plant ; *Salt Tolerance/genetics ; },
abstract = {Salt stress is limiting barley growth, development, and production. Transcription factors (TFs) play a critical role in plant responses to salt stress by modulating gene expression Salinity stress increases over time due to climate change. It represents a major constraint to barley growth, development, and yield. Enhancing salt tolerance to withstand salt stress is crucial for ensuring global food security. We discussed transcription factors (TFs) that play a key role in responding to salt stress by modulating downstream genes and facilitating alterations in physiological and biochemical pathways. TF families strongly associated with the bZIP, DREB, NAC, bHLH, MYB, ERF, and WRKY are particularly involved in regulating ion homeostasis, osmotic adjustment, and signaling under stressful conditions. These discoveries establish a platform for generating salt-tolerant barley genotypes utilizing modern biotechnological methods. CRISPR/Cas and virus-induced gene silencing (VIGS) are broadly used tools to investigate gene function by knocking out or silencing target genes under salt stress. Furthermore, integrating the existing knowledge and foundations of TFs could yield sustainable, salt-resistant barley genotypes for agriculture. Multi-omics and bioinformatics have accelerated the identification of salt-responsive genes and TFs. The review recorded the recent progress in the molecular mechanisms of salinity tolerance in barley and indicates the potential of biotechnology for improving salt tolerance in barley varieties.},
}
@article {pmid40670339,
year = {2025},
author = {Houbaert, A and Denervaud Tendon, V and Hoermayer, L and Morffy, N and Strader, LC and Geldner, N},
title = {Efficient, cell-type-specific production of flavonols by multiplexed CRISPR activation of a suite of metabolic enzymes.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {6559},
pmid = {40670339},
issn = {2041-1723},
support = {ROOBABA 101020794//EC | EU Framework Programme for Research and Innovation H2020 | H2020 Priority Excellent Science | H2020 European Research Council (H2020 Excellent Science - European Research Council)/ ; ALTF 1153-2019//European Molecular Biology Organization (EMBO)/ ; EF-ST 892018//EC | EU Framework Programme for Research and Innovation H2020 | H2020 Priority Excellent Science | H2020 Marie Sk&#x142;odowska-Curie Actions (H2020 Excellent Science - Marie Sk&#x142;odowska-Curie Actions)/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; *Flavonols/biosynthesis/metabolism ; *Arabidopsis/genetics/metabolism ; Plants, Genetically Modified/genetics/metabolism ; Gene Expression Regulation, Plant ; Plant Roots/metabolism/genetics ; Arabidopsis Proteins/genetics/metabolism ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; },
abstract = {Synthetic biology in plants promises to transform basic and applied research by rewiring entire developmental modules, signaling cascades or metabolic pathways. Yet, this requires expression of many genes simultaneously, very difficult with classic transgenic approaches, especially for the generation of stable traits. CRISPR activation systems work in plants and could greatly facilitate multiplexed gene activation. Current CRISPR activation systems are efficient for transient or ubiquitous expression. Yet, to fulfill their potential, CRISPR activation needs to perform robustly in specific organs and tissue types. Here, we present a CRISPR activation system that efficiently drives expression in a cell-type-specific manner in stable lines, which requires assessing expression on a cellular basis using fluorescent reporter lines. Our CRISPR systems consistently re-wire gene expression at the cellular level, inducing genes with cell-type specific expression to efficiently express in a new cell layer, such as root endodermis or epidermis. We demonstrate the power of our system to drive functionally relevant, multiplexed gene activation by achieving endodermis-specific production of wild-type levels of flavonoids, detectable by in-situ fluorescence, in a root-flavonoid deficient myb12 mutant.},
}
@article {pmid40607530,
year = {2025},
author = {Zhang, W and Wu, M and Zhang, Q and Chen, X and Guo, Y},
title = {Targeting liposomes and an accelerated CRISPR system for selective imaging of miR-21 in cells.},
journal = {Analytical methods : advancing methods and applications},
volume = {17},
number = {28},
pages = {5831-5835},
doi = {10.1039/d5ay00619h},
pmid = {40607530},
issn = {1759-9679},
mesh = {*MicroRNAs/genetics/analysis/metabolism ; *Liposomes/chemistry ; Humans ; MCF-7 Cells ; *CRISPR-Cas Systems/genetics ; Mucin-1/genetics ; Aptamers, Nucleotide/chemistry/genetics ; *Molecular Imaging/methods ; Manganese Compounds/chemistry ; },
abstract = {This study demonstrates a liposome-targeted and accelerated CRISPR strategy for specific response of miR-21 within MCF-7 cells. Targeting was achieved with the help of modification of the MUC1 aptamer. MnO2 NS could be used as both a delivery vehicle and an accelerator for the CRISPR system, and finally to achieve spatially selective imaging of miR-21.},
}
@article {pmid40562042,
year = {2025},
author = {DeCarlo, A and MacLeod, G and Fernandes da Silva, C and Shen, LQ and Povilaikaite, J and Deane, M and Aragao, L and Sie, M and Termini, D and Magee, J and Gudenas, B and Sukumaran, S and Charron, F and Marcellus, R and Al-Awar, R and Aman, A and Reynaud, D and Trolat, A and Wybenga-Groot, L and Tabori, U and Nör, C and Harding, SM and Moran, MF and Northcott, PA and Dirks, P and Angers, S and Ramaswamy, V},
title = {Targeting synthetic lethality between non-homologous end joining and radiation in very-high-risk medulloblastoma.},
journal = {Cell reports. Medicine},
volume = {6},
number = {7},
pages = {102202},
doi = {10.1016/j.xcrm.2025.102202},
pmid = {40562042},
issn = {2666-3791},
mesh = {*Medulloblastoma/genetics/radiotherapy/pathology/metabolism ; *DNA End-Joining Repair/radiation effects/genetics ; Humans ; Animals ; Tumor Suppressor Protein p53/genetics/metabolism/deficiency ; DNA-Activated Protein Kinase/metabolism/antagonists &amp; inhibitors ; *Cerebellar Neoplasms/genetics/radiotherapy/pathology ; Mice ; *Synthetic Lethal Mutations/genetics ; Cell Line, Tumor ; CRISPR-Cas Systems/genetics ; Radiation Tolerance/genetics/drug effects ; },
abstract = {Specific and biologically informed treatments for medulloblastoma, especially for the highly lethal TP53-mutant SHH subgroup, remain elusive, where radiotherapy is the primary treatment modality. Leveraging genome-wide CRISPR-Cas9 dropout screening in combination with lethal doses of radiotherapy, we identify loss of p53 as the main driver of radiation resistance in SHH medulloblastoma. A negative-selection CRISPR-Cas9 screen across multiple models of Trp53-deficient SHH medulloblastoma reveals a strong synthetic lethal interaction between components of the non-homologous end-joining pathway and radiation, particularly DNA-dependent protein kinase (DNA-PK) and its binding partners. Both genetic and pharmacological perturbation of DNA-PK enhance radiosensitivity in TP53-deficient SHH medulloblastoma, leading to cell death. In vivo treatment of both somatic and germline TP53-mutant SHH medulloblastoma models with peposertib, a small-molecule inhibitor of DNA-PK, significantly improves survival when combined with radiotherapy, strongly supporting further clinical investigation.},
}
@article {pmid40440427,
year = {2025},
author = {Zhou, L and Chen, Z and Zou, Y and Zhang, X and Wang, Z and Zhu, H and Lin, J and Huang, Z and Zheng, L and Chen, J and Xie, M and Zhang, M and Zhang, R and Zhu, M and Wang, Z and Zhou, H and Gao, S and Yin, Y and Wu, Y and Kang, T},
title = {ASB7 is a negative regulator of H3K9me3 homeostasis.},
journal = {Science (New York, N.Y.)},
volume = {389},
number = {6757},
pages = {309-316},
doi = {10.1126/science.adq7408},
pmid = {40440427},
issn = {1095-9203},
mesh = {*Histones/metabolism ; Homeostasis ; Humans ; *Heterochromatin/metabolism ; Methylation ; *Methyltransferases/metabolism ; Chromobox Protein Homolog 5 ; Cullin Proteins/metabolism/genetics ; Phosphorylation ; Chromosomal Proteins, Non-Histone/metabolism ; *Ubiquitin-Protein Ligases/metabolism ; Epigenesis, Genetic ; Mitosis ; CRISPR-Cas Systems ; HEK293 Cells ; Lysine/metabolism ; Repressor Proteins ; },
abstract = {The maintenance of histone H3 lysine 9 trimethylation (H3K9me3) involves the recognition of preexisting modifications by heterochromatin protein 1 (HP1), which recruits the methyltransferase suppressor of variegation 3-9 homolog 1 (SUV39H1) to methylate the adjacent newly incorporated histones, establishing a positive feedback loop. However, how this positive feedback is restricted to maintain H3K9me3 homeostasis remains largely unknown. We performed an unbiased genome-scale CRISPR-Cas9 screen and identified CUL5[ASB7] E3 ubiquitin ligase as a negative regulator of H3K9me3. ASB7 is recruited to heterochromatin by HP1 and promotes SUV39H1 degradation. During mitosis, cyclin-dependent kinase 1 (CDK1) phosphorylates ASB7, preventing its interaction with SUV39H1, leading to SUV39H1 stabilization and H3K9me3 restoration. Our findings reveal a dynamic circuit involving HP1, SUV39H1, and ASB7 that governs H3K9me3 homeostasis, ensuring faithful epigenetic inheritance and preventing excessive heterochromatin formation.},
}
@article {pmid40399679,
year = {2025},
author = {Gulati, A and Ahn, DH and Suades, A and Hult, Y and Wolf, G and Iwata, S and Superti-Furga, G and Nomura, N and Drew, D},
title = {Stepwise ATP translocation into the endoplasmic reticulum by human SLC35B1.},
journal = {Nature},
volume = {643},
number = {8072},
pages = {855-864},
pmid = {40399679},
issn = {1476-4687},
mesh = {Humans ; *Endoplasmic Reticulum/metabolism/ultrastructure ; *Adenosine Triphosphate/metabolism/analogs &amp; derivatives/chemistry ; Adenosine Diphosphate/metabolism/chemistry ; Cryoelectron Microscopy ; Models, Molecular ; Biological Transport ; *Nucleotide Transport Proteins/metabolism/chemistry/genetics/ultrastructure ; Binding Sites ; Kinetics ; CRISPR-Cas Systems ; },
abstract = {ATP generated in the mitochondria is exported by an ADP/ATP carrier of the SLC25 family[1]. The endoplasmic reticulum (ER) cannot synthesize ATP but must import cytoplasmic ATP to energize protein folding, quality control and trafficking[2,3]. It was recently proposed that a member of the nucleotide sugar transporter family, termed SLC35B1 (also known as AXER), is not a nucleotide sugar transporter but a long-sought-after ER importer of ATP[4]. Here we report that human SLC35B1 does not bind nucleotide sugars but indeed executes strict ATP/ADP exchange with uptake kinetics consistent with the import of ATP into crude ER microsomes. A CRISPR-Cas9 cell-line knockout demonstrated that SLC35B1 clusters with the most essential SLC transporters for cell growth, consistent with its proposed physiological function. We have further determined seven cryogenic electron microscopy structures of human SLC35B1 in complex with an Fv fragment and either bound to an ATP analogue or ADP in all major conformations of the transport cycle. We observed that nucleotides were vertically repositioned up to approximately 6.5 Å during translocation while retaining key interactions with a flexible substrate-binding site. We conclude that SLC35B1 operates by a stepwise ATP translocation mechanism, which is a previously undescribed model for substrate translocation by an SLC transporter.},
}
@article {pmid40383470,
year = {2025},
author = {Varol, A and Klauck, SM and Lees-Miller, SP and Efferth, T},
title = {Comprehensive transcriptomic analysis in wild-type and ATM knockout lung cancer cells: Influence of cisplatin on oxidative stress-induced senescence.},
journal = {Chemico-biological interactions},
volume = {418},
number = {},
pages = {111563},
doi = {10.1016/j.cbi.2025.111563},
pmid = {40383470},
issn = {1872-7786},
mesh = {Humans ; *Ataxia Telangiectasia Mutated Proteins/genetics/deficiency/metabolism ; *Cisplatin/pharmacology ; *Oxidative Stress/drug effects ; *Lung Neoplasms/pathology/genetics/metabolism/drug therapy ; *Cellular Senescence/drug effects ; NF-E2-Related Factor 2/metabolism/genetics ; Cell Line, Tumor ; Reactive Oxygen Species/metabolism ; Gene Expression Profiling ; *Antineoplastic Agents/pharmacology ; Gene Knockout Techniques ; Transcriptome/drug effects ; CRISPR-Cas Systems ; },
abstract = {Genetic mutations and impaired DNA repair mechanisms in cancer not only facilitate tumor progression but also reduce the effectiveness of chemotherapeutic agents, particularly cisplatin. Combination therapy has emerged as a promising strategy to overcome resistance. Comprehensive transcriptomic analyses, supported by integrated comparative bioinformatics and experimental approaches, are essential for identifying biomarkers and novel therapeutic targets underlying drug resistance. In this study, we performed overall survival and mutation analyses, examining 23 double-strand break repair proteins across more than 7500 tumors spanning 23 distinct cancer types. Our findings identify ATM (ataxia-telangiectasia mutated) as a key protein with the highest mutation frequency. Using CRISPR/Cas9, we investigated the effects of ATM mutations on signalling pathways that influence the cellular response to cisplatin. ATM knockout enhanced cisplatin cytotoxicity by activating alternative cell death pathways, including oxidative stress-induced senescence and necroptosis. Microarray analysis revealed a regulatory interplay between ATM and NRF2 in the activation of oxidative stress-induced senescence. Specifically, ATM knockout promoted senescence by increasing reactive oxygen species (ROS) accumulation and downregulating NRF2 expression. To enhance combination therapy, integrating genetic profiling with advanced tools such as CRISPR/Cas9 to target oxidative stress-induced senescence may provide innovative strategies to overcome drug resistance, thereby advancing personalized cancer treatment. These approaches lay the foundation for the development of personalized cancer therapies tailored to the unique mutational landscape of individual patients, offering promising prospects for improving treatment outcomes.},
}
@article {pmid40301074,
year = {2025},
author = {Ogata, T and Koide, K and Kudou, S and Suto, M and Uehara, K and Kaneko, T},
title = {CRISPR-Cas9 genome editing of miso and soy source yeast Zygosaccharomyces sp.},
journal = {The Journal of general and applied microbiology},
volume = {70},
number = {5},
pages = {},
doi = {10.2323/jgam.2025.04.002},
pmid = {40301074},
issn = {1349-8037},
mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems ; *Zygosaccharomyces/genetics/metabolism ; Genome, Fungal ; *Soy Foods/microbiology ; RNA, Guide, CRISPR-Cas Systems/genetics ; Saccharomyces cerevisiae/genetics ; Haploidy ; },
abstract = {Genome modification would be useful for developing breeding techniques for haploid Zygosaccharomyces rouxii and natural hybrid allodiploid Zygosaccharomyces sp. yeast strains used in miso and soy sauce production. In this study, genome editing using CRISPR-Cas9 was attempted in Zygosaccharomyces sp. strains. Based on techniques in Saccharomyces cerevisiae, the Cas9 gene and guide RNA (gRNA) were expressed from the same plasmid. Targeting of the ZygoLEU2 gene of haploid Z. rouxii strain DA2 led to of a single-nucleotide insertion in the ORF, resulting in termination of translation at 10 amino acids. This single-base insertion was 3-bp upstream of the protospacer-associated motif (PAM) sequence, suggesting that it occurred during the repair process following the Cas9-induced double-strand break. The transformant was auxotrophic for leucine, verifying that genome editing using CRISPR-Cas9 had occurred. Application of the CRISPR-Cas9 system to allodiploid Zygosaccharomyces sp. strains, which have T- and P-subgenomes, resulted in transformants with base insertions or deletions upstream of the PAM sequence, or insertions of different subgenome sequences. Leucine-auxotrophic transformants were obtained in which the ORF of the ZygoLEU2 gene in both subgenomes were mutated. In some genome-edited strains, a significant region of one subgenome chromosome was missing. Lastly, we applied CRISPR-Cas9 to the gene encoding Hog1, a protein kinase involved in adaptation to high-salt and high-osmolarity conditions. Mutation of the HOG1 genes of both the T- and P-subgenomes by CRISPR-Cas9 significantly reduced growth in high salt and high osmolarity conditions.},
}
@article {pmid40269250,
year = {2025},
author = {Zhao, K and Yan, Y and Jin, XK and Pan, T and Zhang, SM and Yang, CH and Rao, ZY and Zhang, XZ},
title = {An orally administered gene editing nanoparticle boosts chemo-immunotherapy in colorectal cancer.},
journal = {Nature nanotechnology},
volume = {20},
number = {7},
pages = {935-946},
pmid = {40269250},
issn = {1748-3395},
support = {52333004, 22135005//National Natural Science Foundation of China (National Science Foundation of China)/ ; },
mesh = {*Colorectal Neoplasms/therapy/genetics/pathology/drug therapy/immunology ; *Gene Editing/methods ; Humans ; Animals ; *Nanoparticles/chemistry/administration &amp; dosage ; Mice ; CRISPR-Cas Systems ; *Immunotherapy/methods ; Administration, Oral ; Cell Line, Tumor ; Drug Resistance, Neoplasm/drug effects/genetics ; Tumor Microenvironment/drug effects ; HSP90 Heat-Shock Proteins ; },
abstract = {Chemoresistance and immunosuppression are common obstacles to the efficacy of chemo-immunotherapy in colorectal cancer (CRC) and are regulated by mitochondrial chaperone proteins. Here we show that the disruption of the tumour necrosis factor receptor-associated protein 1 (TRAP1) gene, which encodes a mitochondrial chaperone in tumour cells, causes the translocation of cyclophilin D in tumour cells. This process results in the continuous opening of the mitochondrial permeability transition pore, which enhances chemotherapy-induced cell necrosis and promotes immune responses. On the basis of this discovery we developed an oral CRISPR-Cas9 delivery system based on zwitterionic and polysaccharide polymer-coated nanocomplexes that disrupts the TRAP1 gene in CRC. This system penetrates the intestinal mucus layer and undergoes epithelial transcytosis, accumulating in CRC tissues. It enhances chemotherapeutic efficacy by overcoming chemoresistance and activating the tumour immune microenvironment in orthotopic, chemoresistant and spontaneous CRC models, with remarkable synergistic antitumour effects. This oral CRISPR-Cas9 delivery system represents a promising therapeutic strategy for the clinical management of CRC.},
}
@article {pmid40265707,
year = {2025},
author = {Liu, S and Cao, Z and Lun, J and Zhang, C and Pan, L},
title = {Rapid detection of ACCase W2027C mutation in Leptochloa chinensis (L.) Nees using RAA-CRISPR/Cas12a: a sensitive and visual tool for herbicide resistance diagnosis.},
journal = {Pest management science},
volume = {81},
number = {8},
pages = {4769-4776},
doi = {10.1002/ps.8840},
pmid = {40265707},
issn = {1526-4998},
support = {2023YFD1401100//National Key R&amp;D Program of China/ ; 2025JJ60124//Natural Science Foundation of Hunan Province/ ; 24B0231//Scientific Research Fund of Hunan Provincial Education Department/ ; CARS-01//Earmarked Fund for China Agriculture Research System/ ; CARS-16-E19//Earmarked Fund for China Agriculture Research System/ ; 2022-31//Modern Agricultural Industrial Technology System of Hunan Province/ ; },
mesh = {*Herbicide Resistance/genetics ; *CRISPR-Cas Systems ; Mutation ; *Acetyl-CoA Carboxylase/genetics/metabolism ; *Herbicides/pharmacology ; *Plant Proteins/genetics/metabolism ; *Plant Weeds/genetics/drug effects ; *Nucleic Acid Amplification Techniques/methods ; },
abstract = {BACKGROUND: Prolonged application of ACCase-inhibiting herbicides, such as cyhalofop-butyl, has resulted in the widespread emergence of herbicide-resistant Leptochloa chinensis populations in paddy fields. Among the numerous target-site resistance (TSR) mutations in ACCase, the Trp-2027-Cys (W2027C) mutation is one of the most prevalent in L. chinensis. The increasing prevalence of herbicide-resistant L. chinensis highlights the critical need for rapid and precise diagnostic tools to detect resistance mutations.<br><br>RESULTS: In this study, we present a novel detection system that integrates Recombinase-Aided Amplification (RAA) with CRISPR/Cas12a technology to specifically target the W2027C mutation in the ACCase gene of L. chinensis. The system exhibits high sensitivity, capable of detecting DNA concentrations as low as 2-200&#x2009;fg/&#x3bc;L, and high specificity, facilitating accurate and visual differentiation of resistant from susceptible plants, thereby offering significant potential for rapid field applications.<br><br>CONCLUSION: This report has described the application of the RAA-CRISPR system for mutation detection in herbicide resistant weeds, presenting a promising tool for integrated weed management and enabling more timely decision-making regarding herbicide application and resistance management. &#xa9; 2025 Society of Chemical Industry.},
}
@article {pmid40668918,
year = {2025},
author = {Valcárcel, G and Lazarenkov, A and López-Rubio, AV and Berenguer, C and Calafell-Segura, J and Rodríguez-Ubreva, J and Ballestar, E and Sardina, JL},
title = {Modulating immune cell fate and inflammation through CRISPR-mediated DNA methylation editing.},
journal = {Science advances},
volume = {11},
number = {29},
pages = {eadt1644},
doi = {10.1126/sciadv.adt1644},
pmid = {40668918},
issn = {2375-2548},
mesh = {*DNA Methylation/genetics ; Humans ; *Gene Editing/methods ; *Inflammation/genetics/immunology ; *CRISPR-Cas Systems ; Promoter Regions, Genetic ; DNA Methyltransferase 3A ; Cell Differentiation/genetics ; Epigenesis, Genetic ; DNA (Cytosine-5-)-Methyltransferases/genetics ; Gene Expression Regulation ; Myeloid Cells/metabolism/immunology ; Proto-Oncogene Proteins/genetics ; Mixed Function Oxygenases ; Interleukin 1 Receptor Antagonist Protein ; },
abstract = {Immune cell differentiation and activation are associated with widespread DNA methylation changes; however, the causal relationship between these changes and their impact in shaping cell fate decisions still needs to be fully elucidated. Here, we conducted a genome-wide analysis to investigate the relationship between DNA methylation and gene expression at gene regulatory regions in human immune cells. By using CRISPR-dCas9-TET1 and -DNMT3A epigenome editing tools, we successfully established a cause-and-effect relationship between the DNA methylation levels of the promoter of the interleukin-1 receptor antagonist (IL1RN) gene and its expression. We observed that modifying the DNA methylation status of the IL1RN promoter is sufficient to alter human myeloid cell fate and change the cellular response to inflammatory and pathogenic stimuli. Collectively, our findings demonstrate the potential of targeting specific DNA methylation events to directly modulate immune and inflammatory responses, providing a proof of principle for intervening in a broad range of inflammation-related diseases.},
}
@article {pmid40668108,
year = {2025},
author = {Yang, X and Fan, Y and Xu, X and Shen, T and An, X and Zhang, Y and Zhang, Z and Pan, H and Chang, D},
title = {Direct Testing of Blood Samples to Diagnose Bloodstream Infections.},
journal = {ACS infectious diseases},
volume = {},
number = {},
pages = {},
doi = {10.1021/acsinfecdis.5c00109},
pmid = {40668108},
issn = {2373-8227},
abstract = {Bloodstream infection (BSI) is a critical condition with extremely high mortality. Rapid and accurate diagnosis is crucial for effective treatment. The traditional blood culture (BC) method has issues, such as long testing times and limited sensitivity, making it challenging to meet the need for timely diagnosis. To address this problem, various molecular biology methods for directly detecting blood samples (whole blood, plasma, serum, and positive BC samples) have emerged. These include Raman spectroscopy, mass spectrometry, nucleic acid amplification, and hybridization techniques (such as the CRISPR/Cas system, digital droplet PCR (ddPCR), and T2 magnetic resonance (T2MR)), biosensors, and next-generation sequencing (NGS). These methods can quickly identify pathogens and their drug-resistant markers, significantly reducing diagnostic delays and helping to provide earlier targeted treatment. This article systematically analyzes the principles, advantages, and disadvantages of these advanced techniques, explores their value in revolutionizing the BSI diagnostic model, and looks ahead to future development directions, providing a reference for research and clinical applications in this field.},
}
@article {pmid40667754,
year = {2025},
author = {Wu, X and Luteijn, RD and Lozano-Andrés, E and Marougka, K and Li, W and Narimatsu, Y and van Kuppeveld, FJM and Bosch, BJ and Lebbink, RJ and Vries, E and de Haan, CAM},
title = {Identification of β4GALNT2 as an anti-hPIV3 factor through genome-wide CRISPR/Cas9 library screening.},
journal = {Emerging microbes &amp; infections},
volume = {14},
number = {1},
pages = {2529895},
doi = {10.1080/22221751.2025.2529895},
pmid = {40667754},
issn = {2222-1751},
mesh = {Humans ; *CRISPR-Cas Systems ; *N-Acetylgalactosaminyltransferases/genetics/metabolism ; *Parainfluenza Virus 3, Human/physiology/genetics ; *Respirovirus Infections/virology/genetics ; Polypeptide N-acetylgalactosaminyltransferase ; *Host-Pathogen Interactions ; Glycosylation ; Polysaccharides/metabolism ; Animals ; HEK293 Cells ; },
abstract = {Human respirovirus 3 (also known as human parainfluenza virus 3; hPIV3) is a major cause of severe acute respiratory infections in vulnerable populations. Here we conducted a genome-wide CRISPR/Cas9 library screen to identify key host factors for hPIV3 infection. In addition to identifying several host proteins involved in glycosylation as proviral factors, we identified β-1,4-N-Acetyl-Galactosaminyltransferase 2 (β4GALNT2) as a potent restriction factor. Further investigation demonstrated that the addition of a GalNAc residue to α2-3-sialylated glycans by β4GALNT2, resulting in the Sd[a] glycotope, disrupted the interaction between the viral hemagglutinin-neuraminidase (HN) attachment protein and sialoglycan receptors. Specifically, the additional GalNAc residue interfered with the interaction of residue W371 in HN with sub-terminal glycan moieties. β4GALNT2-mediated Sd[a] epitope expression also negatively affected infection by other respiroviruses, with the strongest effect being observed for hPIV3.},
}
@article {pmid40665763,
year = {2025},
author = {Shao, F and Zhou, Y and Shi, J and Zhang, M and Yang, H and Fei, J},
title = {Establishment and Application of Mice Models for Tracing Gene Expression and Protein Product of TNF.},
journal = {Biotechnology journal},
volume = {20},
number = {7},
pages = {e70080},
doi = {10.1002/biot.70080},
pmid = {40665763},
issn = {1860-7314},
support = {2019YFA0905900//National Key Research and Development Program of China/ ; 19DZ2280500//Science and Technology Commission of Shanghai Municipality/ ; 18DZ2293500//Science and Technology Commission of Shanghai Municipality/ ; 18DZ2290700//Science and Technology Commission of Shanghai Municipality/ ; },
mesh = {Animals ; Mice ; *Tumor Necrosis Factor-alpha/genetics/metabolism ; Mice, Transgenic ; Disease Models, Animal ; Lipopolysaccharides ; Genes, Reporter ; CRISPR-Cas Systems ; Promoter Regions, Genetic ; Luciferases/genetics/metabolism ; Sepsis/genetics/chemically induced/metabolism ; Dexamethasone/pharmacology ; Gene Expression ; Inflammation/genetics ; },
abstract = {Tumor necrosis factor α (TNF-α) is a pleiotropic cytokine crucial for immune function, cellular homeostasis, and disease progression, yet its complex roles in vivo remain unclear. Challenges in studying TNF-α include its widespread gene expression, variability in expression levels, diverse protein forms, and low baseline expression, which complicate traditional detection and tracking methods. To address these challenges, we constructed three distinct transgenic luciferase reporter mouse models (TNF-IRES-Luc, TNF-Nanoluc, and TNF-HiBiT) driven by the endogenous TNF-α gene, using CRISPR/Cas9 technology through homology-directed repair. The firefly luciferase gene, secreted NanoLuc gene, and HiBiT gene were individually integrated into the mouse genome under the control of the endogenous TNF-α promoter. Our results demonstrate that endogenous TNF-α expression can be effectively monitored by measuring luciferase levels in vivo and in vitro, using an in vivo imaging system and a luminometer. This was validated during inflammatory processes such as lipopolysaccharide (LPS)-induced sepsis and phorbol ester (TPA)-induced mouse ear edema. Furthermore, the anti-inflammatory drug dexamethasone (DXM) significantly inhibited TNF-α and luciferase expression in both inflammatory models. Our study demonstrates these mouse models are valuable tools for studying TNF-α expression in inflammatory responses and related diseases, as well as evaluating anti-inflammatory drug efficacy.},
}
@article {pmid40665149,
year = {2025},
author = {Wongborisuth, C and Innachai, P and Saisawang, C and Tubsuwan, A and Jearawiriyapaisarn, N and Kaewprommal, P and Piriyapongsa, J and Chiangjong, W and Anurathapan, U and Songdej, D and Tangprasittipap, A and Hongeng, S},
title = {Disrupting ZBTB7A or BCL11A binding sites reactivates fetal hemoglobin in erythroblasts from healthy and β[0]-thalassemia/HbE individuals.},
journal = {Scientific reports},
volume = {15},
number = {1},
pages = {25580},
pmid = {40665149},
issn = {2045-2322},
support = {Grant ID: BRF1-016/2565//Basic Research Fund: the fiscal year 2022 , Mahidol University/ ; Grant ID: RF_64041//Faculty of Medicine Ramathibodi Hospital, Mahidol University/ ; Grant ID: MRC-IM 02/2565//Mahidol University/ ; },
mesh = {Humans ; *Fetal Hemoglobin/metabolism/genetics ; *beta-Thalassemia/genetics/metabolism ; *Erythroblasts/metabolism ; Binding Sites ; *Transcription Factors/metabolism/genetics ; *DNA-Binding Proteins/metabolism/genetics ; CRISPR-Cas Systems ; Gene Editing ; Repressor Proteins/metabolism/genetics ; Promoter Regions, Genetic ; gamma-Globins/genetics ; *Hemoglobin E/metabolism/genetics ; *Carrier Proteins/metabolism/genetics ; Hematopoietic Stem Cells/metabolism ; *Nuclear Proteins/metabolism/genetics ; },
abstract = {CRISPR/Cas9 genome editing has emerged as a promising treatment for genetic diseases like β-thalassemia. Editing γ-globin promoters to disrupt ZBTB7A/LRF or BCL11A binding sites has shown potential for reactivating fetal hemoglobin and treating sickle cell disease. However, its application to β[0]-thalassemia/HbE disease remains unclear. This study utilized CRISPR/Cas9 to disrupt these sites in mobilized CD34 + hematopoietic stem /progenitor cells from healthy donors and β[0]-thalassemia/HbE patients. The editing efficiency for the BCL11A site (75-92%) was higher than for the ZBTB7A/LRF site (57-60%). Both disruptions similarly increased fetal hemoglobin production in healthy donors (BCL11A 26.2 ± 1.4%, ZBTB7A/LRF 27.9 ± 1.5%) and β[0]-thalassemia/HbE cells (BCL11A 62.7 ± 0.9%, ZBTB7A/LRF 64.0 ± 1.6%). Off-target effects were absent in BCL11A-edited cells but observed at low frequencies in ZBTB7A/LRF-edited cells. Neither disruption significantly affected erythroid differentiation. These findings highlight the comparable contributions of ZBTB7A/LRF and BCL11A binding sites to γ-globin reactivation. CRISPR/Cas9 editing of either site may offer a potential therapeutic strategy for β[0]-thalassemia/HbE disease.},
}
@article {pmid40242936,
year = {2025},
author = {Guler Kara, H and Dogan, E and Bozok, V and Aktan, C and Cakiroglu, E and Eroglu, Z and Aygunes Jafari, D and Korkmaz, KS and Senturk, S and Kosova, B},
title = {The G protein-coupled receptor GPR89A is a novel potential therapeutic target to overcome cisplatin resistance in NSCLC Calu1 cells.},
journal = {The FEBS journal},
volume = {292},
number = {14},
pages = {3755-3770},
doi = {10.1111/febs.70099},
pmid = {40242936},
issn = {1742-4658},
support = {TUB1//T&#xfc;rkiye Bilimsel ve Teknolojik Ara&#x15f;t&#x131;rma Kurumu/ ; TOA-2021-22673//Ege University Research Foundation/ ; },
mesh = {Humans ; *Cisplatin/pharmacology ; *Drug Resistance, Neoplasm/genetics/drug effects ; *Carcinoma, Non-Small-Cell Lung/drug therapy/genetics/pathology/metabolism ; *Receptors, G-Protein-Coupled/genetics/metabolism/antagonists &amp; inhibitors ; *Lung Neoplasms/drug therapy/genetics/pathology/metabolism ; Cell Line, Tumor ; CRISPR-Cas Systems ; Apoptosis/drug effects ; Antineoplastic Agents/pharmacology ; Gene Expression Regulation, Neoplastic/drug effects ; Cell Proliferation/drug effects ; },
abstract = {Lung cancer is the most frequently diagnosed cancer type worldwide and is characterised by its high metastatic potential. Standard therapy for nonsmall cell lung cancer (NSCLC) cases includes chemotherapy with the platinum-based chemotherapeutic agent cisplatin. Although lung cancer cases respond well to cisplatin at the beginning of treatment, ~ 60% develop chemotherapy resistance during this process. In this study, a genome-wide CRISPR-Cas9-based genetic screening approach was employed to identify genes that cisplatin-resistant NSCLC Calu1 cells are more addicted to than sensitive cells. Cisplatin-resistant Calu1 cells were generated by the dose escalation method, and genome-wide CRISPR-Cas9-based genetic screening was performed with the Brunello CRISPR knockout library. Bioinformatics analyses of the obtained next-generation sequencing data revealed 63 potential candidate genes responsible for cisplatin resistance, including G protein-coupled receptor 89A (GPR89A), Poly(U) binding splicing factor 60 (PUF60), NBAS subunit of NRZ tethering complex (NBAS) and GrpE like 1, mitochondrial (GRPEL1). The GPR89A protein is located in the Golgi cisterna and Golgi-associated vesicle membrane, enables voltage-gated anion channel activity, and is involved in intracellular pH reduction. Functional studies carried out with GPR89A-knockout cisplatin-resistant Calu1 cells resulted in cell cycle arrest in the G2/M phase and increased polyploidy, and also prevented colony formation and cell migration. Cisplatin treatment, on the other hand, resulted in increased cell death by apoptosis upon cell cycle arrest in the S phase. In conclusion, this is the first study that identified GPR89A as a potential therapeutic target to overcome cisplatin resistance in NSCLC Calu1 cells.},
}
@article {pmid40664931,
year = {2025},
author = {Besati, M and Safarnejad, MR and Aliahmadi, A and Farzaneh, M and Ruiz, R and Montagud-Martínez, R and Rodrigo, G and Rafati, H},
title = {Detection of tomato brown rugose fruit virus through CRISPR-Cas12a and CRISPR-Cas9 systems.},
journal = {Scientific reports},
volume = {15},
number = {1},
pages = {25638},
pmid = {40664931},
issn = {2045-2322},
mesh = {*Solanum lycopersicum/virology ; *CRISPR-Cas Systems ; *Plant Diseases/virology ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {Tomato brown rugose fruit virus (ToBRFV) is a single-stranded positive-sense RNA virus that targets tomato and pepper plants and is causing significant damage to crops in some regions of the world. ToBRFV is a highly contagious virus that is stable and rapidly spreads by mechanical methods and seeds. As a result, it may spread both locally and over long distances, and it is now recognized as a pandemic in plants. This study investigates the effectiveness of the systems CRISPR-Cas12a and CRISPR-Cas9, in conjugation with recombinase polymerase amplification (RPA), to detect ToBRFV in tomato plant samples collected from the field. The trans-cleavage activity of both nucleases, Cas12a and Cas9, was exploited to process a probe labelled with fluorescein and biotin to be resolved on a lateral flow device, thereby enabling a visual readout. We were able to detect the RNA genome of the virus in about 1 h at a low constant temperature. These results could pave way to offer a rapid, sensitive, and specific method for on-site detection of ToBRFV.},
}
@article {pmid40664653,
year = {2025},
author = {Chen, H and Tan, Q and Li, L and Li, L and Fu, J and Zhu, W and Li, J and Wang, Y and Li, S and Li, H and Sun, Y and Sun, Q and Lu, Z and Liu, Z},
title = {Refined DNA repair manipulation enables a universal knock-in strategy in mouse embryos.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {6502},
pmid = {40664653},
issn = {2041-1723},
mesh = {Animals ; *Gene Knock-In Techniques/methods ; Mice ; *Embryo, Mammalian/metabolism ; CRISPR-Cas Systems/genetics ; *DNA Repair/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; DNA End-Joining Repair/genetics ; Female ; DNA-Directed DNA Polymerase/genetics/metabolism ; Gene Editing/methods ; DNA Breaks, Double-Stranded ; },
abstract = {The design and screening of sgRNA in CRISPR-dependent gene knock-in is always laborious. Therefore, a universal and highly efficient knock-in strategy suitable for different sgRNA target sites is necessary. In our mouse embryo study, we find that the knock-in efficiency guided by adjacent sgRNAs varies greatly, although similar indel frequency. MMEJ-biased sgRNAs usually lead to high knock-in efficiency, whereas NHEJ-biased sgRNAs result in low knock-in efficiency. Blocking MMEJ repair by knocking down Polq can enhance knock-in efficiency, but inhibiting NHEJ repair shows variable effects. We identify a compound, AZD7648, that can shift DSBs repair towards MMEJ. Finally, by combining AZD7648 treatment with Polq knockdown, we develop a universal and highly efficient knock-in strategy in mouse embryos. This approach is validated at more than ten genomic loci, achieving up to 90% knock-in efficiency, marking a significant advancement toward predictable and highly efficient CRISPR-mediated gene integration.},
}
@article {pmid40664139,
year = {2025},
author = {Lau, CH and Liang, QL and Chen, X and Li, J and Shi, Y and Huang, Y and Xia, Q and Zhu, H and Chen, G},
title = {CRISPR/Dx-based colorimetric and electrochemical detection systems for POCT applications.},
journal = {Biosensors &amp; bioelectronics},
volume = {288},
number = {},
pages = {117778},
doi = {10.1016/j.bios.2025.117778},
pmid = {40664139},
issn = {1873-4235},
abstract = {To realize point-of-care testing (POCT) for human diseases, CRISPR/Cas technology has been integrated with commercial analytical devices and chromogenic substrates such as personal glucose meter (PGM), pregnancy test strips (PTS), and horseradish peroxidase (HRP). These integrated detection platforms enable portable diagnostics, economical, universal, rapid, and high throughput detection. The change in color and electrochemical signal enables visible signal readout and intuitive results without specialized equipment and skillful operators. Herein, we comprehensively and critically discuss these advancements and describe the molecular principles of each detection system. The current challenges and technical shortcomings associated with these CRISPR-based diagnostics for POCT applications are discussed. We also provide future perspectives and directions in this field that will improve the performance of CRISPR-PGM, CRISPR-PTS, and CRISPR-HRP for pathogens detection and molecular diagnostics. Their applications in early tumor detection and pan-cancer screening are highlighted. The potential ethical and societal impact of home self-tests for cancer are discussed.},
}
@article {pmid40663463,
year = {2025},
author = {Carmichael, JC and Stevens, CS and Atanasoff, KE and Kowdle, S and Reis, RA and Tortorella, D and Lee, B},
title = {Precision engineering of human cytomegalovirus without BAC constraints: a Sendai virus-delivered CRISPR/Cas9 approach.},
journal = {The Journal of general virology},
volume = {106},
number = {7},
pages = {},
doi = {10.1099/jgv.0.002126},
pmid = {40663463},
issn = {1465-2099},
mesh = {Humans ; *Cytomegalovirus/genetics ; *CRISPR-Cas Systems ; *Sendai virus/genetics ; *Chromosomes, Artificial, Bacterial/genetics ; *Gene Editing/methods ; Genome, Viral ; Fibroblasts/virology ; Genetic Vectors/genetics ; Epithelial Cells/virology ; Cell Line ; },
abstract = {Human cytomegalovirus (HCMV) genetic manipulation traditionally relies on bacterial artificial chromosome (BAC) recombineering, necessitated by its large ~236 kb genome. This approach is limited by the scarcity of HCMV strains engineered into BACs and often requires the deletion of 'non-essential' genes to accommodate the BAC cassette. We developed a novel approach using temperature-sensitive Sendai virus (SeV) vectors to deliver CRISPR/Cas9 for targeted HCMV genome editing without these constraints. This system achieves high editing efficiency (80-90%) in fibroblasts, epithelial cells and endothelial cells without BAC intermediates. As proof of principle, we targeted the HCMV (TB40/E strain) pentamer complex (PC) genes UL128 and UL130, crucial for viral entry into non-fibroblast cells. Edited viruses showed significantly reduced infectivity in epithelial cells, confirming functional disruption of the PC. Plaque purification yielded isogenic clones with phenotypes comparable to AD169, a naturally PC-deficient strain. Furthermore, multiplexed editing created precise 663 bp deletions in over 60% of viral genomes. Importantly, this method enables HCMV editing in physiologically relevant cell types without fibroblast passaging, which typically introduces mutations. This SeV-Cas9 system represents a significant advancement for studying HCMV biology in diverse cell types.},
}
@article {pmid40663383,
year = {2025},
author = {Tao, S and Fang, Y and Zheng, L and Zhang, H and Xu, Y and Liang, W},
title = {Mechanistic study of the immune defense function of the CRISPR1-Cas system in Enterococcus faecalis.},
journal = {Virulence},
volume = {16},
number = {1},
pages = {2530665},
doi = {10.1080/21505594.2025.2530665},
pmid = {40663383},
issn = {2150-5608},
mesh = {*Enterococcus faecalis/genetics/immunology/drug effects ; *CRISPR-Cas Systems ; Plasmids/genetics ; Gene Transfer, Horizontal ; Mutation ; Drug Resistance, Bacterial/genetics ; Transformation, Bacterial ; },
abstract = {Enterococci are Gram-positive cocci that are considered to be one of the causative agents of hospital-acquired infections. CRISPR-Cas is an adaptive immune system with targeted defense functions against foreign invading nucleic acids and plays an important role in antibiotic resistance. In this study, we aimed to investigate II-A CRISPR-Cas-mediated immunity and the molecular mechanism underlying the horizontal transfer of drug resistance genes in Enterococcus faecalis. The mutant strains were constructed by the homologous recombination strategy. The interference of plasmid transformation by the Enterococcus faecalis CRISPR1/Cas system was confirmed through plasmid transformation efficiency. The different mutation positions in the protospacer sequence S1 and PAM region recombinant plasmids were constructed through enzyme digestion and sequencing verification to assess the impact of the CRISPR-encoded immunity. In the wild-type strain, the transformation efficiency of plasmids pAT28-S1-S9 containing protospacers and PAM sites decreased (p < 0.05). Single-base mutations at positions 25 and 28 of the protospacer region eliminated the ability of the wild-type strain to prevent plasmid transformation containing the protospacer and PAM sites (p > 0.05), whereas a single mismatch at protospacer positions 2,10,18,23 did not affect the ability of CRISPR-Cas system-positive strains to interfere with plasmid transformation (p < 0.05). There was no significant difference between the wild-type strain and the mutant strain in the transformation efficiency of the pS1-pΔPAM plasmid without PAM and plasmids containing single mutations (p > 0.05). In conclusion, the CRISPR-Cas system can block the transformation of matching protospacer sequences, and mutations near or within the protospacer adjacent motif (PAM) allow the plasmid to escape CRISPR-encoded immunity.},
}
@article {pmid40663257,
year = {2025},
author = {Yerlikaya, BA and Yerlikaya, S and Gül, B and Yoldaş, H and Kavas, M and Mohamed, HI},
title = {Harnessing CRISPR/Cas9 in engineering biotic stress immunity in crops.},
journal = {Planta},
volume = {262},
number = {3},
pages = {54},
pmid = {40663257},
issn = {1432-2048},
mesh = {*CRISPR-Cas Systems/genetics ; *Crops, Agricultural/genetics/immunology ; Gene Editing/methods ; *Stress, Physiological/genetics ; Disease Resistance/genetics ; Plant Diseases/immunology/genetics ; Plants, Genetically Modified/genetics ; Genetic Engineering/methods ; },
abstract = {There is significant potential for CRISPR/Cas9 to be used in developing crops that can adapt to biotic stresses such as fungal, bacterial, viral, and pest infections and weeds. The increasing global population and climate change present significant threats to food security by putting stress on plants, making them more vulnerable to diseases and productivity losses caused by pathogens, pests, and weeds. Traditional breeding methods are inadequate for the rapid development of new plant traits needed to counteract this decline in productivity. However, modern advances in genome-editing technologies, particularly CRISPR/Cas9, have transformed crop protection through precise and targeted modifications of plant genomes. This enables the creation of resilient crops with improved resistance to pathogens, pests, and weeds. This review examines various methods by which CRISPR/Cas9 can be utilized for crop protection. These methods include knocking out susceptibility genes, introducing resistance genes, and modulating defense genes. Potential applications of CRISPR/Cas9 in crop protection involve introducing genes that confer resistance to pathogens, disrupting insect genes responsible for survival and reproduction, and engineering crops that are resistant to herbicides. In conclusion, CRISPR/Cas9 holds great promise for advancing crop protection and ensuring food security in the face of environmental challenges and increasing population pressures. The most recent advancements in CRISPR technology for creating resistance to bacteria, fungi, viruses, and pests are covered here. We wrap up by outlining the most pressing issues and technological shortcomings, as well as unanswered questions for further study.},
}
@article {pmid40662362,
year = {2025},
author = {Maillard, M and Nishii, R and Vu, HS and Bhattarai, KR and Yang, W and Li, J and Hofmann, U and Savic, D and Bhatia, S and Schwab, M and Ni, M and Yang, JJ},
title = {The NUDIX hydrolase NUDT5 regulates thiopurine metabolism and cytotoxicity.},
journal = {The Journal of clinical investigation},
volume = {135},
number = {14},
pages = {},
doi = {10.1172/JCI190443},
pmid = {40662362},
issn = {1558-8238},
mesh = {Humans ; *Pyrophosphatases/genetics/metabolism ; *Mercaptopurine/pharmacology ; Cell Line, Tumor ; CRISPR-Cas Systems ; *Neoplasm Proteins/genetics/metabolism ; Drug Resistance, Neoplasm ; Nudix Hydrolases ; },
abstract = {Thiopurines are anticancer agents used for the treatment of leukemia and autoimmune diseases. These purine analogs are characterized by a narrow therapeutic index because of the risk of myelosuppression. With the discovery of NUDIX hydrolase 15 (NUDT15) as a major modulator of thiopurine metabolism and toxicity, we sought to comprehensively examine all members of the NUDIX hydrolase family for their effect on the pharmacologic effects of thiopurine. By performing a NUDIX-targeted CRISPR/Cas9 screen in leukemia cells, we identified NUDT5, whose depletion led to drastic thiopurine resistance. NUDT5 deficiency resulted in a nearly complete depletion of active metabolites of thiopurine and the loss of thioguanine incorporation into DNA. Mechanistically, NUDT5 deletion resulted in substantial alteration in purine nucleotide biosynthesis, as determined by steady-state metabolomics profiling. Stable isotope tracing demonstrated that the loss of NUDT5 was linked to a marked suppression of the purine salvage pathway but with minimal effects on purine de novo synthesis. Finally, we comprehensively identified germline genetic variants in NUDT5 associated with thiopurine-induced myelosuppression in 582 children with acute lymphoblastic leukemia. Collectively, these results pointed to NUDT5 as a key regulator of the thiopurine response primarily through its effects on purine homeostasis, highlighting its potential to inform individualized thiopurine therapy.},
}
@article {pmid40661867,
year = {2025},
author = {Mestanza, M and Hernández-Amasifuen, AD and Pineda-Lázaro, AJ and Eriksson, D and Guerrero-Abad, JC},
title = {Genome editing for sustainable agriculture in Peru: advances, potential applications and regulation.},
journal = {Frontiers in genome editing},
volume = {7},
number = {},
pages = {1611040},
pmid = {40661867},
issn = {2673-3439},
abstract = {Peruvian agriculture is characterize by crops such as potato, maize, rice, asparagus, mango, banana, avocado, cassava, onion, oil palm, chili, papikra, blueberry, coffee, cacao, grapes, quinoa, olive, citrus and others. All of them have challenges in production in their specific agroecosystems under stress due to pests, diseases, salinity, drought, cold among others. Gene editing through CRISPR/Cas is a key tool for addressing critical challenges in agriculture by improving resilience to biotic and abiotic stress, increasing yield and enhancing the nutritional value of the crops. This approach allows precise mutation on site-specific gene at the DNA level, obtaining desirable traits when its function is altered. The CRISPR/Cas system could be used as a transgene-free genome editing tool when the ribonucleoprotein (RNP) acts as a carrier to delivered the CRISPR/Cas components into the plant cell protoplasts, or when the tRNA-like sequence (TLS) motifs are fused to single-guide RNA (sgRNA) and Cas mRNA sequence and expressed in transgenic plants rootstock to produce "mobile" CRISPR/Cas components to upper tissue (scion). Those innovations could be a potential approach to strengthen the Peruvian agriculture, food security and gricultural economy, especially in the tropical, Andean and coastal regions. This review article examines the advances and strategies of gene editing, focusing on transgene-free methodologies that could be adopted for research, development and use, and also identifies potential applications in key crops for Peru and analyzes their impact in the productivity and reduction of agrochemicals dependence. Finally, this review highlights the need to establish regulatory policies that strengthen the use of biotechnological precise innovations, ensuring the conservation and valorization of agrobiodiversity for the benefit of Peruvian farmers.},
}
@article {pmid40661507,
year = {2025},
author = {Shentu, TP and Wu, T and Zhou, Z and Yeh, CF and Zhu, J and Li, J and Miao, BA and Lee, TH and Zhang, L and Huang, RT and Harrison, D and Hodonsky, CJ and Auguste, G and Husain, A and Tirrell, MV and Miller, CL and Dickinson, B and Yang, KC and Fang, Y},
title = {Mechanosensitive Endothelial METTL7A Regulates Internal m [7] G mRNA Methylation and Protects Against Atherosclerosis.},
journal = {bioRxiv : the preprint server for biology},
volume = {},
number = {},
pages = {},
doi = {10.1101/2025.05.22.655328},
pmid = {40661507},
issn = {2692-8205},
abstract = {BACKGROUND: Internal N [7] -methylguanosine (m [7] G) is a recently identified chemical modification of mammalian mRNA and a component of the epitranscriptome. While the epitranscriptome plays a key role in regulating RNA metabolism and cellular function, the specific contribution of internal m [7] G to cardiovascular health and disease remains unknown. Atherosclerosis preferentially develops at sites where disturbed blood flow activates endothelial cells, but whether internal m [7] G and its regulatory machinery influence endothelial mechanotransduction and atherogenesis is unclear.<br><br>METHODS: We integrated epitranscriptomic profiling, human tissue analysis, genetically modified mouse models, and targeted nanomedicine approaches to investigate the role of Methyltransferase-like protein 7A (METTL7A), a putative internal m [7] G methyltransferase, in regulating the flow-sensitive endothelial transcriptome and atherosclerosis. Vascular endothelial cells were subjected to well-defined athero-protective and athero-prone flow waveforms in vitro and in vivo . METTL7A function was assessed using RNA sequencing (RNA-seq), liquid chromatography-tandem mass spectrometry (LC-MS/MS), crosslinking immunoprecipitation sequencing (CLIP-seq), RNA stability assays, and a CRISPR-Cas-inspired RNA targeting system (CIRTS). METTL7A expression in human coronary arteries with and without atherosclerosis was evaluated by RNA-seq and immunostaining. In vivo atherosclerosis studies were conducted in both global and endothelial-specific Mettl7a1 knockout mice. Endothelial METTL7A expression was restored using cationic polymer-based nanoparticles delivering CDH5 promoter-driven METTL7A plasmids or VCAM1-targeted lipid nanoparticles delivering N1-methylpseudouridine (m&#xb9;&#x3a8;)-modified METTL7A mRNA.<br><br>RESULTS: Athero-protective unidirectional flow significantly induced METTL7A expression, which promoted internal m [7] G methylation of endothelial transcripts, while other major epitranscriptomic marks and cap-associated m [7] G were not affected by METTL7A. METTL7A preferentially binds to AG-enriched motifs in protein-coding mRNAs and plays a key role in regulating KLF4 and NFKBIA transcripts, enhancing their internal m [7] G and stability and supporting vascular homeostasis. In contrast, endothelial METTL7A expression was significantly reduced by disturbed blood flow and in human atherosclerotic lesions. Global or endothelial-specific loss of METTL7A exacerbated disturbed flow-induced atherosclerosis in mice, independent of serum lipid levels. Restoration of endothelial METTL7A, via nanoparticle-mediated plasmid or m [1] &#x3a8; mRNA delivery, markedly reduced lesion formation in Mettl7a1 [&#x207b;/&#x207b;] and ApoE [&#x207b;/&#x207b;] mice.<br><br>CONCLUSIONS: These findings establish METTL7A as a previously unrecognized mechanosensitive methyltransferase that maintains endothelial homeostasis by stabilizing key anti-inflammatory transcripts, KLF4 and NFKBIA, through internal m [7] G methylation. Loss of METTL7A disrupts endothelial function and accelerates atherogenesis in response to disturbed flow. Therapeutic restoration of endothelial METTL7A, via targeted nanoparticle-mediated gene or m [1] &#x3a8; mRNA delivery, significantly lessens atherosclerosis. Collectively, these results uncover a novel epitranscriptomic mechanism governing vascular health and position METTL7A as a promising target for precision nanomedicine in atherosclerotic cardiovascular disease.},
}
@article {pmid40661421,
year = {2025},
author = {Xiao, R and Hoffmann, FT and Xie, D and Wiegand, T and Palmieri, AI and Sternberg, SH and Chang, L},
title = {Structural basis of RNA-guided transcription by a dCas12f-σ [E] -RNAP complex.},
journal = {bioRxiv : the preprint server for biology},
volume = {},
number = {},
pages = {},
doi = {10.1101/2025.06.10.658880},
pmid = {40661421},
issn = {2692-8205},
abstract = {RNA-guided proteins have emerged as critical transcriptional regulators in both natural and engineered biological systems by modulating RNA polymerase (RNAP) and its associated factors [1-5] . In bacteria, diverse clades of repurposed TnpB and CRISPR-associated proteins repress gene expression by blocking transcription initiation or elongation, enabling non-canonical modes of regulatory control and adaptive immunity [1,6,7] . Intriguingly, a distinct class of nuclease-dead Cas12f homologs (dCas12f) instead activates gene expression through its association with unique extracytoplasmic function sigma factors (σ [E]) [8] , though the molecular basis has remained elusive. Here we reveal a novel mode of RNA-guided transcription initiation by determining cryo-electron microscopy structures of the dCas12f-σ [E] system from Flagellimonas taeanensis . We captured multiple conformational and compositional states, including the DNA-bound dCas12f-σ [E] -RNAP holoenzyme complex, revealing how RNA-guided DNA binding leads to σ [E] -RNAP recruitment and nascent mRNA synthesis at a precisely defined distance downstream of the R-loop. Rather than following the classical paradigm of σ [E] -dependent promoter recognition, these studies show that recognition of the -35 element is largely supplanted by CRISPR-Cas targeting, while the melted -10 element is stabilized through unusual stacking interactions rather than insertion into the typical recognition pocket. Collectively, this work provides high-resolution insights into an unexpected mechanism of RNA-guided transcription, expanding our understanding of bacterial gene regulation and opening new avenues for programmable transcriptional control.},
}
@article {pmid40660068,
year = {2025},
author = {Wang, D and Guo, Y and Liu, M and Liu, H},
title = {The function of TaWOX14 in wheat genetic transformation.},
journal = {Plant cell reports},
volume = {44},
number = {8},
pages = {176},
pmid = {40660068},
issn = {1432-203X},
support = {2021YFF1000203//Ministry of Science and Technology of the People's Republic of China/ ; 232300420189//Natural Science Foundation of Henan Province/ ; 242102111117//Henan Provincial Science and Technology Research Project/ ; },
mesh = {*Triticum/genetics ; *Transformation, Genetic ; *Plant Proteins/genetics/metabolism ; Gene Editing ; Plants, Genetically Modified ; CRISPR-Cas Systems/genetics ; Haploidy ; Gene Expression Regulation, Plant ; *Homeodomain Proteins/genetics/metabolism ; },
abstract = {Overexpression of TaWOX14 can significantly improve the genetic transformation and genome editing efficiencies of some wheat immature embryos, and TaPLD serves as a promising candidate gene for haploid induction in wheat. Genetic transformation and genome editing systems have the potential for accelerating the breeding process in crops. However, their effectiveness is often limited by the regeneration efficiency of explants. The WUSCHEL-related homeobox (WOX) family, a group of plant-specific transcription factors, plays a crucial role in somatic embryogenesis. In wheat, WOX genes are categorized into three clades: ancient, intermediate, and WUS. In this study, we systematically analyzed the function of the WUS clade genes in wheat transformation. Our results demonstrated that overexpression of TaWOX14 significantly improved genetic transformation efficiencies in several wheat genotypes, including Fielder, Kenong199, Zhengmai7698, and Yangmai13. Furthermore, by combining the CRISPR-Cas9 system with TaWOX14, we observed enhanced genome editing efficiency in the wheat variety Fielder. Additionally, we explored the potential of TaPLD as a candidate gene for haploid induction in wheat. Based on the amino acid sequence of Zea mays PHOSPHOLIPASE D3 (ZmPLD3), we edited its homologs in wheat and identified a TaPLD-edited plant with a ploidy level intermediate between haploid and hexaploid. Overall, these findings are expected to accelerate the wheat breeding process by improving genetic transformation efficiency and identifying a potential haploid induction gene. Future research will focus on further characterizing the mechanisms of TaWOX14 and TaPLD, and exploring their applications in breeding programs.},
}
@article {pmid40619644,
year = {2025},
author = {Zhong, J and Chen, Y and Dong, Y and Guo, C and Liu, Y},
title = {SPARC: An Orthogonal Cas12a/Cas13a Dual-Channel CRISPR Platform for Reliable SNV Identification and Mutation Confirmation.},
journal = {Analytical chemistry},
volume = {97},
number = {27},
pages = {14629-14636},
doi = {10.1021/acs.analchem.5c02141},
pmid = {40619644},
issn = {1520-6882},
mesh = {*CRISPR-Cas Systems/genetics ; Mutation ; *Polymorphism, Single Nucleotide ; *Endodeoxyribonucleases/genetics/metabolism ; Humans ; *CRISPR-Associated Proteins/genetics/metabolism ; *Bacterial Proteins/genetics/metabolism ; },
abstract = {Rapid and reliable detection of single nucleotide variants (SNVs) is essential for accurate pathogen diagnostics, genetic mutation screening, and personalized medicine. However, existing CRISPR-based nucleic acid detection platforms frequently suffer from ambiguous signal interpretation, specificity limitations, and complex assay workflows. Herein, we introduce SPARC (specific and precise mutation recognition with Cas12a/Cas13a), a novel orthogonal dual-channel CRISPR assay that significantly enhances the SNV detection reliability. SPARC integrates Acidaminococcus sp. Cas12a (AsCas12a), which specifically detects a conserved region as an internal reference, with our recently identified DNA-activated Leptotrichia buccalis Cas13a (LbuCas13a), which exhibits exceptionally high intrinsic SNV specificity without requiring engineered crRNA mismatches. The orthogonal design uniquely resolves the common diagnostic ambiguity between genuine SNVs and target absence. Combined with recombinase polymerase amplification (RPA) and T7 exonuclease digestion, the SPARC platform achieved a sensitivity as low as 1 aM. We demonstrated the platform's robust clinical applicability through successful detection and accurate differentiation of hepatitis B virus (HBV) and clinically significant YMDD resistance mutations. This work presents an innovative and versatile CRISPR-based solution, highlighting substantial potential for advancing clinical diagnostics and precision medicine.},
}
@article {pmid40590162,
year = {2025},
author = {Xia, L and Yin, J and Wang, Y and Gui, Y and Tong, J and Yin, W and Xiao, D and Mu, Y},
title = {Wide Dynamic Range Multiplex Digital Clustered Regularly Interspaced Short Palindromic Repeat Chip for Rapid Detection and Absolute Quantification of Nucleic Acids.},
journal = {ACS nano},
volume = {19},
number = {27},
pages = {24874-24883},
doi = {10.1021/acsnano.5c03382},
pmid = {40590162},
issn = {1936-086X},
mesh = {CRISPR-Cas Systems ; Limit of Detection ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; *Nucleic Acids/analysis/genetics ; *Lab-On-A-Chip Devices ; Animals ; Nucleic Acid Amplification Techniques ; },
abstract = {Rapid detection and accurate quantification of multiplex nucleic acids are crucial for infectious diagnosis and pathogen identification, yet they still represent a significant challenge that deserves further attention and investigation. Herein, this research developed a wide dynamic range multiplex digital clustered regularly interspaced short palindromic repeat (CRISPR) (WDRM-dCRISPR) chip and established a rapid, sensitive, multiplexed nucleic acid detection (RSMND) platform that can achieve absolute quantification of multiplex nucleic acids with a limit of detection of 90 copies/mL within 30 min. The chip integrates the rapid amplification capability of RPA, the specific cleavage activity of CRISPR/Cas12a, and the portability of microfluidics, enabling precise and reliable digital absolute quantification of multiplex nucleic acids. As a proof of concept, this chip allows for multiplex absolute quantification of foodborne pathogens with a linear dynamic range of 9 × 10[1] to 1.8 × 10[6] copies/mL (R[2] > 0.9999), high sensitivity, high specificity, and strong tolerance to background interference. The platform was successfully used for the analysis of foodborne pathogens in milk. Compared to qPCR, the RSMND platform exhibited superior accuracy and sensitivity in nucleic acid quantification. Overall, this study demonstrates that the platform provides a powerful tool for multiplex nucleic acid detection and holds significant potential for applications in point-of-care testing.},
}
@article {pmid40586733,
year = {2025},
author = {Liu, F and Chen, L and Zhu, L and Liu, J and Li, M and Chen, Y and Yang, G and Qu, L},
title = {Aptamer-Amplified and CRISPR-Cas12a-Assisted Dual-Mode PEC-SERS Biosensor for Ultrasensitive Detection of Inflammatory Bowel Disease Biomarkers.},
journal = {Analytical chemistry},
volume = {97},
number = {27},
pages = {14377-14387},
doi = {10.1021/acs.analchem.5c01481},
pmid = {40586733},
issn = {1520-6882},
mesh = {*Aptamers, Nucleotide/chemistry ; *Biosensing Techniques/methods ; Animals ; *Inflammatory Bowel Diseases/diagnosis/metabolism ; Biomarkers/analysis ; *Electrochemical Techniques/methods ; *Matrix Metalloproteinase 9/analysis ; Humans ; *CRISPR-Cas Systems ; Mice ; *Alkaline Phosphatase/analysis ; Spectrum Analysis, Raman/methods ; Limit of Detection ; *CRISPR-Associated Proteins/metabolism ; Bacterial Proteins ; Endodeoxyribonucleases ; },
abstract = {Inflammatory bowel disease (IBD) has proven to be a critical global health problem characterized by severe life-threatening complications; thus, the development of noninvasive, reliable, and cost-effective diagnostic methods remains an urgent clinical need. Herein, a novel photoelectrochemical (PEC) and surface-enhanced Raman scattering (SERS) dual-mode platform was successfully developed for ultrasensitive detection of IBD-associated biomarkers, matrix metalloproteinases-9 (MMP-9), and intestinal alkaline phosphatase (IAP). A bifunctional covalent organic frameworks/MXene-Au substrate was synthesized with excellent PEC and SERS properties. An aptamer-based amplification strategy was first employed for MMP-9 detection, which was also the basis for the detection of IAP. The magnetic bead-conjugated double-stranded DNA was then designed to generate activator DNA in the presence of IAP, which activated the trans-cleavage activity of the CRISPR-Cas12a system. The resultant Cas12a specifically cleaved the electrode-immobilized single-stranded DNA (ssDNA), triggering the release of methylene blue as a dual-signal reporter, thereby enabling synchronized PEC-SERS detection for MMP-9 and IAP. The biosensor exhibited a wide linear range with detection limits of 0.074 pg/mL (PEC) and 0.016 pg/mL (SERS) for MMP-9, and 0.38 pg/mL (PEC) and 0.16 pg/mL (SERS) for IAP, respectively. Significantly, clinical validation was performed using a murine IBD model and human intestinal inflammation specimens, confirming the practical utility of the PEC-SERS platform. This study establishes a robust dual-mode biosensing strategy with multicomponent detection, enabling advanced biological analysis and precision health monitoring.},
}
@article {pmid39375448,
year = {2025},
author = {Gu, J and Iyer, A and Wesley, B and Taglialatela, A and Leuzzi, G and Hangai, S and Decker, A and Gu, R and Klickstein, N and Shuai, Y and Jankovic, K and Parker-Burns, L and Jin, Y and Zhang, JY and Hong, J and Niu, X and Costa, JA and Pezet, MG and Chou, J and Chen, C' and Paiva, M and Snoeck, HW and Landau, DA and Azizi, E and Chan, EM and Ciccia, A and Gaublomme, JT},
title = {Mapping multimodal phenotypes to perturbations in cells and tissue with CRISPRmap.},
journal = {Nature biotechnology},
volume = {43},
number = {7},
pages = {1101-1115},
pmid = {39375448},
issn = {1546-1696},
support = {1DP2CA281605//U.S. Department of Health &amp; Human Services | NIH | National Cancer Institute (NCI)/ ; 5R01CA197774-04//U.S. Department of Health &amp; Human Services | NIH | National Cancer Institute (NCI)/ ; 5R01CA227450-04//U.S. Department of Health &amp; Human Services | NIH | National Cancer Institute (NCI)/ ; 1R21HG012639-01A1//U.S. Department of Health &amp; Human Services | NIH | National Human Genome Research Institute (NHGRI)/ ; 1R01HG012875-01//U.S. Department of Health &amp; Human Services | NIH | National Human Genome Research Institute (NHGRI)/ ; Lloyd J. Old STAR award//Cancer Research Institute (CRI)/ ; },
mesh = {Humans ; Phenotype ; *CRISPR-Cas Systems/genetics ; Cell Line, Tumor ; Breast Neoplasms/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Induced Pluripotent Stem Cells ; Mice ; },
abstract = {Unlike sequencing-based methods, which require cell lysis, optical pooled genetic screens enable investigation of spatial phenotypes, including cell morphology, protein subcellular localization, cell-cell interactions and tissue organization, in response to targeted CRISPR perturbations. Here we report a multimodal optical pooled CRISPR screening method, which we call CRISPRmap. CRISPRmap combines in situ CRISPR guide-identifying barcode readout with multiplexed immunofluorescence and RNA detection. Barcodes are detected and read out through combinatorial hybridization of DNA oligos, enhancing barcode detection efficiency. CRISPRmap enables in situ barcode readout in cell types and contexts that were elusive to conventional optical pooled screening, including cultured primary cells, embryonic stem cells, induced pluripotent stem cells, derived neurons and in vivo cells in a tissue context. We conducted a screen in a breast cancer cell line of the effects of DNA damage repair gene variants on cellular responses to commonly used cancer therapies, and we show that optical phenotyping pinpoints likely pathogenic patient-derived mutations that were previously classified as variants of unknown clinical significance.},
}
@article {pmid39349835,
year = {2025},
author = {Lei, X and Huang, A and Chen, D and Wang, X and Ji, R and Wang, J and Zhang, Y and Zhang, Y and Lu, S and Zhang, K and Chen, Q and Zhang, Y and Yin, H},
title = {Rapid generation of long, chemically modified pegRNAs for prime editing.},
journal = {Nature biotechnology},
volume = {43},
number = {7},
pages = {1156-1167},
pmid = {39349835},
issn = {1546-1696},
mesh = {Humans ; *Gene Editing/methods ; Ribonucleoproteins/genetics/metabolism ; *RNA, Guide, CRISPR-Cas Systems/genetics ; CRISPR-Cas Systems/genetics ; HEK293 Cells ; },
abstract = {The editing efficiencies of prime editing (PE) using ribonucleoprotein (RNP) and RNA delivery are not optimal due to the challenges in solid-phase synthesis of long PE guide RNA (pegRNA) (>125 nt). Here, we develop an efficient, rapid and cost-effective method for generating chemically modified pegRNA (125-145 nt) and engineered pegRNA (epegRNA) (170-190 nt). We use an optimized splint ligation approach and achieve approximately 90% production efficiency for these RNAs, referred to as L-pegRNA and L-epegRNA. L-epegRNA demonstrates enhanced editing efficiencies across various cell lines and human primary cells with improvements of up to more than tenfold when using RNP delivery and several hundredfold with RNA delivery of PE, compared to epegRNA produced by in vitro transcription. L-epegRNA-mediated RNP delivery also outperforms plasmid-encoded PE in most comparisons. Our study provides a solution to obtaining high-quality pegRNA and epegRNA with desired chemical modifications, paving the way for the use of PE in therapeutics and various other fields.},
}
@article {pmid39134754,
year = {2025},
author = {Karasu, ME and Toufektchan, E and Chen, Y and Albertelli, A and Cullot, G and Maciejowski, J and Corn, JE},
title = {Removal of TREX1 activity enhances CRISPR-Cas9-mediated homologous recombination.},
journal = {Nature biotechnology},
volume = {43},
number = {7},
pages = {1168-1176},
pmid = {39134754},
issn = {1546-1696},
mesh = {Humans ; *Exodeoxyribonucleases/genetics/metabolism ; *CRISPR-Cas Systems/genetics ; *Phosphoproteins/genetics/metabolism ; *Homologous Recombination/genetics ; Gene Editing/methods ; },
abstract = {CRISPR-Cas9-mediated homology-directed repair (HDR) can introduce desired mutations at targeted genomic sites, but achieving high efficiencies is a major hurdle in many cell types, including cells deficient in DNA repair activity. In this study, we used genome-wide screening in Fanconi anemia patient lymphoblastic cell lines to uncover suppressors of CRISPR-Cas9-mediated HDR. We found that a single exonuclease, TREX1, reduces HDR efficiency when the repair template is a single-stranded or linearized double-stranded DNA. TREX1 expression serves as a biomarker for CRISPR-Cas9-mediated HDR in that the high TREX1 expression present in many different cell types (such as U2OS, Jurkat, MDA-MB-231 and primary T cells as well as hematopoietic stem and progenitor cells) predicts poor HDR. Here we demonstrate rescue of HDR efficiency (ranging from two-fold to eight-fold improvement) either by TREX1 knockout or by the use of single-stranded DNA templates chemically protected from TREX1 activity. Our data explain why some cell types are easier to edit than others and indicate routes for increasing CRISPR-Cas9-mediated HDR in TREX1-expressing contexts.},
}
@article {pmid40659848,
year = {2025},
author = {Karimi, M and Ghorbani, A and Niazi, A and Rostami, M and Tahmasebi, A},
title = {Integrating AI and CRISPR Cas13a for rapid detection of tomato brown rugose fruit virus.},
journal = {Scientific reports},
volume = {15},
number = {1},
pages = {25422},
pmid = {40659848},
issn = {2045-2322},
mesh = {*Solanum lycopersicum/virology ; *Plant Diseases/virology ; *CRISPR-Cas Systems ; *Plant Viruses/genetics/isolation &amp; purification ; Computational Biology/methods ; RNA, Viral/genetics ; },
abstract = {The Tomato Brown Rugose Fruit Virus (ToBRFV) has recently emerged as a serious threat to global tomato production, underscoring the need for rapid and sensitive diagnostic tools. Here, we present an AI-driven CRISPR-Cas13a pipeline for designing crRNAs with high specificity to enable the detection of ToBRFV. A computational pipeline that retrieves viral sequences, aligns them in multiple sequence alignments, analyzes their conservation, and screens for off-targets-all coupled with machine learning to optimize crRNA sequences. Experimentally validated crRNAs were evaluated with a fluorescence-based Cas13a assay and showed better sensitivity than RT-PCR, RT-qPCR, and RT-LAMP. By the CRISPR-Cas13a system, ToBRFV was detected at 1:200 (1 ng/µL) dilutions, which performed superior to conventional methods. Integrating bioinformatics with experimental workflows, this pipeline provides a powerful framework for rapid diagnostics that can be deployed in the field, addressing significant challenges in plant virus surveillance and management.},
}
@article {pmid40659683,
year = {2025},
author = {Li, P and Wu, Z and Liu, T and Deng, C and Liu, Q and Ni, J},
title = {The defensome of prokaryotes in aquifers.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {6482},
pmid = {40659683},
issn = {2041-1723},
support = {U2240205//National Natural Science Foundation of China (National Science Foundation of China)/ ; 51721006//National Natural Science Foundation of China (National Science Foundation of China)/ ; },
mesh = {*Groundwater/microbiology ; *Bacteria/genetics/immunology/virology ; China ; *Prokaryotic Cells ; Metagenomics ; Bacteriophages ; CRISPR-Cas Systems/genetics ; Ecosystem ; Metagenome ; Phylogeny ; },
abstract = {Groundwater harbors a pristine biosphere where microbes co-evolve with less human interference, yet the ancient and ongoing arms race between prokaryotes and viruses remains largely unknown in such ecosystems. Based on our recent nationwide groundwater monitoring campaign across China, we construct a metagenomic groundwater prokaryotic defensome catalogue (GPDC), encompassing 190,810 defense genes, 90,824 defense systems, 139 defense families, and 669 defense islands from 141 prokaryotic phyla. Over 94% of the defense genes in GPDC are novel and contribute vast microbial immune resources in groundwater. We find that candidate phyla radiation (CPR) bacteria possess higher defense system density and diversity against intense phage infection, while microbes as a whole exhibit an inverse relationship between defense systems and adaptive traits like resistance genes in groundwater. We further identify five first-line defense families covering 69.2% of the total defense systems, and high-turnover accessory immune genes are mostly conveyed to defense islands by mobile genetic elements. Our study also reveals viral resistance to microbial defense through co-localized anti-defense genes and interactions between CRISPR-Cas9 and anti-CRISPR protein. These findings expand our understanding of microbial immunity in pristine ecosystems and offer valuable immune resources for potential biotechnological applications.},
}
@article {pmid40659672,
year = {2025},
author = {Viswanatha, R and Entwisle, S and Hu, Y and Kim, AR and Reap, K and Butnaru, M and Qadiri, M and Mohr, SE and Perrimon, N},
title = {Higher resolution pooled genome-wide CRISPR knockout screening in Drosophila cells using integration and anti-CRISPR (IntAC).},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {6498},
pmid = {40659672},
issn = {2041-1723},
mesh = {Animals ; *CRISPR-Cas Systems/genetics ; *Drosophila melanogaster/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; *Gene Knockout Techniques/methods ; Cell Line ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Gene Editing/methods ; Genome, Insect ; Humans ; Drosophila Proteins/genetics ; Plasmids/genetics ; },
abstract = {CRISPR screens enable systematic, scalable genotype-to-phenotype mapping. We previously developed a CRISPR screening method for Drosophila melanogaster and mosquito cell lines using plasmid transfection and site-specific integration to introduce single guide (sgRNA) libraries. The method relies on weak sgRNA promoters to avoid early CRISPR-Cas9 activity causing discrepancies between genome edits and integrated sgRNAs. To address this issue and utilize higher strength sgRNA expression, we introduce a method to co-transfect a plasmid expressing anti-CRISPR protein to suppress early CRISPR-Cas9 activity which we term "IntAC" (integrase with anti-CRISPR). IntAC dramatically improves precision-recall of fitness genes across the genome, allowing us to generate the most comprehensive list of cell fitness genes yet assembled for Drosophila. Drosophila fitness genes show strong correlation with human fitness genes and underscore the effects of paralogs on gene essentiality. We also perform a resistance screen to proaerolysin, a glycosylphosphatidylinositol-(GPI)-binding pore-forming toxin, retrieving 18/23 expected and one previously uncharacterized GPI synthesis gene. We also demonstrate that an IntAC sublibrary enables precise positive selection of a transporter under solute overload. IntAC represents a straightforward enhancement to existing Drosophila CRISPR screening methods, dramatically increasing accuracy, and might also be broadly applicable to virus-free CRISPR screens in other cell and species types.},
}
@article {pmid40659638,
year = {2025},
author = {Sewgoolam, B and Jim, KK and de Bakker, V and Bock, FP and Gibson, PS and Veening, JW},
title = {Genome-wide antibiotic-CRISPRi profiling identifies LiaR activation as a strategy to resensitize fluoroquinolone-resistant Streptococcus pneumoniae.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {6491},
pmid = {40659638},
issn = {2041-1723},
support = {310030_192517//Schweizerischer Nationalfonds zur F&#xf6;rderung der Wissenschaftlichen Forschung (Swiss National Science Foundation)/ ; 310030_200792//Schweizerischer Nationalfonds zur F&#xf6;rderung der Wissenschaftlichen Forschung (Swiss National Science Foundation)/ ; NCCR AntiResist 51NF40_180541//Schweizerischer Nationalfonds zur F&#xf6;rderung der Wissenschaftlichen Forschung (Swiss National Science Foundation)/ ; },
mesh = {*Streptococcus pneumoniae/genetics/drug effects/metabolism ; *Fluoroquinolones/pharmacology ; *Anti-Bacterial Agents/pharmacology ; Animals ; *Bacterial Proteins/genetics/metabolism ; *Drug Resistance, Bacterial/genetics/drug effects ; Zebrafish ; Regulon/genetics ; Gene Expression Regulation, Bacterial/drug effects ; Humans ; Bacitracin/pharmacology ; Ciprofloxacin/pharmacology ; Microbial Sensitivity Tests ; Pneumococcal Infections/drug therapy/microbiology ; Genome, Bacterial ; CRISPR-Cas Systems ; },
abstract = {Streptococcus pneumoniae is a human pathogen that has become increasingly resistant to synthetic fluoroquinolone antibiotics that target bacterial topoisomerases. To identify pathways essential under fluoroquinolone stress and potential novel targets to revitalize use of this antibiotic class, we perform genome-wide CRISPRi-seq screens and generate antibiotic-gene essentiality signatures. Expectedly, genes involved in DNA recombination and repair become more important under fluoroquinolone-induced DNA damage, including recA, recJ, recF, recO, rexAB, and ruvAB. Surprisingly, specific downregulation of the gene encoding the histidine kinase LiaS caused fluoroquinolone hypersensitivity. LiaS is part of the LiaFSR (VraTSR) three-component regulatory system involved in cell envelope homeostasis. We show that LiaS keeps the response regulator LiaR inactive, and that liaS deletion causes LiaR hyperphosphorylation and upregulation of the LiaR regulon. We use RNA-seq to refine the LiaR regulon, highlighting the role of heat-shock response and pleiotropic regulator SpxA2 in fluoroquinolone sensitivity. Activating the LiaR-regulon by the cell envelope-targeting antibiotic bacitracin synergized with ciprofloxacin and levofloxacin, restoring sensitivity in fluoroquinolone-resistant strains in vitro. Furthermore, bacitracin/levofloxacin combination therapy is effective in vivo and improved treatment of fluoroquinolone-resistant S. pneumoniae infection in a zebrafish meningitis model. These findings offer a starting point for identification and validation of potent combination therapies to treat antibiotic-resistant pneumococcal infections.},
}
@article {pmid40659249,
year = {2025},
author = {Storz, U},
title = {The CRISPR Cas patent files, part 3: Prime Editing and integrase-based variants.},
journal = {Journal of biotechnology},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.jbiotec.2025.07.006},
pmid = {40659249},
issn = {1873-4863},
abstract = {The epic patent disputes regarding CRISPR Cas9 and, to a lesser extent, CRISPR Cas12a, have somehow overcast a new patent debate that is materializing at the horizon, namely the one that relates to Prime Editing and its integrase-based siblings. While these techniques offer great promise for the precise integration of large DNA stretches into a host genome, it appears that a new chain of dependencies is about to unfold that makes the establishment of Freedom to Operate complicated for interested parties.[[1]].},
}
@article {pmid40658808,
year = {2025},
author = {Sekiba, K and Miyake, N and Miyakawa, Y and Shibata, C and Seimiya, T and Kishikawa, T and Fujishiro, M and Otsuka, M},
title = {CRISPR-mediated proximity labeling unveils ABHD14B as a host factor to regulate HBV cccDNA transcriptional activity.},
journal = {Hepatology communications},
volume = {9},
number = {8},
pages = {},
doi = {10.1097/HC9.0000000000000757},
pmid = {40658808},
issn = {2471-254X},
mesh = {Humans ; *Hepatitis B virus/genetics ; *DNA, Circular/genetics/metabolism ; *DNA, Viral/genetics/metabolism ; Virus Replication ; CRISPR-Cas Systems ; RNA, Viral/genetics ; Hep G2 Cells ; Transcription, Genetic ; *Viral Transcription ; Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {BACKGROUND: The long-term goal of chronic hepatitis B research is a functional cure (HBsAg seroclearance). Although currently used nucleos(t)ide analogs can efficiently inhibit viral replication, they do not reduce viral RNAs or proteins produced from covalently closed circular DNA (cccDNA), and rarely achieve a functional cure. To overcome this situation, revealing the mode of the existence of cccDNA is required, including identifying the interreacting proteins with cccDNA. Here, we aimed to identify novel proteins that interact with cccDNA.<br><br>METHODS: Using an in vitro HBV infection model and a sequence-specific proximity labelling method consisting of dead Cas9 and biotin identification (BioID2), we comprehensively determined proteins that possibly interact with cccDNA. After identifying the candidate proteins, the HBV RNA transcription levels were examined by knocking out the associated genes.<br><br>RESULTS: We identified ABHD14B as a protein that interacts with cccDNA and inhibits HBV RNA transcription from cccDNA. ABHD14B decreases the acetylation levels of histone proteins that control the transcription levels of HBV RNA in cccDNA. Moreover, ABHD14B interacts with TFII-I, which binds directly to cccDNA in a sequence-dependent manner. These results suggest that the host protein, ABHD14B, is recruited to cccDNA via the TFII-I protein, inhibiting HBV RNA transcription from cccDNA by deacetylating cccDNA histones.<br><br>CONCLUSIONS: ABHD14B was newly identified as a suppressor of HBV RNA transcription from cccDNA, which may improve our understanding of the mode of existence of cccDNA, providing a basis for development of a functional cure.},
}
@article {pmid40657403,
year = {2025},
author = {Akram, F and Safdar, M and Shabbir, I and Fatima, T and Ikram-Ul-Haq, },
title = {Insight into the eminent biotechnological applications of xylanolytic enzymes for sustainable bioprocessing.},
journal = {3 Biotech},
volume = {15},
number = {8},
pages = {249},
pmid = {40657403},
issn = {2190-572X},
abstract = {Xylan is one of the most abundant polysaccharides in nature and presents a structural complexity characterized by a heterogeneous polymer composition. Comprising various sugar subunits and associated acids linked through a diverse array of bonds, xylan poses challenges for complete degradation. This review article provides a comprehensive overview of xylan's structure, the role of xylanolytic enzymes in its degradation, and the industrial applications of xylanases in sectors, such as paper and pulp, food, textiles, and pharmaceuticals. Furthermore, it also discusses the use of advanced biotechnology tools, such as nano-biotechnology and genetic engineering, particularly through CRISPR/CAS technology, for enhancing the thermostability of xylanases. This article also provides insights into emerging trends in xylanase research, including bioprospecting novel thermostable xylanases from metagenomes, protein engineering, synthetic biology, and the integration of biorefinery. Finally, it highlights the importance of regulatory frameworks and standardization initiatives for ensuring the quality and the sustainability of xylanase-based technologies. Overall, this review offers valuable insights into the multifaceted role of xylanases in biotechnology and industrial bioprocessing while outlining future directions for research and innovation in this field.},
}
@article {pmid40655875,
year = {2025},
author = {Okesanya, OJ and Ayeni, RA and Amadin, P and Ngwoke, I and Amisu, BO and Ukoaka, BM and Ahmed, MM and Oso, TA and Musa, SS and Lucero-Prisno, DE},
title = {Advances in HIV Treatment and Vaccine Development: Emerging Therapies and Breakthrough Strategies for Long-Term Control.},
journal = {AIDS research and treatment},
volume = {2025},
number = {},
pages = {6829446},
pmid = {40655875},
issn = {2090-1240},
abstract = {Since its identification in 1981, HIV has posed a global public health challenge, witnessing transformative advancements in treatment and prevention. This review summarizes recent novel therapeutic and preventive approaches for long-term HIV control, management, and elimination, and how global collaboration and technological innovations may advance HIV control efforts. This study highlights the progress and challenges in HIV treatment, emphasizing the effectiveness of current antiretroviral therapy (ART) in suppressing viral replication, reducing transmission, and preventing end-organ damage. However, adherence remains a significant barrier due to pill burden, side effects, and psychosocial factors affecting patients. ART-related toxicities include neuropathy, hepatotoxicity, metabolic disorders, and neuropsychiatric effects. Long-acting ART (LA-ART) offers a promising alternative to daily dosing; however, challenges such as injection site reactions persist. Broadly neutralizing antibodies (bNAbs) have shown enhanced efficacy in viral suppression and immune response activation, offering potential for treatment and vaccine design. Innovative gene-editing tools, such as CRISPR-Cas systems, are being explored for their ability to excise or silence proviral DNA; however, their clinical application is limited by off-target effects and delivery challenges. Latency-targeting strategies like "shock and kill" and "block and lock" remain experimental with limited clinical success, and nanotechnology-based drug delivery systems offer targeted, sustained, and less toxic treatment options. Despite the challenges posed by the virus's rapid mutation rate and immune evasion mechanisms, novel vaccine approaches, such as mRNA technology, vector-based platforms, and epitope-targeting strategies, are being explored. In addition, artificial intelligence and machine learning are enhancing the design of vaccines, predictive modeling, and fast-tracking progress in this area. Socio-economic bottlenecks in HIV control, such as stigma, gender disparities, and inequitable healthcare access, exacerbate the epidemic, particularly in sub-Saharan Africa. Enhancing global collaboration, providing sustainable funding, and integrating emerging and innovative technologies are critical for advancing HIV prevention and management. Achieving an AIDS-free generation and ultimately eliminating the epidemic will depend on effectively addressing the social, structural, and scientific barriers that hinder progress in this regard. Trial Registration: ClinicalTrials.gov identifier: NCT02120352, NCT02938520, NCT03639311, NCT03497676, NCT03635788.},
}
@article {pmid40655388,
year = {2025},
author = {Jhalora, V and Bist, R},
title = {A Comprehensive Review of Molecular Mechanisms Leading to the Emergence of Multidrug Resistance in Bacteria.},
journal = {Indian journal of microbiology},
volume = {65},
number = {2},
pages = {844-865},
pmid = {40655388},
issn = {0046-8991},
abstract = {UNLABELLED: Multidrug resistance (MDR) in bacteria poses a serious global health threat, compromising the effectiveness of antibiotics. MDR causes approximately 700,000 deaths annually, with MDR tuberculosis alone claiming 230,000 lives. While bacteria inherently possess intrinsic resistance, acquired resistance stands out as the primary culprit in MDR development. Acquired resistance mechanisms mediated by the bacterial cell wall, nucleic acids, and proteins play a pivotal role in the genesis of MDR. Bacteria can modify their cell wall structure, produce resistant enzymes, exhibit mutations in antibiotic-targeted genes, and acquire resistant genes through horizontal gene transfer. Bacteria can produce proteins that act as enzymes, chemically modifying or directly degrading the antibiotic molecules, leading to the loss of their functionality. Apart from these mechanisms, biofilms also play a pivotal role in MDR expansion. Despite the development of several antibiotics since the discovery of penicillin, continuous structural and molecular modifications in bacteria render these antibiotics ineffective against MDR. The most recent approaches such as clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated proteins (CRISPR-Cas), nanotechnology, a combination of CRISPR-Cas, and nanoparticles, show promise in treating MDR. Thus, this review delves deep into the molecular mechanisms of MDR, emphasizing the limitations of current antibiotics due to bacterial evolution and highlighting current strategies in the fight against MDR bacteria. This will drive comprehensive research to uncover additional resistance mechanisms and develop innovative strategies to combat resistant bacteria effectively.<br><br>SUPPLEMENTARY INFORMATION: The online version supplementary material available at 10.1007/s12088-024-01384-6.},
}
@article {pmid40654865,
year = {2025},
author = {Taranenko, D and Kotovskaya, O and Kuznedelov, K and Yanovskaya, D and Demkina, A and Fardeeva, S and Mamontov, V and Vierra, K and Burman, N and Li, D and Wang, M and Wiedenheft, B and Severinov, K and Semenova, E and Isaev, A},
title = {A census of anti-CRISPR proteins reveals AcrIE9 as an inhibitor of Escherichia coli K12 Type IE CRISPR-Cas system.},
journal = {bioRxiv : the preprint server for biology},
volume = {},
number = {},
pages = {},
doi = {10.1101/2025.05.07.652737},
pmid = {40654865},
issn = {2692-8205},
abstract = {CRISPR-Cas adaptive immunity systems provide defense against mobile genetic elements and are often countered by diverse anti-CRISPR (Acr) proteins. The Type IE CRISPR-Cas of Escherichia coli K12 has been a model for structural and functional studies and is a part of the species' core genome. However, this system is transcriptionally silent, which has fueled questions about its true biological function. To clarify the role of this system in defense, we carried out a census of Acr proteins found in Enterobacterales and identified AcrIE9 as a potent inhibitor of the E. coli K12 Type IE CRISPR-Cas system. While sharing little sequence identity, AcrIE9 proteins from Pseudomonas and Escherichia both interact with the Cas7 subunit of the Cascade complex, thus preventing its binding to DNA. We further show that AcrIE9 is genetically linked to AcrIE10, forming the most widespread anti-CRISPR cluster in Enterobacterales , and this module often co-occurs with a novel HTH-like protein with unusual architecture.},
}
@article {pmid40654372,
year = {2025},
author = {Xu, Y and Le, H and Wu, Q and Wang, N and Gong, C},
title = {Advancements in CRISPR/Cas systems for disease treatment.},
journal = {Acta pharmaceutica Sinica. B},
volume = {15},
number = {6},
pages = {2818-2844},
pmid = {40654372},
issn = {2211-3835},
abstract = {The clustered regularly interspaced short palindromic repeats (CRISPR)-Cas (CRISPR-associated proteins) is an adaptive immune system present in most bacteria and archaea, protecting them from infection by exogenous genetic elements. Due to its simplicity, cost-effectiveness, and precise gene editing capabilities, CRISPR/Cas technology has emerged as a promising tool for treating diseases. The continuous refinement of derivative systems has further broadened its scope in disease treatment. Nevertheless, the heterogeneous physiopathological nature of diseases and variations in disease onset sites pose significant challenges for in vivo applications of CRISPR systems. The efficiency of CRISPR systems in disease treatment is directly influenced by the performance of the delivery system. Additionally, concerns such as off-target effects present crucial hurdles in the clinical implementation of CRISPR systems. This review provides a comprehensive overview of the development of CRISPR systems, vector technologies, and their applications in disease treatment, while also addressing the challenges encountered in clinical settings. Furthermore, future research directions are outlined to pave the way for advancements in CRISPR-based therapies.},
}
@article {pmid40204636,
year = {2025},
author = {Steiner, KK and Young, AC and Patterson, AR and Sugiura, A and Watson, MJ and Preston, SEJ and Zhelonkin, A and Jennings, EQ and Chi, C and Heintzman, DR and Pahnke, AP and Toudji, YT and Hatem, Z and Madden, MZ and Arner, EN and Sewell, AE and Blount, AK and Okparaugo, R and Fallman, E and Krystofiak, ES and Sheldon, RD and Gibson-Corley, KN and Voss, K and Nowinski, SM and Jones, RG and Mogilenko, DA and Rathmell, JC},
title = {Mitochondrial fatty acid synthesis and MECR regulate CD4+ T cell function and oxidative metabolism.},
journal = {Journal of immunology (Baltimore, Md. : 1950)},
volume = {214},
number = {5},
pages = {958-976},
pmid = {40204636},
issn = {1550-6606},
support = {S10 OD034315/OD/NIH HHS/United States ; P30EY008126/NH/NIH HHS/United States ; T32 CA009582/CA/NCI NIH HHS/United States ; R01 AI165722/AI/NIAID NIH HHS/United States ; R01 AI153167/AI/NIAID NIH HHS/United States ; U2C DK059637/DK/NIDDK NIH HHS/United States ; R38HL143619//Breakthrough T1D Advanced Postdoctoral Fellowship/ ; P30CA068485//National Institutes of Health Cancer Center/ ; P30DK058404/NH/NIH HHS/United States ; P30CA068485/NH/NIH HHS/United States ; //Distinguished Innovator/ ; T32GM007347//Breakthrough T1D Advanced Postdoctoral Fellowship/ ; F31 DK139660/NH/NIH HHS/United States ; R01 HL136664/HL/NHLBI NIH HHS/United States ; //Lupus Research Alliance/ ; P30DK020593/NH/NIH HHS/United States ; U2CDK059637/NH/NIH HHS/United States ; F31 DK139660/DK/NIDDK NIH HHS/United States ; //The Vanderbilt Cell Imaging Shared Resource/ ; K00CA253718//Breakthrough T1D Advanced Postdoctoral Fellowship/ ; T32CA009582//Breakthrough T1D Advanced Postdoctoral Fellowship/ ; R21AR080233/NH/NIH HHS/United States ; P30 DK058404/DK/NIDDK NIH HHS/United States ; K00 CA253718/CA/NCI NIH HHS/United States ; R01AI165722//Paul G. Allen Frontiers Group Distinguished Investigator Program/ ; R01 DK105550/DK/NIDDK NIH HHS/United States ; //The Translational Pathology Shared Resource/ ; R21 AR080233/AR/NIAMS NIH HHS/United States ; 1S10OD034315/NH/NIH HHS/United States ; R01 AI153167/NH/NIH HHS/United States ; R38 HL143619/HL/NHLBI NIH HHS/United States ; 2T32CA009592-32/NH/NIH HHS/United States ; R01DK105550/NH/NIH HHS/United States ; F30CA239367//Breakthrough T1D Advanced Postdoctoral Fellowship/ ; DRG-2495-23//Breakthrough T1D Advanced Postdoctoral Fellowship/ ; P30 EY008126/EY/NEI NIH HHS/United States ; R01HL136664/NH/NIH HHS/United States ; F30 CA239367/CA/NCI NIH HHS/United States ; T32 GM007347/GM/NIGMS NIH HHS/United States ; P30 DK020593/DK/NIDDK NIH HHS/United States ; P30 CA068485/CA/NCI NIH HHS/United States ; P30CA068485//Breakthrough T1D Advanced Postdoctoral Fellowship/ ; T32 CA009592/CA/NCI NIH HHS/United States ; //Mark Foundation Endeavor/ ; R01 CA217987/CA/NCI NIH HHS/United States ; R01CA217987/NH/NIH HHS/United States ; },
mesh = {*CD4-Positive T-Lymphocytes/immunology/metabolism ; *Mitochondria/metabolism ; *Fatty Acids/biosynthesis ; Animals ; Mice ; CRISPR-Cas Systems ; *Oxidoreductases Acting on CH-CH Group Donors/genetics/metabolism ; Mice, Knockout ; Ferroptosis ; Male ; Female ; *Oxygen/metabolism ; Colitis/physiopathology ; },
abstract = {Imbalanced effector and regulatory CD4+ T cell subsets drive many inflammatory diseases. These T cell subsets rely on distinct metabolic programs, modulation of which differentially affects T cell fate and function. Lipid metabolism is fundamental yet remains poorly understood across CD4+ T cell subsets. Therefore, we performed targeted in vivo CRISPR/Cas9 screens to identify lipid metabolism genes and pathways essential for T cell functions. These screens established mitochondrial fatty acid synthesis genes Mecr, Mcat, and Oxsm as key metabolic regulators. Of these, the inborn error of metabolism gene Mecr was most dynamically regulated. Mecrfl/fl; Cd4cre mice had normal naïve CD4+ and CD8+ T cell numbers, demonstrating that MECR is not essential in homeostatic conditions. However, effector and memory T cells were reduced in Mecr knockout and MECR-deficient CD4+ T cells and proliferated, differentiated, and survived less well than control T cells. Interestingly, T cells ultimately showed signs of mitochondrial stress and dysfunction in the absence of MECR. Mecr-deficient T cells also had decreased mitochondrial respiration, reduced tricarboxylic acid intermediates, and accumulated intracellular iron, which appeared to contribute to increased cell death and sensitivity to ferroptosis. Importantly, MECR-deficient T cells exhibited fitness disadvantages and were less effective at driving disease in an in vivo model of inflammatory bowel disease. Thus, MECR-mediated metabolism broadly supports CD4+ T cell proliferation and survival in vivo. These findings may also provide insight to the immunological state of MECR- and other mitochondrial fatty acid synthesis-deficient patients.},
}
@article {pmid40653316,
year = {2025},
author = {Kouvela, A and Jaramillo Ponce, JR and Giarimoglou, N and Chicher, J and Marzi, S and Stathopoulos, C and Stamatopoulou, V},
title = {Coupling tRNAGly gene redundancy with staphylococcal cell wall integrity, antibiotic susceptibility, and virulence potential.},
journal = {Nucleic acids research},
volume = {53},
number = {13},
pages = {},
doi = {10.1093/nar/gkaf599},
pmid = {40653316},
issn = {1362-4962},
support = {//University of Patras/ ; },
mesh = {*Cell Wall/genetics/drug effects/metabolism ; *Staphylococcus aureus/genetics/pathogenicity/drug effects ; Humans ; Anti-Bacterial Agents/pharmacology ; Virulence/genetics ; CRISPR-Cas Systems ; Biofilms/growth &amp; development/drug effects ; Gene Expression Regulation, Bacterial ; Riboswitch ; Staphylococcal Infections/microbiology ; Protein Biosynthesis ; Gene Editing ; },
abstract = {Redundancy in transfer RNA (tRNA) gene copies across species remains poorly understood and, in many cases, largely unexplored. In Staphylococcus aureus, multiple tRNAGly genes encode isoacceptors involved in protein synthesis and cell wall formation, aminoacylated by a sole glycyl-tRNA synthetase (GlyRS) which is under the transcription regulation of a species-specific glyS T-box riboswitch. The T-box can interact with all tRNAGly isoacceptors to adopt species-specific conformations and affect both pathways. Using CRISPR/Cas9 editing, we ablated a gene copy corresponding to the proteinogenic P1 tRNAGlyGCC. Surprisingly, the growth and the overall translational activity of the edited strain were found unaffected, suggesting functional compensation by the remaining tRNAGly genes. On the other hand, transcriptomics and proteomics analyses combined with functional assays revealed nutrient-dependent stress responses with surprisingly impaired cell wall integrity and increased susceptibility to cell wall-targeting antibiotics. Additionally, the edited strain displayed reduced biofilm formation but retained the ability to invade human cells in vitro. Overall, the present study underscores the critical role of tRNA gene redundancy in the physiology of S. aureus and highlights tRNAs as regulators of metabolic homeostasis in pathogenic bacteria.},
}
@article {pmid40652883,
year = {2025},
author = {Zhang, S and Kim, JC and Ahn, J},
title = {Phage engineering strategies to expand host range for controlling antibiotic-resistant pathogens.},
journal = {Microbiological research},
volume = {300},
number = {},
pages = {128278},
doi = {10.1016/j.micres.2025.128278},
pmid = {40652883},
issn = {1618-0623},
abstract = {Bacteriophages are known as a promising alternative to control rising bacterial resistance. The adsorption phase is critical for the successful infection of phages, as it determines their ability to recognize and attach to specific bacterial host cells. However, their limited host ranges due to narrow host specificity significantly limit their potential applications and overall effectiveness. Receptor-binding proteins (RBPs) are crucial in the recognition process, and modifying these proteins provides a valuable opportunity to broaden host ranges and enhance adsorption rates. Therefore, gaining a more comprehensive understanding of the interactions between phages and their bacterial hosts is essential. To overcome this challenge, various in vivo and in vitro engineering platforms have been developed, including recombineering, clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated proteins (Cas) systems, yeast-based technologies, and cell-free systems. These methods provide diverse strategies and flexibility for constructing customized phage genomes with desired characteristics, ultimately enhancing phage application efficiency. This review discusses different types of RBPs in phages and their mechanisms of adsorption, highlighting their relevance for adaptable engineering strategies. We also summarize various phage engineering platforms and explore the design of synthetic phages with expanded host ranges. Finally, we highlight the advantages and limitations of current engineering methods, providing insights to guide future research efforts.},
}
@article {pmid40652308,
year = {2025},
author = {Oliver-Caldes, A and Mañe Pujol, J and Battram, AM and Perez-Amill, L and Bachiller, M and Calderon, H and Castella, M and Carpio, J and Salsench, SV and Tovar, N and Cardus, O and Urbano-Ispizua, A and Moreno, DF and Rodríguez-Lobato, LG and Lozano, E and Rosiñol, L and Juan, M and Martín-Antonio, B and Fernández de Larrea, C},
title = {TIGIT blockade in the context of BCMA-CART cell therapy does not augment efficacy in a multiple myeloma mouse model.},
journal = {Oncoimmunology},
volume = {14},
number = {1},
pages = {2529632},
doi = {10.1080/2162402X.2025.2529632},
pmid = {40652308},
issn = {2162-402X},
mesh = {Animals ; *Multiple Myeloma/therapy/immunology/pathology ; *Receptors, Immunologic/antagonists &amp; inhibitors/immunology/genetics ; Mice ; *Immunotherapy, Adoptive/methods ; Humans ; Disease Models, Animal ; *Receptors, Chimeric Antigen/immunology ; *B-Cell Maturation Antigen/immunology ; Cell Line, Tumor ; Xenograft Model Antitumor Assays ; CRISPR-Cas Systems ; Female ; },
abstract = {BCMA-directed CAR-T therapies have shown promising results in multiple myeloma (MM). However, patients continue to relapse. T cell exhaustion with increased TIGIT expression is a resistance mechanism which was confirmed in CAR-T cells from ARI0002h trial, an academic CAR-T developed in our institution. We aimed to analyze the impact of blocking TIGIT on the efficacy of ARI0002h. We used three different strategies to block TIGIT: (1) Addition of an external blocking anti-TIGIT-antibody (Ab), (2) Modify ARI0002h into a 4[th] generation CAR-T, named ARITIGIT, capable of secreting a soluble TIGIT-blocking scFv and (3) TIGIT knock-out in ARI0002h using CRISPR/Cas9. Each strategy was evaluated in vitro and in vivo. Adding a TIGIT-blocking Ab to ARI0002h improved in vitro cytotoxicity, but failed to enhance mice survival. The new 4[th] generation CAR-T, ARITIGIT, was also unable to achieve better survival outcomes despite favoring the in vivo model by using a myeloma cell line with high expression of the TIGIT ligand PVR. Interestingly, when mice were challenged with a second infusion of tumor cells, mimicking a relapse model, a trend for improved survival with ARITIGIT was observed (p = 0.11). Finally, TIGIT-knock-out on ARI0002h (KO-ARI0002h) using CRISPR/Cas9 showed similar in vitro activity to ARI0002h. In an in vivo stress model, TIGIT KO-ARI0002h prolonged survival (p = 0.02). However, this improvement was not significant compared to ARI0002h (p = 0.07). This study failed to demonstrate a significant benefit of TIGIT-blockade on ARI0002h cells despite using three different approaches, suggesting that targeting a single immune checkpoint may be insufficient.},
}
@article {pmid40651052,
year = {2025},
author = {Arutselvan, R and Kumar, S and Akash, AU and Greeshma, K and Sinha, SS and Khan, AS and Pati, K and Chauhan, VBS and Gowda, KH and Pradhan, S and Jeeva, ML and Veena, SS and Makeshkumar, T and Meena, M and Sangeetha, BG and Laxminarayana, K and Nedunchezhiyan, M},
title = {Deciphering the complex signaling networks in phytophthora infected plants: Insights into microbiome interactions and plant defense mechanisms.},
journal = {Plant physiology and biochemistry : PPB},
volume = {228},
number = {},
pages = {110222},
doi = {10.1016/j.plaphy.2025.110222},
pmid = {40651052},
issn = {1873-2690},
abstract = {Phytophthora species are destructive plant pathogens that cause severe economic losses in agriculture and natural ecosystems, known for their rapid spread through soil and water and resistance to conventional control methods. Understanding the complex signaling networks activated in plants during Phytophthora infection is crucial for developing effective management strategies. This review summarizes research findings on Phytophthora-plant interactions, with special emphasis on Phytophthora-plant microbiome interactions. Initially, molecular mechanisms involved in the plant response to Phytophthora infection are discussed, further emphasizing key signaling pathways activated by Phytophthora in host plants. The role of phytohormones in imparting resistance to Phytophthora infections is explored in depth. Additionally, the interaction and effects of Phytophthora and the plant immune system with the plant microbiome are examined, highlighting how these interactions facilitate disease and/or aid in plant defense. Various biotechnological approaches for enhancing plant resistance to Phytophthora, including recent technologies like CRISPR-Cas systems, are also reviewed. The conclusion addresses the need for further research into signaling networks within Phytophthora-plant-microbiome interactions and their future implications for crop protection.},
}
@article {pmid40650977,
year = {2025},
author = {Faber, A and Politan, RJ and Stukenberg, D and Morris, KM and Kim, R and Jeon, E and Inckemann, R and Becker, A and Thuronyi, B and Fritz, G},
title = {Expanding genetic engineering capabilities in Vibrio natriegens with the Vnat Collection.},
journal = {Nucleic acids research},
volume = {53},
number = {13},
pages = {},
doi = {10.1093/nar/gkaf580},
pmid = {40650977},
issn = {1362-4962},
support = {FT230100283//Australian Research Council/ ; //Forrest Research Foundation/ ; //Forrest Research Foundation Scholarship/ ; GRK 2937//Deutsche Forschungsgemeinschaft/ ; //Williams College/ ; //Australian Research Council/ ; },
mesh = {*Vibrio/genetics/metabolism ; *Genetic Engineering/methods ; Promoter Regions, Genetic ; Synthetic Biology/methods ; Metabolic Engineering/methods ; Gene Editing/methods ; CRISPR-Cas Systems ; Operon ; Cloning, Molecular ; },
abstract = {Vibrio natriegens, with its exceptionally fast growth rate, has great promise as a revolutionary chassis for synthetic biology, yet the realization of its full potential has been limited by the lack of robust, standardized genetic tools. Here, we present the Vnat Collection, a comprehensive, modular toolkit specifically engineered to overcome these limitations. Leveraging optimized Golden Gate cloning strategies, we introduce improved junction sequences and a highly efficient dropout part system, achieving up to a 300-fold increase in assembly efficiency. Our toolkit significantly expands the synthetic biology toolbox by providing a wide array of characterized inducible promoters, enabling precise, orthogonal gene regulation, and novel operon connectors to streamline the construction of multi-gene pathways critical for metabolic engineering. Furthermore, we enhance genome editing workflows through refined NT-CRISPR methods, incorporating homology-flanked targeting constructs and demonstrating a simplified protocol that eliminates intermediate purification steps. With over 220 rigorously validated modular components, the Vnat Collection establishes an advanced standard for genetic engineering of V. natriegens, empowering researchers to efficiently harness this organism's unparalleled potential for diverse biotechnology applications.},
}
@article {pmid40650973,
year = {2025},
author = {Chi, H and McMahon, S and Daniel-Pedersen, L and Graham, S and Gloster, TM and White, MF},
title = {SAM-AMP lyases in type III CRISPR defence.},
journal = {Nucleic acids research},
volume = {53},
number = {13},
pages = {},
doi = {10.1093/nar/gkaf655},
pmid = {40650973},
issn = {1362-4962},
support = {BB/T004789/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; 101018608//European Research Council Advanced Grant/ ; //University of St Andrews/ ; },
mesh = {S-Adenosylmethionine/metabolism ; *CRISPR-Cas Systems ; Escherichia coli/genetics ; *Bacterial Proteins/chemistry/metabolism/genetics ; Thionucleosides/metabolism/chemistry ; Models, Molecular ; *Lyases/chemistry/metabolism/genetics ; Deoxyadenosines/metabolism/chemistry ; Crystallography, X-Ray ; },
abstract = {Type III CRISPR systems detect non-self RNA and activate the enzymatic Cas10 subunit, which generates nucleotide second messengers for activation of ancillary effectors. Although most signal via cyclic oligoadenylate, an alternative class of signalling molecule SAM-AMP, formed by conjugating ATP and S-adenosylmethionine, was described recently. SAM-AMP activates a trans-membrane effector of the CorA magnesium transporter family to provide anti-phage defence. Intriguingly, immunity also requires SAM-AMP degradation by means of a specialized CRISPR-encoded NrN family phosphodiesterase in Bacteroides fragilis. In Clostridium botulinum, the nrn gene is replaced by a gene encoding a SAM-AMP lyase. Here, we investigate the structure and activity of C. botulinum SAM-AMP lyase, which can substitute for the nrn gene to provide CorA-mediated immunity in Escherichia coli. The structure of SAM-AMP lyase bound to its reaction product 5'-methylthioadenosine-AMP reveals key details of substrate binding and turnover by this PII superfamily protein. Bioinformatic analysis revealed a phage-encoded SAM-AMP lyase that degrades SAM-AMP efficiently in vitro, consistent with an anti-CRISPR function.},
}
@article {pmid40650969,
year = {2025},
author = {Madariaga-Marcos, J and Baltramonaitis, M and Henkel-Heinecke, S and Kauert, DJ and Irmisch, P and Bigelyte-Stankeviciene, G and Silanskas, A and Karvelis, T and Siksnys, V and Sasnauskas, G and Seidel, R},
title = {Structural and mechanistic insights into the sequential dsDNA cleavage by SpCas12f1.},
journal = {Nucleic acids research},
volume = {53},
number = {13},
pages = {},
doi = {10.1093/nar/gkaf588},
pmid = {40650969},
issn = {1362-4962},
support = {S-MIP-19-32//Research Council of Lithuania/ ; 02-002-P-0001//Mission-driven Implementation of Science and Innovation Programmes/ ; //New Generation Lithuania/ ; GA 724863/ERC_/European Research Council/International ; SPP2141//Deutsche Forschungsgemeinschaft/ ; SE 1646/8-1//Deutsche Forschungsgemeinschaft/ ; //Alexander von Humboldt Foundation/ ; //Leipzig University/ ; //German Research Foundation/ ; },
mesh = {*DNA Cleavage ; *DNA/metabolism/chemistry/genetics ; *CRISPR-Associated Proteins/chemistry/metabolism/genetics ; CRISPR-Cas Systems ; Models, Molecular ; Kinetics ; Catalytic Domain ; R-Loop Structures ; Protein Binding ; *Bacterial Proteins/chemistry/metabolism/genetics ; },
abstract = {Miniature CRISPR-Cas12f1 effector complexes have recently attracted considerable interest for genome engineering applications due to their compact size. Unlike other Class 2 effectors, Cas12f1 functions as a homodimer bound to a single ∼200 nt RNA. While the basic biochemical properties of Cas12f1, such as its use of a single catalytic center for catalysis, have been characterized, the orchestration of the different events occurring during Cas12f1 reactions remained little explored. To gain insights into the dynamics and mechanisms involved in DNA recognition and cleavage by Cas12f1 from Syntrophomonas palmitatica (SpCas12f1), we solved the structure of SpCas12f1 bound to target DNA and employed single-molecule magnetic tweezers measurements in combination with ensemble kinetic measurements. Our data indicate that SpCas12f1 forms 18 bp R-loops, in which local contacts of the protein to the R-loop stabilize R-loop intermediates. DNA cleavage is catalyzed by a single SpCas12f1 catalytic center, which first rapidly degrades a ∼11 bp region on the nontarget strand by cutting at random sites. Subsequent target strand cleavage is slower and requires at least a nick in the nontarget strand.},
}
@article {pmid40650785,
year = {2025},
author = {Samuels, M and Besta, S and Betrán, AL and Nia, RS and Xie, X and Gu, X and Shu, Q and Giamas, G},
title = {CRISPR screening approaches in breast cancer research.},
journal = {Cancer metastasis reviews},
volume = {44},
number = {3},
pages = {59},
pmid = {40650785},
issn = {1573-7233},
mesh = {Humans ; *Breast Neoplasms/genetics/diagnosis/pathology/therapy ; Female ; *CRISPR-Cas Systems ; Animals ; *Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {The emergence of CRISPR-Cas9 technology has transformed functional genomics, offering unmatched opportunities to dissect and understand biological pathways and identify novel therapeutic targets in cancer. Breast cancer is a complex, heterogeneous disease and remains a major cause of morbidity and mortality in women, particularly when diagnosed at advanced or metastatic stages where effective treatments are limited. High-throughput CRISPR screening is undoubtedly a powerful tool to discover novel drug targets, uncover synthetic lethal interactions, and identify vulnerabilities in cancer. This review focuses on advances in our understanding of breast cancer developed through CRISPR-based screening technology, particularly in identifying drivers of breast cancer progression, growth, and metastasis, as well as in identifying potential new therapeutic targets and combination therapies. We discuss recent discoveries, current challenges, and limitations of this approach and explore how advancements in CRISPR technology could have a profound impact on the future of breast cancer treatment.},
}
@article {pmid40650287,
year = {2025},
author = {Gao, Z and Long, T and Guo, P and Luo, J and Nie, X and Xie, Q and Chen, G and Hu, Z},
title = {The Nuclear Transcription Factor SlNF-YC9 Regulates the Protrusion of Tomato Fruit Tip.},
journal = {International journal of molecular sciences},
volume = {26},
number = {13},
pages = {},
doi = {10.3390/ijms26136511},
pmid = {40650287},
issn = {1422-0067},
support = {31872121//National Natural Science Foundation of China/ ; },
mesh = {*Solanum lycopersicum/genetics/metabolism/growth &amp; development ; *Fruit/genetics/growth &amp; development/metabolism ; Gene Expression Regulation, Plant ; *Plant Proteins/genetics/metabolism ; *Transcription Factors/genetics/metabolism ; Promoter Regions, Genetic ; *CCAAT-Binding Factor/genetics/metabolism ; Indoleacetic Acids/metabolism ; CRISPR-Cas Systems ; },
abstract = {NF-Y transcriptional regulators play crucial roles in diverse biological processes in plants, primarily through the formation of NF-Y complexes that bind to specific DNA motifs. These complexes modulate the expression of downstream genes, which influence plant development and growth. In our research, the function of the NF-Y family C subunit member SlNF-YC9 gene in tomato was investigated with the CRISPR/Cas9 method. In contrast to the WT (wild type), the mutant CR-SlNF-YC9 exhibited a prominent protrusion at the fruit tip. The quantitative PCR analysis displayed that the transcription levels of genes associated with auxin transport (PIN4, PIN5, and PIN9) as well as auxin response genes (ARF7 and LAX3) were enhanced in the CR-SlNF-YC9 fruits than in the WT. Analysis of dual-luciferase reporter and EMSA assays showed that the SlNF-YC9-YB13b-YA7a trimer specifically binds the FUL2 promoter and represses its expression. In conclusion, our results suggest that SlNF-YC9 is crucial in influencing tomato fruit shape by the formation of NF-Y heterotrimeric complexes.},
}
@article {pmid40650152,
year = {2025},
author = {Kovalev, MA and Mamaeva, NY and Kristovskiy, NV and Feskin, PG and Vinnikov, RS and Oleinikov, PD and Sosnovtseva, AO and Yakovlev, VA and Glukhov, GS and Shaytan, AK},
title = {Epigenome Engineering Using dCas Systems for Biomedical Applications and Biotechnology: Current Achievements, Opportunities and Challenges.},
journal = {International journal of molecular sciences},
volume = {26},
number = {13},
pages = {},
doi = {10.3390/ijms26136371},
pmid = {40650152},
issn = {1422-0067},
support = {075-15-2024-539//Russian Ministry of Science and Higher Education/ ; },
mesh = {Humans ; *CRISPR-Cas Systems ; *Gene Editing/methods ; *Epigenome/genetics ; *Biotechnology/methods ; Animals ; *Epigenesis, Genetic ; *Epigenomics/methods ; Epigenome Editing ; },
abstract = {Epigenome engineering, particularly utilizing CRISPR/dCas-based systems, is a powerful strategy to modulate gene expression and genome functioning without altering the DNA sequence. In this review we summarized current achievements and prospects in dCas-mediated epigenome editing, primarily focusing on its applications in biomedicine, but also providing a wider context for its applications in biotechnology. The diversity of CRISPR/dCas architectures is outlined, recent innovations in the design of epigenetic editors and delivery methods are highlighted, and the therapeutic potential across a wide range of diseases, including hereditary, neurodegenerative, and metabolic disorders, is examined. Opportunities for the application of dCas-based tools in animal, agricultural, and industrial biotechnology are also discussed. Despite substantial progress, challenges, such as delivery efficiency, specificity, stability of induced epigenetic modifications, and clinical translation, are emphasized. Future directions aimed at enhancing the efficacy, safety, and practical applicability of epigenome engineering technologies are proposed.},
}
@article {pmid40650151,
year = {2025},
author = {Jung, YJ and Kim, JY and Cho, YG and Kang, KK},
title = {CRISPR/Cas9-Mediated Knockout of OsbZIP76 Reveals Its Role in ABA-Associated Immune Signaling in Rice.},
journal = {International journal of molecular sciences},
volume = {26},
number = {13},
pages = {},
doi = {10.3390/ijms26136374},
pmid = {40650151},
issn = {1422-0067},
support = {RS-2024-00322378//New Breeding Technologies Development Program/ ; },
mesh = {*Oryza/genetics/immunology/microbiology/metabolism ; *Abscisic Acid/metabolism/pharmacology ; *CRISPR-Cas Systems ; *Plant Proteins/genetics/metabolism ; Signal Transduction ; Plant Diseases/microbiology/genetics/immunology ; Gene Expression Regulation, Plant ; Gene Knockout Techniques ; Xanthomonas/pathogenicity ; Disease Resistance/genetics ; *Basic-Leucine Zipper Transcription Factors/genetics/metabolism ; *Plant Immunity/genetics ; Ascomycota ; },
abstract = {The basic leucine zipper (bZIP) transcription factors are involved in a wide range of physiological processes in plants, including hormone signaling, stress responses, and growth and development regulation. They play a key role in abscisic acid (ABA)-mediated immune regulation. However, the immune-related function of OsbZIP76 in rice remains poorly understood. In this study, we generated OsbZIP76 knockout (KO) lines using CRISPR/Cas9-mediated genome editing and examined their phenotypic responses to the bacterial pathogen Xanthomonas oryzae pv. oryzae (Xoo) and the fungal pathogen Magnaporthe oryzae. The KO lines showed increased susceptibility to both pathogens compared to wild-type (WT) plants. Furthermore, qRT-PCR analysis revealed that, upon pathogen infection, the expression of pathogenesis-related genes such as PR1a, PR5, and NPR1 was significantly suppressed in the KO lines. ABA treatment experiments showed that KO lines were hypersensitive to exogenous ABA, indicating a role for OsbZIP76 in ABA perception and signaling. Notably, the expression of the OsbZIP76 gene itself was strongly induced by both ABA treatment and pathogen infection, supporting its role as a positive regulator in ABA-associated immune signaling. Overall, this study demonstrates that OsbZIP76 functions as an important immune regulator by integrating defense gene expression with ABA signaling, providing new insights into the molecular crosstalk between hormonal signaling and pathogen defense mechanisms.},
}
@article {pmid40650142,
year = {2025},
author = {Becchi, G and Whitehead, M and Harvey, JP and Sladen, PE and Dushti, M and Chapple, JP and Yu-Wai-Man, P and Cheetham, ME},
title = {CRISPRa-Mediated Increase of OPA1 Expression in Dominant Optic Atrophy.},
journal = {International journal of molecular sciences},
volume = {26},
number = {13},
pages = {},
doi = {10.3390/ijms26136364},
pmid = {40650142},
issn = {1422-0067},
support = {5167/5168//Fight for Sight/ ; },
mesh = {*GTP Phosphohydrolases/genetics/metabolism ; Humans ; *Optic Atrophy, Autosomal Dominant/genetics/metabolism/therapy/pathology ; HEK293 Cells ; *CRISPR-Cas Systems ; Mitochondria/metabolism/genetics ; Gene Editing/methods ; Gene Expression Regulation ; Retinal Ganglion Cells/metabolism/pathology ; RNA, Guide, CRISPR-Cas Systems/genetics ; },
abstract = {Dominant Optic Atrophy (DOA) is the most common inherited optic neuropathy and presents as gradual visual loss caused by the loss of retinal ganglion cells (RGCs). Over 60% of DOA cases are caused by pathogenic variants in the OPA1 gene, which encodes a mitochondrial GTPase essential in mitochondrial fusion. Currently, there are no treatments for DOA. Here, we tested the therapeutic potential of an approach to DOA using CRISPR activation (CRISPRa). Homology directed repair was used to introduce a common OPA1 pathogenic variant (c.2708_2711TTAGdel) into HEK293T cells as an in vitro model of DOA. Heterozygous c.2708_2711TTAGdel cells had reduced levels of OPA1 mRNA transcript, OPA1 protein, and mitochondrial network alterations. The effect of inactivated Cas9 fused to an activator (dCas9-VPR) was tested with a range of guide RNAs (gRNA) targeted to the promotor region of OPA1. gRNA3 and dCas9-VPR increased OPA1 expression at the RNA and protein level towards control levels. Importantly, the correct ratio of OPA1 isoform transcripts was maintained by CRISPRa. CRISPRa-treated cells showed an improvement in mitochondrial networks compared to untreated cells, indicating partial rescue of a disease-associated phenotype. Collectively, these data support the potential application of CRISPRa as a therapeutic intervention in DOA.},
}
@article {pmid40650127,
year = {2025},
author = {El Hazzouri, S and Al-Rifai, R and Surges, N and Rath, M and Singer, H and Oldenburg, J and El-Maarri, O},
title = {FVIII Trafficking Dynamics Across Subcellular Organelles Using CRISPR/Cas9 Specific Gene Knockouts.},
journal = {International journal of molecular sciences},
volume = {26},
number = {13},
pages = {},
doi = {10.3390/ijms26136349},
pmid = {40650127},
issn = {1422-0067},
support = {2019-7-05; 2020-7-05; 2022-7-02//University Hospital Bonn/ ; IISR-2023-DE-200366//Takeda (Germany)/ ; 91724372//German Academic Exchange Service/ ; Internal Funding//Institute of Experimental Hematology and Transfusion Medicine - University Hospital Bonn/ ; },
mesh = {Humans ; *CRISPR-Cas Systems ; HEK293 Cells ; Protein Transport ; Gene Knockout Techniques ; *Factor VIII/metabolism/genetics ; Calnexin/metabolism/genetics ; Endoplasmic Reticulum/metabolism ; rab GTP-Binding Proteins/metabolism ; *Organelles/metabolism ; rab7 GTP-Binding Proteins ; Calreticulin/metabolism/genetics ; Golgi Apparatus/metabolism ; },
abstract = {Factor VIII (FVIII) interacts with Endoplasmic Reticulum (ER) chaperones Calnexin (CANX) and Calreticulin (CALR) and with ER-Golgi Intermediate Compartment (ERGIC) transporters, Lectin, mannose-binding 1 (LMAN1) and Multiple Coagulation Deficiency 2 (MCFD2). We previously reported that the Gamma-aminobutyric Acid Receptor-associated proteins (GABARAPs) also influence FVIII secretion. Here, we further investigated the intracellular dynamics of FVIII using single and double CRISPR/Cas9 Knockout (KO) models of the abovementioned chaperones as well as the GABARAP proteins in HEK293 cells expressing FVIII. Cellular pathways were manipulated by Brefeldin A (BFA), Chloroquine (CQ), a Rab7 inhibitor, and subjected to glucose starvation. The effect of each KO on FVIII secretion and organelle distribution was assessed by a two-stage chromogenic assay and immunofluorescence (IF) microscopy, prior and upon cell treatments. Using these approaches, we first observed distinct effects of each studied protein on FVIII trafficking. Notably, intracellular localization patterns revealed clustering of FVIII phenotypes in GABARAP[KO], CANX[KO], and CALR[KO] cells together under both basal and treated conditions, an observation that was also reflected in their respective double KO combinations. Besides, a clear involvement of additional components of the endomembrane system was evident, specifically at the trans-Golgi space, as marked by FVIII colocalization with the Ras-like proteins in brain (Rab8 and Rab7) and with the Vesicle-Associated Membrane Protein (VAMP8), along with the observed impact of the selected cell treatments on FVIII phenotypes. These outcomes enhance our understanding of the molecular mechanisms regulating FVIII and pave the way for new perspectives, which could be further projected into FVIII replacement, cell and gene therapies.},
}
@article {pmid40650046,
year = {2025},
author = {Cattaneo, M and Giagnorio, E and Lauria, G and Marcuzzo, S},
title = {Therapeutic Approaches for C9ORF72-Related ALS: Current Strategies and Future Horizons.},
journal = {International journal of molecular sciences},
volume = {26},
number = {13},
pages = {},
doi = {10.3390/ijms26136268},
pmid = {40650046},
issn = {1422-0067},
support = {//Italian Ministry of Health (RRC)/ ; T4-AN-09 prog. ZRPOS2//CALabria HUB per Ricerca Innovativa ed Avanzata- CALHUB.RIA "Creazione di Hub delle Sci-enze della Vita"/ ; prog. ZRA124//AriSLA foundation, "Bulb-Omics"/ ; PNRR-MCNT2-2023-12377336//the European Union - Next Generation EU - NRRP M6C2 - Investment 2.1 Enhancement and strengthening of biomedical research in the NHS,/ ; },
mesh = {Humans ; *C9orf72 Protein/genetics/metabolism ; *Amyotrophic Lateral Sclerosis/genetics/therapy/metabolism/pathology ; Animals ; DNA Repeat Expansion ; Gene Editing ; Genetic Therapy/methods ; Mutation ; Oligonucleotides, Antisense/therapeutic use ; },
abstract = {Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by the loss of upper and lower motor neurons. One of its major genetic causes is C9ORF72, where mutations lead to hexanucleotide repeat expansions in the C9ORF72 gene. These expansions drive disease progression through mechanisms, including the formation of toxic RNAs and the accumulation of damaged proteins such as dipeptide repeats (DPRs). This review highlights these pathogenic mechanisms, focusing on RNA foci formation and the accumulation of toxic DPRs, which contribute to neuronal damage. It also discusses promising targeted therapies, including small molecules and biological drugs, designed to counteract these specific molecular events. Small molecules such as G-quadruplex stabilizers, proteasome and autophagy modulators, and RNase-targeting chimeras show potential in reducing RNA foci and DPR accumulation. Furthermore, targeting enzymes involved in repeat-associated non-AUG (RAN) translation and nucleocytoplasmic transport, which are crucial for disease pathogenesis, opens new therapeutic avenues. Even some anti-viral drugs show encouraging results in preclinical studies. Biological drugs, such as antisense oligonucleotides and gene-editing technologies like CRISPR-Cas, were explored for their potential to specifically target C9ORF72 mutations and modify the disease's molecular foundations. While preclinical and early clinical data show promise, challenges remain in optimizing delivery methods, ensuring long-term safety, and improving efficacy. This review concludes by emphasizing the importance of continued research and the potential for these therapies to alter the disease trajectory and improve patient outcomes.},
}
@article {pmid40649992,
year = {2025},
author = {Reddy, KD and Rathnayake, SNH and Idrees, S and Boedijono, F and Xenaki, D and Padula, MP and Berge, MVD and Faiz, A and Oliver, BGG},
title = {A Novel Regulatory Role for RPS4Y1 in Inflammatory and Fibrotic Processes.},
journal = {International journal of molecular sciences},
volume = {26},
number = {13},
pages = {},
doi = {10.3390/ijms26136213},
pmid = {40649992},
issn = {1422-0067},
support = {GNT1158186//National Health and Research Council, Australia (NHMRC)/ ; },
mesh = {Humans ; *Asthma/genetics/metabolism/pathology ; *Ribosomal Proteins/genetics/metabolism ; Female ; Male ; *Inflammation/genetics/metabolism/pathology ; Fibrosis ; Interleukin-6/genetics/metabolism ; Tenascin/genetics/metabolism ; Animals ; CRISPR-Cas Systems ; },
abstract = {Asthma is a chronic inflammatory respiratory disease well-known to demonstrate sexual dimorphism in incidence and severity, although the mechanisms causing these differences remain incompletely understood. RPS4X and RPS4Y1 are X and Y-chromosome-linked genes coding ribosomal subunits previously associated with inflammation, airway remodelling and asthma medication efficacy. Particularly, RPS4Y1 has been under-investigated within the context of disease, with little examination of molecular mechanisms and pathways regulated by this gene. The ribosome, a vital cellular machinery, facilitates the translation of mRNA into peptides and then proteins. Imbalance or dysfunction in ribosomal components may lead to malfunctioning proteins. Using CRISPR-Cas9 knockout cellular models for RPS4Y1 and RPS4X, we characterised the function of RPS4Y1 in the context of the asthma-relevant processes, inflammation and fibrosis. No viable RPS4X knockouts could be generated. We highlight novel molecular mechanisms such as specific translation of IL6 and tenascin-C mRNA by RPS4Y1 containing ribosomes. Furthermore, an RPS4Y1-centric gene signature correlates with clinical lung function measurements, specifically in adult male asthma patients. These findings inform the current understanding of sex differences in asthma, as females do not produce the RPS4Y1 protein. Therefore, the pathologically relevant functions of RPS4Y1 may contribute to the complex sexually dimorphic pattern of asthma susceptibility and progression.},
}
@article {pmid40649886,
year = {2025},
author = {Chang, TY and Lin, LC and Kao, CY and Lu, JJ},
title = {Study of lug Operon, SCCmec Elements, Antimicrobial Resistance, MGEs, and STs of Staphylococcus lugdunensis Clinical Isolates Through Whole-Genome Sequencing.},
journal = {International journal of molecular sciences},
volume = {26},
number = {13},
pages = {},
doi = {10.3390/ijms26136106},
pmid = {40649886},
issn = {1422-0067},
support = {TCRD-TPE-NSTC-113-18 and TCRD-TPE-114-04(1/3)//Taipei Tzu Chi Hospital, Buddhist Tzu Chi Medical Foundation/ ; NSTC 113-2320-B-303-006//National Science and Technology Council, Taiwan/ ; },
mesh = {*Staphylococcus lugdunensis/genetics/drug effects/isolation &amp; purification ; Whole Genome Sequencing ; *Operon ; Humans ; *Staphylococcal Infections/microbiology ; Multilocus Sequence Typing ; Genome, Bacterial ; Anti-Bacterial Agents/pharmacology ; *Drug Resistance, Bacterial/genetics ; Drug Resistance, Multiple, Bacterial/genetics ; Interspersed Repetitive Sequences ; Phylogeny ; Microbial Sensitivity Tests ; *Bacterial Proteins/genetics ; CRISPR-Cas Systems ; },
abstract = {Staphylococcus lugdunensis is a coagulase-negative staphylococcus known for its significant pathogenic potential, often causing severe infections such as endocarditis and bacteremia, with virulence comparable to S. aureus. Despite general susceptibility to most antibiotics, the emergence of oxacillin-resistant strains is increasingly concerning. This study conducted whole-genome sequencing on 20 S. lugdunensis isolates from Chang Gung Memorial Hospital to explore their genetic diversity, antimicrobial resistance mechanisms, and mobile genetic elements. The lugdunin biosynthetic operon, essential for antimicrobial peptide production, was present in multilocus sequence typing (MLST) types 1, 3, and 6 but absent in STs 4, 27, and 29. Additionally, IS256 insertion elements, ranging from 7 to 17 copies, were identified in four strains and linked to multidrug resistance. CRISPR-Cas systems varied across STs, with type III-A predominant in ST1 and ST6 and type IIC in ST4, ST27, and ST29; notably, ST3 lacked CRISPR systems, correlating with a higher diversity of SCCmec elements and an increased potential for horizontal gene transfer. Phage analysis revealed stable phage-host associations in ST1, ST6, and ST29, whereas ST4 displayed a varied prophage profile. Phenotypic resistance profiles generally aligned with genomic predictions, although discrepancies were observed for aminoglycosides and clindamycin. These findings highlight the complex genetic landscape and evolutionary dynamics of S. lugdunensis, emphasizing the need for genomic surveillance to inform clinical management and prevent the spread of resistant strains.},
}
@article {pmid40648011,
year = {2025},
author = {Fan, X and Zhang, Y and Gu, P and Naz, M},
title = {Epitranscriptomic Control of Drought Tolerance in Rice: The Role of RNA Methylation.},
journal = {Plants (Basel, Switzerland)},
volume = {14},
number = {13},
pages = {},
pmid = {40648011},
issn = {2223-7747},
support = {(2023-BS-209 and 2024-BS-281)//Liaoning Provincial Department of Science and Technology Doctoral Research Initiation Fund Program/ ; K20241057//Wuxi's "Taihu Light" Scientific and Technological Innovation Initiative/ ; JC2024019//Natural science foundation of Nantong/ ; },
abstract = {Drought stress is a predominant abiotic constraint adversely affecting global rice (Oryza sativa) production and threatening food security. While the transcriptional and post-transcriptional regulation of drought-responsive pathways has been widely investigated, the emerging field of epitranscriptomics, particularly RNA chemical modifications such as N6-methyladenosine (m[6]A), adds a new dimension to gene regulation under stress. The most prevalent internal modification in eukaryotic messenger RNA influences RNA metabolism by interacting dynamically with enzymes that add, remove, or recognize the modification. Recent studies in rice reveal that m[6]A deposition is not static but dynamically regulated in response to water-deficit conditions, influencing transcript stability, splicing, nuclear export, and translation efficiency of key drought-responsive genes. This review critically synthesizes current findings on the distribution and functional implications of m[6]A and other epitranscriptomic marks (e.g., 5-methylcytosine [m[5]C], pseudouridine [Ψ]) in modulating rice responses to drought. We discuss the regulatory circuitry involving m[6]A effectors such as OsMTA, OsFIP37, and YTH domain proteins and their integration with known drought-signaling pathways including ABA and reactive oxygen species (ROS) cascades. We also highlight emerging high-resolution technologies such as m[6]A-seq, direct RNA sequencing, and nanopore-based detection that facilitate epitranscriptomic profiling in rice. Finally, we propose future directions for translating epitranscriptomic knowledge into crop improvement, including CRISPR/Cas-based modulation of RNA modification machinery to enhance drought tolerance.},
}
@article {pmid40647992,
year = {2025},
author = {Sutula, M and Tussipkan, D and Kali, B and Manabayeva, S},
title = {Molecular Mechanisms Underlying Defense Responses of Potato (Solanum tuberosum L.) to Environmental Stress and CRISPR/Cas-Mediated Engineering of Stress Tolerance.},
journal = {Plants (Basel, Switzerland)},
volume = {14},
number = {13},
pages = {},
pmid = {40647992},
issn = {2223-7747},
support = {BR21882180//Ministry of Science and Higher Education of the Republic of Kazakhstan/ ; },
abstract = {Environmental stresses, such as drought, salinity, and pathogen attacks, significantly affect potato growth, development, and yield by disrupting key physiological and biochemical processes. Plant responses to these stresses are mediated by changes in gene expression, transcriptional regulation, and the activity of various functional proteins, all of which contribute to the molecular mechanisms of stress tolerance. Genome editing using the CRISPR/Cas9 system has been effectively used to enhance the resistance of potato to environmental stresses and to improve its nutritional value. This article provides a comprehensive review of recent studies retrieved from academic databases focusing on the effects of various environmental stressors on potato growth, yield, and postharvest storage. It also examines the influence of these stresses on the production of secondary metabolites and their associated molecular pathways. Finally, the review highlights advancements in the application of CRISPR/Cas-based genome editing technologies between 2021 and 2025 to improve stress tolerance and nutritional traits in potato plants.},
}
@article {pmid40647524,
year = {2025},
author = {Khan, R and Phely, L and Ehrenfeld, S and Schmitz, T and Veratti, P and Wolfes, J and Shoumariyeh, K and Andrieux, G and Martens, US and Bra, S and Auer, M and Schilling, O and Boerries, M and Speicher, M and Illert, AL and Duyster, J and Miething, C},
title = {Modeling the t(2;5) Translocation of Anaplastic Large Cell Lymphoma Using CRISPR-Mediated Chromosomal Engineering.},
journal = {Cancers},
volume = {17},
number = {13},
pages = {},
doi = {10.3390/cancers17132226},
pmid = {40647524},
issn = {2072-6694},
abstract = {BACKGROUND/OBJECTIVES: ALK+ Anaplastic Large Cell Lymphoma (ALCL) is an aggressive T-cell lymphoma that is characterized by expression of the Anaplastic Lymphoma Kinase (ALK), which is induced by the t(2;5) chromosomal rearrangement, leading to the expression of the NPM-ALK fusion oncogene. Most previous preclinical models of ALK+ ALCL were based on overexpression of the NPM-ALK cDNA from heterologous promoters. Due to the enforced expression, this approach is prone to artifacts arising from synthetic overexpression, promoter competition and insertional variation.<br><br>METHODS: To improve the existing ALCL models and more closely recapitulate the oncogenic events in ALK+ ALCL, we employed CRISPR/Cas-based chromosomal engineering to selectively introduce translocations between the Npm1 and Alk gene loci in murine cells.<br><br>RESULTS: By inducing precise DNA cleavage at the syntenic loci on chromosome 11 and 17 in a murine IL-3-dependent Ba/F3 reporter cell line, we generated de novo Npm-Alk translocations in vivo, leading to IL-3-independent cell growth. To verify efficient recombination, we analyzed the expression of the NPM-ALK fusion protein in the recombined cells and could also show the t(11;17) in the IL-3 independent Ba/F3 cells. Subsequent functional testing of these cells using an Alk-inhibitor showed exquisite responsiveness towards Crizotinib, demonstrating strong dependence on the newly generated ALK fusion oncoprotein. Furthermore, a comparison of the gene expression pattern between Ba/F3 cells overexpressing the Npm-Alk cDNA with Ba/F3 cells transformed by CRISPR-mediated Npm-Alk translocation indicated that, while broadly overlapping, a set of pathways including the unfolded protein response pathway was increased in the Npm-Alk overexpression model, suggesting increased reactive changes induced by exogenous overexpression of Npm-Alk. Furthermore, we observed clustered expression changes in genes located in chromosomal regions close to the breakpoint in the new CRISPR-based model, indicating positional effects on gene expression mediated by the translocation event, which are not part of the older models.<br><br>CONCLUSIONS: Thus, CRISPR-mediated recombination provides a novel and more faithful approach to model oncogenic translocations, which may lead to an improved understanding of the molecular pathogenesis of ALCL and enable more accurate therapeutic models of malignancies driven by oncogenic fusion proteins.},
}
@article {pmid40647454,
year = {2025},
author = {Sidabraite, A and Mosert, PL and Ahmed, U and Jones, SK and Gulla, A},
title = {Advancing Cholangiocarcinoma Diagnosis: The Role of Liquid Biopsy and CRISPR/Cas Systems in Biomarker Detection.},
journal = {Cancers},
volume = {17},
number = {13},
pages = {},
doi = {10.3390/cancers17132155},
pmid = {40647454},
issn = {2072-6694},
support = {101078247/ERC_/European Research Council/International ; IG 5728-2024//European Molecular Biology Organization (EMBO)/ ; P-SV-24-447//Research Council of Lithuania/ ; P-LT-TW-24-21//Research Council of Lithuania/ ; },
abstract = {Background/Objectives: Cholangiocarcinoma (CCA) is a highly heterogeneous malignancy of the biliary tract with limited diagnostic tools for early detection. Current serum markers, such as CA19-9, lack specificity and sensitivity, particularly in early-stage disease, which hinders the effectiveness of curative interventions. This narrative review evaluates the limitations of existing diagnostic approaches and explores the potential of combining liquid biopsy (LB) technologies with CRISPR/Cas-based systems for precise, minimally invasive biomarker detection. Methods: A narrative review was conducted, synthesizing literature from 2018 to 2025 across PubMed, MDPI, Web of Science, Google Scholar, and Embase using MeSH terms such as "cholangiocarcinoma," "liquid biopsy," "miRNA," and "CRISPR/Cas." Results: Circulating microRNAs (e.g., miR-21, miR-16, miR-877) exhibit high diagnostic accuracy. The RACE (Rolling Circle Amplification-assisted CRISPR/Cas9 Cleavage) platform shows promise for detecting extracellular vesicle (EV)-derived miRNAs with high sensitivity and single-nucleotide specificity. When paired with liquid biopsy, CRISPR-based assays enable real-time, cost-effective, and multiplexed detection of tumor-specific biomarkers. Conclusions: The introduction of LB combined with CRISPR/Cas systems could potentially revolutionize the early and accurate diagnosis of CCA, thereby advancing the overall treatment strategy. However, this method is still under development and requires further testing before it can be incorporated into routine diagnostics.},
}
@article {pmid40647071,
year = {2025},
author = {Nascimento, APS and Barros, AN},
title = {Sustainable Innovations in Food Microbiology: Fermentation, Biocontrol, and Functional Foods.},
journal = {Foods (Basel, Switzerland)},
volume = {14},
number = {13},
pages = {},
pmid = {40647071},
issn = {2304-8158},
abstract = {The growing demand for more sustainable food systems has driven the development of solutions based on food microbiology, capable of integrating safety, functionality, and environmental responsibility. This paper presents a critical and up-to-date review of the most relevant advances at the interface between microbiology, sustainability, and food innovation. The analysis is structured around three main axes: (i) microbial fermentation, with a focus on traditional practices and precision technologies aimed at valorizing agro-industrial waste and producing functional foods; (ii) microbial biocontrol, including the use of bacteriocins, protective cultures, bacteriophages, and CRISPR-Cas (Clustered Regularly Interspaced Short Palindromic Repeats-CRISPR-associated)-based tools as alternatives to synthetic preservatives; and (iii) the development of functional foods containing probiotics, prebiotics, synbiotics, and postbiotics, with the potential to modulate the gut microbiota and promote metabolic, immune, and cognitive health. In addition to reviewing the microbiological and technological mechanisms involved, the paper discusses international regulatory milestones, scalability challenges, and market trends related to consumer acceptance and clean labeling. Finally, emerging trends and research gaps are addressed, including the use of omics technologies, artificial intelligence, and unexplored microbial resources. Food microbiology, by incorporating sustainable practices and advanced technologies, is positioned as a strategic pillar for building a healthy, circular, science-based food model.},
}
@article {pmid40646930,
year = {2025},
author = {Han, P and Wang, Y and Sun, H},
title = {Impact of Temperature Stresses on Wheat Quality: A Focus on Starch and Protein Composition.},
journal = {Foods (Basel, Switzerland)},
volume = {14},
number = {13},
pages = {},
pmid = {40646930},
issn = {2304-8158},
abstract = {With climate change, maintaining wheat quality has become essential for the functional properties, end-use, commodity value, and nutritional benefits of wheat flour. Temperature indirectly influences wheat quality by modulating grain size, starch and protein content, and the balance between these components. This review systematically analyzes temperature-mediated alterations in wheat grain quality, with particular emphasis on the two core components: starch and protein. Specifically, daytime warming generally increases protein content while reducing starch accumulation; however, temperatures exceeding 30 °C diminish key protein quality parameters (UPP%, Glu/Gli ratio, HMW-GS/LMW-GS ratio). Nighttime warming enhances protein quality but compromises starch content and yield potential. Conversely, under low-temperature conditions, starch content declines, whereas protein content is primarily influenced by genotypes and treated temperatures. Furthermore, the underlying mechanisms driving temperature-induced changes in wheat quality traits are discussed. However, the mechanisms of temperature effects have not been fully elucidated, and the results often vary between regions or over years. Thus, identifying conserved high/low-temperature resistance genes, QTLs, epialleles, and epiQTL, as well as developing corresponding molecular markers and epi-markers, is an urgent priority. Meanwhile, genome-editing tools such as CRISPR/Cas could serve as a powerful approach for creating new wheat germplasm with durable high/low-temperature resistance.},
}
@article {pmid40646801,
year = {2025},
author = {Han, H and Zhang, D and Hao, W and Liu, A and Xia, N and Cui, M and Luo, J and Jiang, S and Zheng, W and Chen, N and Gu, J and Bai, J and Zhu, J},
title = {Parallel and Visual Detections of ASFV by CRISPR-Cas12a and CRISPR-Cas13a Systems Targeting the Viral S273R Gene.},
journal = {Animals : an open access journal from MDPI},
volume = {15},
number = {13},
pages = {},
pmid = {40646801},
issn = {2076-2615},
support = {32473040; 32172867//Natural Science Foundation of China/ ; },
abstract = {African swine fever virus (ASFV) causes a highly contagious and lethal hemorrhagic disease and significantly threatens the pig industry. There is no commercially effective vaccine available currently, making the detection of ASFV critical for control and prevention. Previously, we established the CRISPR-LbCas12a and LwCRSIRP-Cas13a visual detections of ASFV, separately, targeting the structural p17 gene D117L. In this study, we performed the parallel detections of ASFV based on the conserved viral protease gene S273R using CRISPR-LbCas12a and CRISPR-LbuCas13a systems. Our results showed that both systems are able to specifically detect ASFV as low as two copies of the S273R gene, and effectively detect clinical samples with minimal DNA purification. The work promotes CRISPR-Cas systems for the application of on-site detection in the field.},
}
@article {pmid40600900,
year = {2025},
author = {Hou, Y and Li, Y and Zheng, R and Zhang, F and Guo, F and Li, M and Zeng, M},
title = {Leveraging protein language models for cross-variant CRISPR/Cas9 sgRNA activity prediction.},
journal = {Bioinformatics (Oxford, England)},
volume = {41},
number = {7},
pages = {},
pmid = {40600900},
issn = {1367-4811},
support = {2022YFC3400300//National Key Research and Development Program of China/ ; 62472445//National Natural Science Foundation of China/ ; 2023JJ40763//Hunan Provincial Natural Science Foundation of China/ ; },
mesh = {*CRISPR-Cas Systems ; *RNA, Guide, CRISPR-Cas Systems/genetics/metabolism ; *Gene Editing/methods ; *CRISPR-Associated Protein 9/genetics/metabolism ; Deep Learning ; Computational Biology/methods ; },
abstract = {MOTIVATION: Accurate prediction of single-guide RNA (sgRNA) activity is crucial for optimizing the CRISPR/Cas9 gene-editing system, as it directly influences the efficiency and accuracy of genome modifications. However, existing prediction methods mainly rely on large-scale experimental data of a single Cas9 variant to construct Cas9 protein (variants)-specific sgRNA activity prediction models, which limits their generalization ability and prediction performance across different Cas9 protein (variants), as well as their scalability to the continuously discovered new variants.<br><br>RESULTS: In this study, we proposed PLM-CRISPR, a novel deep learning-based model that leverages protein language models to capture Cas9 protein (variants) representations for cross-variant sgRNA activity prediction. PLM-CRISPR uses tailored feature extraction modules for both sgRNA and protein sequences, incorporating a cross-variant training strategy and a dynamic feature fusion mechanism to effectively model their interactions. Extensive experiments demonstrate that PLM-CRISPR outperforms existing methods across datasets spanning seven Cas9 protein (variants) in three real-world scenarios, demonstrating its superior performance in handling data-scarce situations, including cases with few or no samples for novel variants. Comparative analyses with traditional machine learning and deep learning models further confirm the effectiveness of PLM-CRISPR. Additionally, motif analysis reveals that PLM-CRISPR accurately identifies high-activity sgRNA sequence patterns across diverse Cas9 protein (variants). Overall, PLM-CRISPR provides a robust, scalable, and generalizable solution for sgRNA activity prediction across diverse Cas9 protein (variants).<br><br>The source code can be obtained from https://github.com/CSUBioGroup/PLM-CRISPR.},
}
@article {pmid40490045,
year = {2025},
author = {Ye, X and Jia, H and Zu, Y},
title = {lmod2a mutations affect F-actin and SRF pathway leading to cardiac dysfunction in zebrafish.},
journal = {Developmental biology},
volume = {525},
number = {},
pages = {306-316},
doi = {10.1016/j.ydbio.2025.06.002},
pmid = {40490045},
issn = {1095-564X},
mesh = {Animals ; *Zebrafish/genetics/embryology/metabolism ; *Actins/metabolism/genetics ; *Serum Response Factor/metabolism/genetics ; *Zebrafish Proteins/genetics/metabolism ; Mutation/genetics ; Signal Transduction/genetics ; Cardiomyopathy, Dilated/genetics ; *Heart/embryology/physiopathology ; *Muscle Proteins/genetics/metabolism ; CRISPR-Cas Systems ; *Microfilament Proteins/genetics/metabolism ; Disease Models, Animal ; },
abstract = {Leiomodin 2 (LMOD2), a critical pathogenic gene associated with human dilated cardiomyopathy (DCM), is essential in regulating thin filament length during cardiac development. This study generated a homozygous knockout zebrafish line (lmod2a[-/-]) using CRISPR/Cas9 genome editing. lmod2a[-/-] embryos exhibited impaired locomotor activity alongside irregular heart rhythms, reduced cardiac output, compromised contractility, and delayed calcium transients, as revealed by high-speed imaging and calcium optical mapping. Immunofluorescence staining demonstrated a marked reduction in filamentous actin (F-actin), corroborated by QPCR data showing downregulation of the F-actin marker gene acta1b. Moreover, expression levels of key downstream targets of the serum response factor (SRF) signaling pathway were markedly reduced in mutants. These findings indicate that lmod2a deficiency disrupts F-actin homeostasis and SRF-mediated gene regulation, ultimately leading to defective cardiac performance. This study establishes a novel zebrafish model for investigating LMOD-associated cardiomyopathies and provides valuable insights for future therapeutic interventions targeting actin-related cardiac disorders.},
}
@article {pmid40480611,
year = {2025},
author = {Huang, L and Song, Y and Li, N and Gao, J and Zhang, B and Liu, Z and Zheng, Y},
title = {Deletion of bikaverin and fusarubin biosynthesis gene clusters via CRISPR/Cas9 system in Fusarium fujikuroi and its effect on GA3 biosynthesis.},
journal = {Journal of biotechnology},
volume = {405},
number = {},
pages = {229-237},
doi = {10.1016/j.jbiotec.2025.06.004},
pmid = {40480611},
issn = {1873-4863},
mesh = {*Fusarium/genetics/metabolism ; *Gibberellins/metabolism ; *CRISPR-Cas Systems/genetics ; Multigene Family/genetics ; Gene Deletion ; Fungal Proteins/genetics/metabolism ; Xanthones ; },
abstract = {Gibberellic acid (GA3) is a critical plant hormone with significant agricultural applications, yet its production in Fusarium fujikuroi is constrained by competition for metabolic precursors, particularly acetyl-CoA, which is essential for GA3 biosynthesis. The genome of F. fujikuroi harbors numerous secondary metabolite biosynthetic gene clusters that divert acetyl-CoA away from the GA3 pathway, thereby limiting its yield. To address this challenge, we employed the CRISPR/Cas9 system to delete the bikaverin and fusarubin biosynthesis gene clusters, which are known to compete with GA3 biosynthesis for acetyl-CoA. This genetic intervention resulted in a substantial increase in GA3 production, with the ΔBIKΔFSR strain yielding 31.67 % more GA3 compared to the wild-type strain. Notably, the deletion of these gene clusters not only enhanced GA3 biosynthesis but also improved mycelial growth and carbon assimilation, as evidenced by increased consumption of reducing sugars during fermentation. We further employed qRT-PCR to assess comparative expression levels of genes associated with the glycohydrolysis, glycolysis, and the TCA pathway in engineered strain. Results indicated that removing by-product gene clusters enhances the glycohydrolase system, accelerating carbon assimilation. Given the presence of dozens of secondary metabolite biosynthetic gene clusters in the F. fujikuroi genome, the strategy reported here offers a promising avenue for further enhancing GA3 production by targeting additional non-essential gene clusters.},
}
@article {pmid40472724,
year = {2025},
author = {Shimon, O and Dean, AM and Cohen, S and Moser, AL and Dacso, CC and Gilad, Y and Lonard, DM and O'Malley, BW},
title = {CRISPR-Cas9 engineering of human T regulatory cells - Design and optimization of a manufacturing process.},
journal = {Molecular immunology},
volume = {184},
number = {},
pages = {13-21},
doi = {10.1016/j.molimm.2025.05.019},
pmid = {40472724},
issn = {1872-9142},
mesh = {Humans ; *T-Lymphocytes, Regulatory/immunology ; *CRISPR-Cas Systems/genetics/immunology ; Animals ; *Gene Editing/methods ; Mice ; Immunotherapy, Adoptive/methods ; },
abstract = {Regulatory T cells (Tregs) are a subset of CD4 + T cells that comprise 5-10 % of the total CD4 + T cell population. Tregs, which are critically important for the maintenance of immune tolerance and immune homeostasis, are distinguished from other subtypes of CD4 + T cells by the expression of the transcription factor FOXP3. Because of the centrality to immunoregulation, Tregs have gained increasing attention as promising targets for clinical applications in autoimmune diseases, transplant rejection and graft-versus-host disease (GvHD). However, the essential role of Tregs in the complex network of the immune system implies their targeting as a promising therapeutic approach also in other medical indications, such as neurodegenerative diseases and cancer. Our group recently published a study showing that genetically modified Tregs are capable of clearing solid malignancies in various mice models, including an aggressive triple negative breast cancer (TNBC) and prostate cancer, which provides the impetus to develop an adoptive cell therapy using Steroid Receptor Coactivator 3 (SRC-3) knock out (KO) Tregs. It is well known that isolation, genetic editing and the expansion of Tregs as a homogenous and healthy population present specific technical challenges. In this context, here we outline the development of a process for the production of SRC-3 KO human Tregs (hTregs), which can subsequently be adapted for Current Good Manufacturing Practice (cGMP) settings to facilitate clinical-scale production.},
}
@article {pmid40449665,
year = {2025},
author = {Gomes, D and Rodrigues, JL and Scrutton, NS and Rodrigues, LR},
title = {De novo production of prenylnaringenin compounds by a metabolically engineered Escherichia coli.},
journal = {Journal of biotechnology},
volume = {405},
number = {},
pages = {215-228},
doi = {10.1016/j.jbiotec.2025.05.017},
pmid = {40449665},
issn = {1873-4863},
mesh = {*Escherichia coli/genetics/metabolism ; *Metabolic Engineering/methods ; *Flavanones/metabolism ; Dimethylallyltranstransferase/metabolism/genetics ; Biosynthetic Pathways/genetics ; CRISPR-Cas Systems ; Organophosphorus Compounds/metabolism ; Hemiterpenes ; },
abstract = {Prenylnaringenin (PN) compounds, namely 8-prenylnaringenin (8-PN), 3'-prenylnaringenin (3'-PN), and 6-prenylnaringenin (6-PN), are reported to have several interesting bioactivities. This study aimed to validate a biosynthetic pathway for de novo production of PN in Escherichia coli. A previously optimized E. coli chassis capable of efficiently de novo producing naringenin was used to evaluate eleven prenyltransferases (PTs) for the production of PN compounds. As PT reaction requires dimethylallyl pyrophosphate (DMAPP) as extended substrate that has limited availability inside the cells, clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein 12a (Cas12a) (CRISPR-Cas12a) was used to construct ten boosted DMAPP-E. coli strains. All the PTs, in combination with the naringenin biosynthetic pathway, were tested in these strains. Experiments in 96-well deep well plates identified twelve strains capable of producing PN. E. coli M-PAR-121 with the integration of the 1-deoxy-D-xylulose-5-phosphate synthase (DXS) gene from E. coli (EcDXS) into the lacZ locus of the genome (E. coli M-PAR-121:EcDXS) expressing the soluble aromatic PT from Streptomyces roseochromogenes (CloQ) and the naringenin biosynthetic pathway was selected as the best producer strain. After optimizing the production media in shake flasks, 160.57 µM of 3'-PN, 4.4 µM of 6-PN, and 2.66 µM of 8-PN were obtained. The production was then evaluated at the bioreactor scale and 397.57 µM of 3'-PN (135.33 mg/L) and 25.61 µM of 6-PN (8.72 mg/L) were obtained. To the best of our knowledge, this work represents the first report of de novo production of PN compounds using E. coli as a chassis.},
}
@article {pmid40404079,
year = {2025},
author = {Ferrari, K and Gurung, S and Loges, LN and Batta, SPR and Hammond, MA and Griciunaite, M and DeMoya, R and Restrepo, NK and Sumanas, S},
title = {Zebrafish Kelch-like family member 4 is required for vasculogenesis and hematopoiesis.},
journal = {Developmental biology},
volume = {525},
number = {},
pages = {1-12},
pmid = {40404079},
issn = {1095-564X},
mesh = {Animals ; *Zebrafish/embryology/genetics/metabolism ; *Hematopoiesis/physiology/genetics ; *Zebrafish Proteins/genetics/metabolism ; *Neovascularization, Physiologic/genetics/physiology ; Gene Expression Regulation, Developmental ; Hematopoietic Stem Cells/metabolism/cytology ; Endothelial Cells/metabolism ; Apoptosis ; Embryo, Nonmammalian/metabolism ; CRISPR-Cas Systems ; Cell Proliferation ; Transcription Factors/metabolism/genetics ; Mutation ; },
abstract = {Molecular mechanisms regulating vascular development and hematopoiesis are still incompletely understood. The KLHL (Kelch-like) family of proteins function as adapters to target proteins for ubiquitination. However, their role in vascular development has not been previously analyzed. Here we have characterized a novel regulator of vascular development, kelch-like family member 4 (klhl4) in zebrafish. We show that zebrafish klhl4 is expressed in early vascular endothelial and hematopoietic progenitors, while its expression is restricted to vascular endothelial cells during later developmental stages. To determine the functional role of klhl4, we generated loss-of-function zebrafish mutants using CRISPR/Cas9 genome editing. klhl4 mutant embryos were viable, yet they exhibited delayed sprouting of intersegmental vessels (ISVs), which correlated with reduced expression of vascular endothelial and erythroid specific molecular markers. Time-lapse imaging showed that vascular endothelial and hematopoietic progenitor cells exhibit delayed migration towards the midline and undergo increased apoptosis and reduced proliferation in klhl4 mutants. Expression of npas4l and etv2/etsrp, two master regulators of endothelial and hematopoietic development, was reduced in klhl4 mutants, suggesting that some vascular defects could be caused by the reduction of npas4l and etv2 expression. However, npas4l or etv2 overexpression failed to rescue ISV sprouting defects in klhl4 mutants, suggesting that klhl4 may promote vasculogenesis by additional mechanisms. In summary, our findings demonstrate a novel role for zebrafish klhl4 in regulating vascular endothelial and hematopoietic development during embryogenesis. Because the Klhl4 protein sequence is highly conserved between different vertebrates, it is likely that it may play a similar role in other organisms.},
}
@article {pmid40646370,
year = {2025},
author = {Liu, Y and Liu, H and Wang, G and Jia, X and Zhou, J and Dong, X and Li, H and Chang, H and Li, Z and Jin, Z and Li, K},
title = {Dual-Check CRISPR-SERS strategy for sensitively detecting Monkeypox DNA and its single-base mutated DNA.},
journal = {Mikrochimica acta},
volume = {192},
number = {8},
pages = {497},
pmid = {40646370},
issn = {1436-5073},
support = {2025GXNSFHA069082//Guangxi Natural Science Foundation Project/ ; AD22035215//Guangxi Science and Technology Project/ ; 2022YFE0134600//National Key Research and Development Program of China/ ; AB22080055//Key Research and Development Program of Guangxi/ ; },
mesh = {*Spectrum Analysis, Raman/methods ; Metal Nanoparticles/chemistry ; Gold/chemistry ; *CRISPR-Cas Systems ; *DNA, Viral/genetics/analysis ; DNA, Single-Stranded/chemistry/genetics ; Limit of Detection ; Plasmids/genetics ; CRISPR-Associated Proteins/metabolism ; Endodeoxyribonucleases/metabolism ; Carbocyanines/chemistry ; Silver/chemistry ; Bacterial Proteins/metabolism ; },
abstract = {This study presents a convenient and efficient dual-check strategy for detecting Monkeypox DNA utilizing the SERS (Surface-enhanced Raman Spectroscopy)-CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) system. The Monkeypox plasmid DNA (mpDNA) is recognized by the CRISPR RNA (crRNA)-Cas12a protein complex, where crRNA encompasses a targeting complementary sequence. Upon recognition, the trans-cleavage activity of Cas12a is activated and trans-cleaves the probe DNA (Cy3-ssDNA) which is modified on Au nanoparticles (AuNPs). As the ssDNA strand is cleaved, Cy3 molecules are released in the solution, while the amount of Cy3 modified on the AuNPs decreases. The free Cy3 molecules are collected from the supernatant, and their SERS intensities are measured using the silver nanopillar (AgNRs) substrate. The mpDNA with varying concentration from 5 nM to 0.5 fM can be quantitatively determined based on the SERS signals of free Cy3 and the collected nanotag. This strategy allows the detection of mpDNA with a concentration of 50 fM within 60 min. Moreover, the strategy can successfully detect single-base mutated mpDNA. Owing to the non-specific trans-cleavage activity of the protein, this strategy can be adapted to various nucleic acid detection scenarios by designing complementary RNA and DNA sequences.},
}
@article {pmid40646080,
year = {2025},
author = {Jung, Y and Seo, E and Yang, S and Bae, S and Kim, JH and Jang, HK and Jo, DH},
title = {Establishment and rescue of fibroblast cell lines carrying a nonsense mutation of RB1 by CRISPR-based base editing.},
journal = {Scientific reports},
volume = {15},
number = {1},
pages = {25074},
pmid = {40646080},
issn = {2045-2322},
support = {18-2023-0010//Seoul National University Hospital/ ; GTL24021-000//Ministry of Science and ICT, South Korea/ ; RS-2024-00400829//Ministry of Science and ICT, South Korea/ ; RS-2023-00260351//Ministry of Science and ICT, South Korea/ ; RS-2023-00301850//Ministry of Education/ ; RS-2024-00438476//Ministry of Health &amp; Welfare/ ; },
mesh = {*Codon, Nonsense/genetics ; Humans ; *Gene Editing/methods ; *Fibroblasts/metabolism/cytology ; *CRISPR-Cas Systems ; *Retinoblastoma Binding Proteins/genetics/metabolism ; Cell Line ; Cell Proliferation/genetics ; Retinoblastoma/genetics/pathology ; Ubiquitin-Protein Ligases ; },
abstract = {Pathogenic variants of the RB1 gene have commonly been found in many cancer types, including retinoblastoma. Nonsense mutations are the most common mutation type in retinoblastoma; however, few cell lines mimic nonsense mutations in the RB1 gene that are commonly observed in patients. Here, we established retinoblastoma-like cell lines carrying mono- and bi-allelic nonsense mutations in the RB1 gene. We introduced the R552X mutation using target activation-induced cytidine deaminase base editing and successfully constructed cell lines carrying mono- and bi-allelic mutations. The model cell lines showed decreased RB1 expression at both the mRNA and protein levels, and increased cell proliferation. Furthermore, we rescued the nonsense mutation in the RB1 gene in model cell lines by converting stop codon 552 to tryptophan using an adenine base editor. This approach may be applicable for establishing cell lines with pathogenic variants found in patients and suggests a strong potential for the application of gene editing as a therapeutic strategy.},
}
@article {pmid40645983,
year = {2025},
author = {Ching, RW and Świst-Rosowska, KM and Erikson, G and Koschorz, B and Engist, B and Jenuwein, T},
title = {Forced expression of MSR repeat transcripts above a threshold limit breaks heterochromatin organisation.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {6420},
pmid = {40645983},
issn = {2041-1723},
support = {CRC992 'MEDEP&apos;//Deutsche Forschungsgemeinschaft (German Research Foundation)/ ; },
mesh = {*Heterochromatin/metabolism/genetics ; Animals ; Mice ; *DNA, Satellite/genetics/metabolism ; Fibroblasts/metabolism ; Transcription, Genetic ; CRISPR-Cas Systems ; Chromosome Segregation/genetics ; },
abstract = {Mouse heterochromatin is characterised by transcriptionally competent major satellite repeat (MSR) sequences and it has been proposed that MSR RNA contributes to the integrity of heterochromatin. We establish an inducible dCas9-effector system in mouse embryonic fibroblasts, where we can modulate MSR transcription through the targeting of a dCas9-Repressor or a dCas9-Activator. With this system, we can define a threshold limit of >300-fold deregulation of MSR transcript levels, above which the structural organisation of heterochromatin becomes disrupted. MEF cells expressing MSR RNA above this threshold limit are not viable and the defects in heterochromatin organisation and chromosome segregation cannot be reverted. This study highlights the importance of restricting MSR RNA output to maintain heterochromatin integrity and relates MSR transcript levels to either physiological or pathological conditions. It also reveals that the structural organisation of heterochromatin is governed by the transcriptional chromatin state and associated MSR RNA of the MSR repeats.},
}
@article {pmid40393529,
year = {2025},
author = {Hegeman, CV and Elsharkasy, OM and Driedonks, TAP and Friesen, KRJ and Vader, P and de Jong, OG},
title = {Modulating binding affinity of aptamer-based loading constructs enhances extracellular vesicle-mediated CRISPR/Cas9 delivery.},
journal = {Journal of controlled release : official journal of the Controlled Release Society},
volume = {384},
number = {},
pages = {113853},
doi = {10.1016/j.jconrel.2025.113853},
pmid = {40393529},
issn = {1873-4995},
mesh = {*Extracellular Vesicles/metabolism ; *CRISPR-Cas Systems ; *Aptamers, Nucleotide/administration &amp; dosage/genetics/metabolism ; Humans ; *Ribonucleoproteins/administration &amp; dosage ; RNA, Guide, CRISPR-Cas Systems/genetics/administration &amp; dosage ; *CRISPR-Associated Protein 9 ; HEK293 Cells ; Gene Editing ; },
abstract = {The CRISPR/Cas9 toolbox consists of modular nucleases that can be employed to efficiently modify genomic sequences with high specificity. However, delivery of the large Cas9-sgRNA ribonucleoprotein (RNP) complexes remains challenging due to their immunogenicity, size, and overall negative charge. An approach to overcome these limitations is the use of extracellular vesicles (EVs) as intracellular delivery vehicles. EVs exhibit the natural ability to carry and deliver RNA and proteins across biological barriers, and can be engineered to load and deliver a variety of biotherapeutic molecules. Previous studies have shown that efficient EV-mediated cargo delivery does not only require active loading strategies, but also benefits from strategies to release cargo from the EV membrane. Here, we load Cas9 RNP complexes into EVs by expressing sgRNAs containing MS2 aptamers (MS2-sgRNAs), alongside Cas9 and a fusion protein of CD63 and tandem MS2 coat proteins (MCPs). We demonstrate that efficient Cas9 RNP delivery can also be facilitated by modulating the binding affinity between MS2 aptamers and the MCPs. To study the effect of altering the binding affinity between the MS2 hairpin and the MCP on Cas9 RNP delivery, various mutations affecting the binding affinity were made in both the interacting MS2-hairpin and the RNA-binding domain of the MCPs. Comparing Cas9 RNP delivery of the modulated MS2-sgRNAs revealed that adapting binding affinity highly affects functional RNP delivery. Mutations resulting in high affinity did not facilitate efficient RNP delivery unless combined with a photo-inducible release strategy, showing that cargo release was a limiting factor in RNP delivery. Mutations that decreased affinity resolved this issue, resulting in Cas9 RNP delivery without the requirement of additional release strategies. However, further decreasing affinity resulted in decreased Cas9 gene-editing efficiency due to decreased levels of Cas9 RNP loading into EVs. A similar effect on functional delivery was seen after modification of the RNA-binding domain of the MCPs. Our results demonstrate that EVs are capable of functional Cas9-sgRNA complex delivery, and that modulation of binding affinity can be used to increase efficient functional delivery with non-covalent loading constructs, without the need for additional engineering strategies for cargo release.},
}
@article {pmid40644543,
year = {2025},
author = {Cao, D and Zhu, J and Guo, Y and Zhou, Y and Zeng, J and Tu, Y and Zhao, Z and Xie, L and Song, E and Zhu, M and Yin, L},
title = {Dynamically covalent lipid nanoparticles mediate CRISPR-Cas9 genome editing against choroidal neovascularization in mice.},
journal = {Science advances},
volume = {11},
number = {28},
pages = {eadj0006},
doi = {10.1126/sciadv.adj0006},
pmid = {40644543},
issn = {2375-2548},
mesh = {Animals ; *Choroidal Neovascularization/genetics/therapy/pathology ; *Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; Mice ; *Nanoparticles/chemistry ; Vascular Endothelial Growth Factor A/genetics/antagonists &amp; inhibitors ; RNA, Guide, CRISPR-Cas Systems/genetics ; *Lipids/chemistry ; Disease Models, Animal ; Humans ; RNA, Messenger/genetics ; Retinal Pigment Epithelium/metabolism ; CRISPR-Associated Protein 9 ; Liposomes ; },
abstract = {As an important modality for choroidal neovascularization (CNV) treatment, intravitreal injection of vascular endothelial growth factor A (VEGFA) inhibitors suffers from undesired response rate, low patient compliance, and ocular damage. Here, dynamically covalent lipid nanoparticles (LNPs) were engineered to mediate VEGFA gene editing and CNV treatment by codelivering Cas9 mRNA (mCas9) and single guide RNA (sgRNA) targeting VEGFA (sgVEGFA). A library of lipidoids bearing iminoboronate ester linkage was developed via facile "one-pot" synthesis, and the top-performing lipidoid-A4B3C7 was formulated into LNP-A4B3C7 with the highest mRNA transfection efficiency. Inside the diseased retinal pigment epithelial cells, LNPs were dissociated upon H2O2-triggered lipidoid degradation, facilitating mRNA/sgRNA release to potentiate the gene editing efficiency. In laser-induced CNV mice, mCas9/sgVEGFA@LNP-A4B3C7 after single intravitreal injection led to pronounced VEGFA disruption and CNV area reduction, outperforming the clinical anti-VEGF drug in eliciting sustained therapeutic effect. This study establishes a robust nonviral platform for mRNA delivery and genome editing and renders a promising strategy for CNV treatment.},
}
@article {pmid40644365,
year = {2025},
author = {Yan, J and Dong, H and Tian, T and Xiao, C and Sun, Y and Gong, J and Xia, Q and Hou, Y},
title = {Doublesex and GATAβ4 synergistically regulate the sex-dimorphic expression of storage protein 1 in Bombyx mori.},
journal = {PLoS genetics},
volume = {21},
number = {7},
pages = {e1011762},
doi = {10.1371/journal.pgen.1011762},
pmid = {40644365},
issn = {1553-7404},
mesh = {Animals ; *Bombyx/genetics ; Female ; Male ; *Insect Proteins/genetics/metabolism ; Promoter Regions, Genetic ; CRISPR-Cas Systems ; Sex Characteristics ; *GATA Transcription Factors/genetics/metabolism ; Gene Expression Regulation ; Transcription Factors/genetics ; DNA-Binding Proteins ; },
abstract = {Sexually dimorphic traits are widespread in organisms and are crucial for reproduction and behavior. These traits are typically controlled by sex-specific genes. However, their regulatory mechanisms are complex and incompletely understood. In Bombyx mori, a group of sex-differential storage proteins (SPs) exists, with storage protein 1 (SP1) expressed exclusively in females. In this study, we used the CRISPR/Cas9 system to knock out the doublesex gene and found that SP1 expression was sharply upregulated in male doublesex mutants and downregulated in female doublesex mutants, which suggests that doublesex is a key factor in the sex-differential expression of SP1. Then, we revealed that the female-specific doublesex isoform (dsxF) bound to and activated the SP1 promoter more strongly than the male-specific isoform (dsxM). Meanwhile, a transcription factor named GATAβ4 was found to be involved in the regulation by doublesex. Overexpression of GATAβ4 in Bombyx mori larvae affected adult reproductive behavior and dramatically upregulated SP1 expression in males. Furthermore, GATAβ4 interacted with both dsxF and dsxM, promoting nuclear translocation of dsxM, which in turn inhibited GATAβ4 binding to the SP1 promoter. In total, we found that dsxM did not directly repress SP1 expression in males but instead cooperated with other transcription factors to regulate downstream gene expression. These findings provide new insights into the regulation of sex-specific genes and the mechanisms controlling dimorphic traits.},
}
@article {pmid40643795,
year = {2025},
author = {Kim, YM and Na, HJ and Kwon, DH and Lee, JH and Park, BM and Lee, S and Nam, TW and Park, MY and Park, SH and Kim, SJ and Choi, B and Lee, HW},
title = {Generation of NOD SCID mice with near-complete deletions of Il2rg and Prkdc for human cancer and HSC engraftment.},
journal = {Transgenic research},
volume = {34},
number = {1},
pages = {35},
pmid = {40643795},
issn = {1573-9368},
support = {RS-2024-00359509//National Research Foundation of Republic of Korea and Ministry of Science and ICT/ ; RS-2023-00224201//National Research Foundation of Republic of Korea and Ministry of Science and ICT/ ; RS-2023-00224201//National Research Foundation of Republic of Korea and Ministry of Science and ICT/ ; RS-2023-00224201//National Research Foundation of Republic of Korea and Ministry of Science and ICT/ ; RS-2023-00224201//National Research Foundation of Republic of Korea and Ministry of Science and ICT/ ; 20012378//the Ministry of Trade, Industry &amp; Energy (MOTIE, Korea)/ ; 20012378//the Ministry of Trade, Industry &amp; Energy (MOTIE, Korea)/ ; 20012378//the Ministry of Trade, Industry &amp; Energy (MOTIE, Korea)/ ; 20012378//the Ministry of Trade, Industry &amp; Energy (MOTIE, Korea)/ ; 20012378//the Ministry of Trade, Industry &amp; Energy (MOTIE, Korea)/ ; 20012378//the Ministry of Trade, Industry &amp; Energy (MOTIE, Korea)/ ; S3142545//the Ministry of SMEs and Startups (MSS, Korea)/ ; S3142545//the Ministry of SMEs and Startups (MSS, Korea)/ ; S3142545//the Ministry of SMEs and Startups (MSS, Korea)/ ; S3142545//the Ministry of SMEs and Startups (MSS, Korea)/ ; S3142545//the Ministry of SMEs and Startups (MSS, Korea)/ ; },
mesh = {Animals ; Humans ; Mice ; *Interleukin Receptor Common gamma Subunit/genetics ; Mice, Inbred NOD ; Mice, SCID ; CRISPR-Cas Systems ; *DNA-Activated Protein Kinase/genetics ; *Neoplasms/genetics/therapy/pathology ; *Hematopoietic Stem Cell Transplantation ; Disease Models, Animal ; Mice, Knockout ; },
abstract = {Immunodeficient mouse models are invaluable tools for preclinical research, particularly for cancer therapies and studies of the human immune system. Notably, strains with combined Prkdc (scid) and Il2rg (null) mutations-such as NOG and NSG mice- are widely used due to their profound immunodeficiency, allowing efficient engraftment of various human cells. However, these models were generated by disrupting the Il2rg gene through replacement with a neomycin resistance (Neo) cassette in embryonic stem cells. Incomplete excision of this cassette can inadvertently alter the expression of neighboring genes, thereby introducing potential confounding variables. In addition, they may still express mutant mRNAs that escape nonsense-mediated decay (NMD) and/or produce truncated proteins with residual activity, potentially compromising the interpretation of experimental outcomes. To address this, we developed the N2G mouse strain (NOD-2-Genes KO) where almost all genomic loci of both Prkdc and Il2rg genes are deleted via CRISPR/Cas9 genome editing. N2G mice exhibited tumor growth comparable to NOG mice following the transplantation with several human cancer cell lines. Moreover, human CD34[+] cord blood (CB) cells engrafted into N2G mice showed robust reconstitution of human immune cells, especially T cells in peripheral blood, spleen and bone marrow, compared to NSG mice. These results suggest that N2G mice, lacking residual mutant mRNA and the exogenous Neo resistant gene, offer an advanced model for preclinical studies.},
}
@article {pmid40643739,
year = {2025},
author = {Friberg, M and Sharma, S and Sitbon, F and Andersson, M and Hofvander, P},
title = {Modifying the potato tuber storage protein patatin targeting improved thermal stability.},
journal = {Planta},
volume = {262},
number = {2},
pages = {46},
pmid = {40643739},
issn = {1432-2048},
support = {2018-01846//Svenska Forskningsr&#xe5;det Formas/ ; "Breeding of starch potatoes"//SLU Grogrund/ ; "Potato tuber//C4F/ ; sink//C4F/ ; starch development"//C4F/ ; },
mesh = {*Solanum tuberosum/genetics/metabolism ; *Plant Proteins/genetics/metabolism/chemistry ; *Plant Tubers/genetics/metabolism ; Gene Editing/methods ; Protein Stability ; CRISPR-Cas Systems ; Multigene Family ; Carboxylic Ester Hydrolases ; },
abstract = {Gene editing of the patatin gene cluster using a single-guide RNA sequence consistently modifies over 10% of the targeted genes in modified individuals. Patatins have gained recent attention, as a group of highly nutritious proteins with excellent functional properties. Some techniques have been suggested for industrial-scale patatin purification, mostly as a by-product from potato starch processing. The purification process has proved to be a challenge due to the low thermostability of patatins, especially under acidic conditions. One strategy to make patatin more accessible for extraction would be to stabilize the protein structure through the introduction of point mutations. Here, we show that the tuber expression of patatin genes is dominated by a few genes from the extended gene family, most of which were predicted to be catalytically inactive. We have further evaluated the suitability of the patatin gene cluster as a target for clustered regularly interspaced repeat (CRISPR)/Cas9-based mutagenesis. In the mutation study, we show that targeting using a single single-stranded guide RNA (sgRNA) can lead to mutations in over 10% of all alleles. Finally, four patatin variants with amino acid substitutions were designed based on in silico analysis of patatin protein structure. These modified patatins were then heterologously expressed in bacteria and evaluated for increased thermostability. While none of the mutant proteins performed better than a wild-type variant, with regard to their thermal properties, one candidate proved to be less sensitive to shifting pH, making it an interesting candidate for further optimizations.},
}
@article {pmid40642869,
year = {2025},
author = {Li, Y and Tang, Y and Li, X and Høyland-Kroghsbo, NM and Ingmer, H and Jiao, X and Li, Q},
title = {Quorum Sensing Inhibits Type III-A CRISPR-Cas System Activity Through Repressing Positive Regulators SarA and ArcR in Staphylococcus Aureus.},
journal = {Advanced science (Weinheim, Baden-Wurttemberg, Germany)},
volume = {},
number = {},
pages = {e06049},
doi = {10.1002/advs.202506049},
pmid = {40642869},
issn = {2198-3844},
support = {2024YFE0198800//National Key Research and Development Program of China/ ; 2023YFD1800503//National Key Research and Development Program of China/ ; 2022YFC2604200//National Key Research and Development Program of China/ ; BK20190883//Natural Science Foundation of Jiangsu Province/ ; 32072821//National Natural Science Foundation of China/ ; 31730094//National Natural Science Foundation of China/ ; KYCX21_3267//Postgraduate Research &amp; Practice Innovation Program of Jiangsu Province/ ; R264-2017-3936//Lundbeck Foundation/ ; 2024ZB284//Jiangsu Funding Program for Excellent Postdoctoral Talent/ ; JX103SYL2024060102//Open Co-operation Project of Center for Global Health/ ; //Priority Academic Program Development of Jiangsu Higher Education Institutions/ ; },
abstract = {CRISPR-Cas is an adaptive immune system that protects prokaryotes from the invasion of foreign genetic elements. The components and immunity mechanisms of CRISPR-Cas have been extensively studied, but the regulation of this system in Staphylococci remains unclear. Here, it is shown that the cell-cell communication, known as quorum sensing (QS), inhibits the expression and activity of the type III-A CRISPR-Cas system in S. aureus. The QS regulator, AgrA, directly binds to the promoters of two transcriptional regulators encoding the genes sarA and arcR to inhibit their expression. However, both SarA and ArcR act as positive regulators that promote the transcription of cas genes by directly binding to a novel promoter Pcas. Furthermore, the Pcas of 300 bp located in cas1 displays as a critical regulatory node to initiate the transcription of cas10 and csm3. Our data reveal a new regulatory mechanism for QS-mediated repression of the Type III-A CRISPR-Cas system, which may allow S. aureus to acquire foreign genetic elements encoding antibiotic resistance or virulence factors specifically at high cell density.},
}
@article {pmid40641638,
year = {2025},
author = {Hua, G and He, C and Zuo, E},
title = {SuperDecode: A versatile toolkit for mutation analysis in genome editing.},
journal = {aBIOTECH},
volume = {6},
number = {2},
pages = {377-380},
pmid = {40641638},
issn = {2662-1738},
abstract = {The CRISPR-Cas system has revolutionized modern life sciences, enabling groundbreaking applications ranging from functional genomics to therapeutic development. Despite its transformative potential, significant technical limitations persist in current computational tools for quantifying editing efficiency - particularly concerning data processing capabilities, analytical throughput, and operational flexibility. This research presents SuperDecode, a novel computational framework designed to address these methodological constraints. The SuperDecode offers key advantages, including local processing capabilities, large-size sequencing files, batch-processing, and diversified operational functions.},
}
@article {pmid40641119,
year = {2025},
author = {Du, J and Gong, X and Huang, R and Zheng, B and Chen, C and Yang, Z},
title = {Harnessing CRISPR/Cas9 to overcome targeted therapy resistance in non‑small cell lung cancer: Advances and challenges (Review).},
journal = {Oncology reports},
volume = {54},
number = {3},
pages = {},
doi = {10.3892/or.2025.8944},
pmid = {40641119},
issn = {1791-2431},
mesh = {Humans ; *Carcinoma, Non-Small-Cell Lung/genetics/drug therapy/therapy ; *CRISPR-Cas Systems/genetics ; *Drug Resistance, Neoplasm/genetics ; *Lung Neoplasms/genetics/drug therapy/therapy ; *Gene Editing/methods ; Molecular Targeted Therapy/methods ; Genetic Therapy/methods ; },
abstract = {Targeted therapy has markedly improved outcomes for patients with non‑small cell lung cancer (NSCLC). However, the emergence of drug resistance remains a major clinical challenge, limiting long‑term treatment efficacy. Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9, a revolutionary gene‑editing technology, offers precise and efficient genetic modifications, providing new insights into the mechanisms of drug resistance in NSCLC. The present review explored the application of CRISPR/Cas9 in overcoming resistance associated with key oncogenic drivers, including EGFR, KRAS, ALK, ROS1, MET and BRAF. It summarized recent advances in CRISPR‑based strategies to reverse resistance, enhance targeted therapy effectiveness and develop potential therapeutic interventions. Additionally, it discussed current limitations, including off‑target effects, delivery challenges and ethical concerns, while highlighting future directions for clinical translation. Using CRISPR/Cas9 technology may pave the way for novel, personalized treatment approaches in NSCLC, ultimately improving patient outcome.},
}
@article {pmid40457715,
year = {2025},
author = {Bourel, C and Souza-Fonseca-Guimaraes, F and Lesage, S},
title = {Highlights of 2024: unleashing the power of NK cells-cancer's worst nightmare.},
journal = {Immunology and cell biology},
volume = {103},
number = {6},
pages = {526-529},
doi = {10.1111/imcb.70037},
pmid = {40457715},
issn = {1440-1711},
mesh = {Humans ; *Killer Cells, Natural/immunology ; *Neoplasms/therapy/immunology ; *Immunotherapy, Adoptive/methods ; Animals ; CRISPR-Cas Systems ; Cytokines/metabolism ; Lymphocyte Activation ; Cytotoxicity, Immunologic ; },
abstract = {In this article for the Highlights of 2024 Series, we discuss strategies to enhance NK cell-based cancer therapies. These include (1) cytokine expression on bacterial membranes to boost NK cell activation in tumors, (2) optimizing CAR-NK cell manufacturing for improved efficacy, (3) using CRISPR-Cas9 to identify and target inhibitory genes, and (4) using tetraspecific engagers to enhance cytotoxicity and cytokine memory-like NK cells strengthening anti-tumor responses. This year's progress holds much promise for cancer treatments exploiting NK cells.},
}
@article {pmid40640739,
year = {2025},
author = {Dai, P and Huang, T and Ye, X and Mi, S and Zhang, J and Luo, X and Hu, D and Zhang, H},
title = {K1 Klebsiella pneumoniae is more conserved than K2 for both virulence plasmid and chromosome.},
journal = {BMC genomics},
volume = {26},
number = {1},
pages = {652},
pmid = {40640739},
issn = {1471-2164},
support = {2023KY1326//Zhejiang Provincial Health Commission/ ; 82172332//National Natural Science Foundation of China/ ; GSWS2019039//Gusu Health Youth Talent of Suzhou/ ; SKY2021007//Science and Technology Program of Suzhou/ ; XKTJ-TD202001//Discipline Construction of the Second Affiliated Hospital of Soochow University/ ; },
abstract = {OBJECTIVE: To illustrate the differences between K1 and K2 Klebsiella pneumoniae strains.<br><br>METHODS: Totally 68 K1 and 99 K2 K. pneumoniae strains from GenBank were analyzed for virulence genes, sequence types (STs), restriction-modification (R-M) systems, and clustered regularly interspaced short palindromic repeat (CRISPR)-Cas systems. Phylogenetic trees of the virulence plasmids and chromosomes in the strains were built using kSNP4.<br><br>RESULTS: Virulence genes peg-344, allS, p-rmpA, p-rmpA2, c-rmpA, iroN, and iucA were more prevalent in K1 strains than K2. K1 strains were categorized into 7 STs with 79.41% being ST23 while K2 strains were categorized into 14 STs with 38.38% being ST14. K1 strains showed higher rates of CRISPR-Cas systems than K2 while lower rates of Type I and II R-M systems were found in K1 strains than K2. More rates of virulence plasmids (52/68 vs. 24/99) were found in K1 strains than K2. Based upon the phylogenetic tree of virulence plasmids, 46 in K1 strains belonged to the same clade while 11 and 7 virulence plasmids in K2 strains constituted the 2 major clades. For the chromosomes, 61 K1 strains belonged to the same clade while 99 K2 strains could be categorized into 4 major clades.<br><br>CONCLUSIONS: K1 K. pneumoniae strains are more conserved than K2 for both virulence plasmids and chromosomes. K1 strains are deficient in R-M systems but rich in CRISPR-Cas, which is contrary to K2.},
}
@article {pmid40640156,
year = {2025},
author = {Imburgia, C and Organick, L and Zhang, K and Cardozo, N and McBride, J and Bee, C and Wilde, D and Roote, G and Jorgensen, S and Ward, D and Anderson, C and Strauss, K and Ceze, L and Nivala, J},
title = {Random access and semantic search in DNA data storage enabled by Cas9 and machine-guided design.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {6388},
pmid = {40640156},
issn = {2041-1723},
mesh = {*CRISPR-Cas Systems/genetics ; *DNA/genetics ; *Information Storage and Retrieval/methods ; *CRISPR-Associated Protein 9/metabolism/genetics ; Machine Learning ; Semantics ; Sequence Analysis, DNA/methods ; },
abstract = {DNA is a promising medium for digital data storage due to its exceptional data density and longevity. Practical DNA-based storage systems require selective data retrieval to minimize decoding time and costs. In this work, we introduce CRISPR-Cas9 as a user-friendly tool for multiplexed, low-latency molecular data extraction. We first present a one-pot, multiplexed random access method in which specific data files are selectively cleaved using a CRISPR-Cas9 addressing system and then sequenced via nanopore technology. This approach was validated on a pool of 1.6 million DNA sequences, comprising 25 unique data files. We then developed a molecular similarity-search approach combining machine learning with Cas9-based retrieval. Using a deep neural network, we mapped a database of 1.74 million images into a reduced-dimensional embedding, encoding each embedding as a Cas9 target sequence. These target sequences act as molecular addresses, capturing clusters of semantically related images. By leveraging Cas9's off-target cleavage activity, query sequences cleave both exact and closely related targets, enabling high-fidelity retrieval of molecular addresses corresponding to in silico image clusters similar to the query. These approaches move towards addressing key challenges in molecular data retrieval by offering simplified, rapid isothermal protocols and new DNA data access capabilities.},
}
@article {pmid40640155,
year = {2025},
author = {Wang, J and Ye, X and Liu, Y and Li, W and Zhang, X and Zhang, W and Yi, C and Liu, C},
title = {Regulating cleavage activity and enabling microRNA detection with split sgRNA in Cas12b.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {6392},
pmid = {40640155},
issn = {2041-1723},
support = {22307150//National Natural Science Foundation of China (National Science Foundation of China)/ ; },
mesh = {Humans ; *MicroRNAs/genetics/metabolism ; *CRISPR-Cas Systems/genetics ; *RNA, Guide, CRISPR-Cas Systems/genetics/metabolism ; Herpesvirus 4, Human/genetics/isolation &amp; purification ; Colorectal Neoplasms/blood/genetics ; *CRISPR-Associated Proteins/genetics/metabolism ; HEK293 Cells ; },
abstract = {The CRISPR-Cas12b system has revolutionized molecular diagnostics, yet its reliance on single guide RNAs (sgRNAs) exceeding 100 nt limits precise regulation and applications. We present a split sgRNA strategy for Cas12b, utilizing universal components with customizable Spacer to detect various nucleic acid targets by simply replacing Spacer. Glyoxal labeling of the universal split direct repeat (DR) region represses Cas12b activity, which is restored by elevated temperatures or prolonged incubation, enabling dynamic regulation. Incorporating a photo-cleavable linker into the DR allows UV-mediated modulation, ensuring compatibility with recombinase polymerase amplification. Successful detection of Epstein-Barr virus in clinical plasma samples matched the sensitivity of traditional qPCR. Importantly, microRNAs can replace the Spacer, enabling direct detection without reverse transcription or amplification. Supported by evidence from cultured cells and plasma from healthy individuals and colorectal cancer patients, this method yields consistent results with RT-qPCR while simplifying protocols. This split strategy enhances Cas12b systems, offering a promising approach for clinical nucleic acid analysis.},
}
@article {pmid40639408,
year = {2025},
author = {Ille, K and Melzer, S},
title = {Efficient and versatile rapeseed transformation for new breeding technologies.},
journal = {The Plant journal : for cell and molecular biology},
volume = {123},
number = {1},
pages = {e70330},
doi = {10.1111/tpj.70330},
pmid = {40639408},
issn = {1365-313X},
support = {Enable FKZ: 031B0801D//Bundesministerium f&#xfc;r Bildung und Forschung/ ; Epibrass FKZ:031B1223B//Bundesministerium f&#xfc;r Bildung und Forschung/ ; },
mesh = {*Brassica napus/genetics ; *Transformation, Genetic ; Plants, Genetically Modified/genetics ; *Plant Breeding/methods ; CRISPR-Cas Systems ; Gene Editing ; Plant Proteins/genetics/metabolism ; Phenotype ; },
abstract = {Many gene functions are widely studied and understood in Arabidopsis; however, the lack of efficient transformation systems often limits the application and verification of this knowledge in crop plants. Brassica napus L., a member of the Brassicaceae family, is usually transformed by Agrobacterium-mediated hypocotyl transformation, but not all growth types are equally amenable to transformation. In particular, winter rapeseed, which requires vernalization to initiate flowering, is recalcitrant to in vitro regeneration and transformation. The analysis of gene functions in rapeseed is further complicated by the allotetraploid nature of its genome and the genome triplication within the Brassica genus, which has led to the presence of a large number of gene homologs for each Arabidopsis ortholog. We have established a transformation method that facilitates the regeneration of winter rapeseed by using the WUSCHEL gene from Beta vulgaris. This allowed us to efficiently transform a winter and spring rapeseed genotype in small-scale experiments. As proof of principle, we targeted BnCLV3 and BnSPL9/15 with CRISPR/Cas9 and showed that entire gene families are effectively edited using this transformation protocol. This allowed us to simultaneously study many redundantly acting homologous genes in rapeseed. We observed mutant phenotypes for BnCLV3 and BnSPL9/15 in primary transformants, indicating that biallelic knockouts were obtained for up to eight genes. This allowed an initial phenotypic characterization to be performed already a few months after starting the experiment.},
}
@article {pmid40637794,
year = {2025},
author = {Hajian, M and Pirali, A and Moghaddam, SHH and Moradi-Hajidavaloo, R and Varnosfaderani, SR and Jozi, M and Izadi, T and Tanhaie-Vash, N and Kues, W and Nasr-Esfahani, MH and Jafarpour, F and Eghbalsaied, S},
title = {Efficient gene editing of BMP15, GDF9, and MSTN-but not the imprinted CLPG gene-in goat embryos via electrotransfection and handmade cloning.},
journal = {Functional &amp; integrative genomics},
volume = {25},
number = {1},
pages = {150},
pmid = {40637794},
issn = {1438-7948},
support = {CT1402091612//Iranian Vice President for Science/ ; Ref 3.4-IRN-1191261-GF-E//Alexander von Humboldt-Stiftung/ ; },
mesh = {Animals ; *Gene Editing/methods ; *Myostatin/genetics ; *Bone Morphogenetic Protein 15/genetics ; *Goats/genetics/embryology ; *Growth Differentiation Factor 9/genetics/metabolism ; CRISPR-Cas Systems ; Electroporation ; Nuclear Transfer Techniques ; Genomic Imprinting ; Female ; Cloning, Organism ; Embryo, Mammalian/metabolism ; },
abstract = {CRISPR/Cas9 technology represents a powerful tool for advancing livestock breeding by enabling precise, on-target gene edits without the genomic mixing associated with traditional introgression methods. In this study, we employed a dual gRNA-based CRISPR/Cas9 strategy to induce targeted deletions and indel mutations in both reproductive and growth-related genes. These included the metacentric genes bone morphogenetic protein 15 (BMP15) and growth differentiation factor 9 (GDF9), which are associated with increased ovulation rate and litter size, as well as the telomeric genes myostatin (MSTN) and callipyge (CLPG), which are linked to muscle development and enhanced meat production. We employed an optimized electrotransfection protocol consisting of 10-20 µg of each plasmid DNA, 250 µL OptiMEM-GlutaMAX, and one million goat fibroblast cells. The electroporation was performed using a Bio-Rad system in a 4-mm cuvette, with two 10-millisecond pulses at 270 volts, separated by a 10-second interval. This protocol enabled efficient genome editing of goat embryonic fibroblast cells, which were subsequently used to generate cloned embryos via handmade somatic cell nuclear transfer (SCNT), involving manual enucleation and cell-oocyte fusion steps. Sequencing revealed high mutation rates (78-97%) and a predominance of biallelic edits in BMP15, GDF9, and MSTN. Notably, MSTN gRNAs with a 7-bp overlapping sequence at their 3' ends showed a high editing efficiency. In contrast, the imprinted CLPG gene exhibited a significantly lower mutation rate (~ 30%), likely due to epigenetic constraints. While overall mutation rates did not differ significantly between metacentric and telomeric genes, on-target deletions were more frequent in metacentric genes (43%) than in telomeric ones (20%). Embryo development rates from gene-edited cells were comparable to those from non-edited controls. These findings underscore the utility of combining electrotransfection with SCNT for efficient editing of non-imprinted genes and highlight the need for improved strategies to overcome barriers in editing imprinted loci.},
}
@article {pmid40637230,
year = {2025},
author = {Amistadi, S and Fontana, L and Magnoni, C and Felix, T and Charvin, MK and Martinucci, P and Gautier, C and Greau, L and Bessières, B and Antoniou, P and Romano, O and Allemand, E and Mussolino, C and Miccio, A},
title = {Dissecting the epigenetic regulation of the fetal hemoglobin genes to unravel a novel therapeutic approach for β-hemoglobinopathies.},
journal = {Nucleic acids research},
volume = {53},
number = {13},
pages = {},
doi = {10.1093/nar/gkaf637},
pmid = {40637230},
issn = {1362-4962},
support = {//French National Research Agency/ ; ANR-10-IAHU-01//Agence Nationale de la Recherches/ ; ANR-21-CE18-0066-01//Agence Nationale de la Recherches/ ; CA21113//European Cooperation in Science and Technology/ ; //Institut Imagine/ ; PLP202110014595//Fondation pour la Recherche M&#xe9;dicale/ ; },
mesh = {Humans ; *Fetal Hemoglobin/genetics/metabolism ; *Epigenesis, Genetic ; DNA Methylation ; gamma-Globins/genetics ; *beta-Globins/genetics ; *Anemia, Sickle Cell/genetics/therapy ; Promoter Regions, Genetic ; Gene Editing/methods ; *Hemoglobinopathies/genetics/therapy ; CRISPR-Cas Systems ; Erythroid Cells/metabolism ; },
abstract = {Beta-hemoglobinopathies are severe genetic diseases caused by mutations affecting the production of the adult β-globin chain. The clinical severity is mitigated by the co-inheritance of mutations that reactivate the production of the fetal β-like γ-globin in adults. However, the epigenetic mechanisms underlying the adult-to-fetal hemoglobin (HbA-to-HbF) switching are still not fully understood. Here, we used epigenome editing technologies to dissect the molecular mechanisms underlying γ- and β-globin gene regulation and to develop novel potential therapeutics for β-hemoglobinopathies. Targeted removal of DNA methylation by dCas9-Tet1 (alone or together with the deposition of histone acetylation by CBP-dCas9) at the fetal promoters led to efficient and durable γ-globin reactivation, demonstrating that DNA methylation is a driver for HbF repression. This strategy, characterized by high specificity and a good safety profile, led to a substantial correction of the pathological phenotype in erythroid cells from patients with sickle cell disease.},
}
@article {pmid40636259,
year = {2025},
author = {Zheng, H and Wang, B and Dong, X and Wu, J and Shi, L and Zhang, J and Chen, H and Zhou, A},
title = {SIRT7 deletion inhibits Glaesserella parasuis-mediated inflammatory responses in porcine alveolar macrophages.},
journal = {Frontiers in cellular and infection microbiology},
volume = {15},
number = {},
pages = {1589199},
pmid = {40636259},
issn = {2235-2988},
mesh = {Animals ; Swine ; *Sirtuins/genetics/metabolism/deficiency ; *Macrophages, Alveolar/microbiology/immunology ; *Haemophilus parasuis/immunology/pathogenicity ; *Inflammation/microbiology ; *Haemophilus Infections/veterinary/immunology/microbiology/pathology ; Signal Transduction ; *Swine Diseases/microbiology/immunology ; Gene Deletion ; CRISPR-Cas Systems ; },
abstract = {Glaesserella parasuis (GPS) infection causes severe inflammatory disorder, resulting in lung injury. SIRT7 is an NAD[+]-dependent deacetylase known to regulate inflammatory responses, but its role in GPS infection remains unclear. Here we found that GPS infection increased SIRT7 expression and induced inflammatory responses. Deficiency of SIRT7 by CRISPR/Cas9 technology significantly inhibited GPS-induced cytopathic effects and inflammatory responses. In addition, RNA-seq analysis showed that differentially expressed genes(DEGs) induced by SIRT7 deficiency were enriched in biological processes such as cell proliferation, actin cytoskeleton formation, lipid synthesis, protein kinase activation regulation, and GTPase activity regulation. Functional enrichment analysis further indicated the involvement of these DEGs in tight junction pathway, PI3K-Akt signaling pathway, actin cytoskeleton regulation, cGMP-PKG signaling pathway, Hippo signaling pathway, and TNF signaling pathway. Finally, we identified some hub genes (GNAI3, GNAI1, JAK1, NDUFS8, CYC1) related to oxidative phosphorylation. In summary, our results demonstrate that SIRT7 is pivotal for GPS-induced inflammatory responses, which represents a promising target resistant to GPS infection.},
}
@article {pmid40635088,
year = {2025},
author = {Wulff, JP and Laminack, RK and Scott, MJ},
title = {Genetic and behavioral analyses suggest that larval and adult stages of Lucilia cuprina employ different sensory systems to detect rotten beef.},
journal = {Parasites &amp; vectors},
volume = {18},
number = {1},
pages = {270},
pmid = {40635088},
issn = {1756-3305},
support = {DEB-2030345//National Science Foundation/ ; },
mesh = {Animals ; Larva/genetics/physiology/growth &amp; development ; Female ; Cattle ; Receptors, Odorant/genetics ; *Calliphoridae/genetics/physiology/growth &amp; development ; Insect Proteins/genetics/metabolism ; CRISPR-Cas Systems ; Lucilia cuprina ; },
abstract = {BACKGROUND: The blowfly Lucilia cuprina is a destructive parasite of sheep that causes flystrike or myiasis. Larvae consume the animal's living flesh, producing large wounds that can lead to death. The main aim of this study was to identify genes that may play important roles in the behavior and physiology of L. cuprina larvae.<br><br>METHODS: An RNA-Seq analysis of RNA from whole larvae at different developmental stages and third-instar head and gut tissues was used to identify sensory receptors and other genes relevant to the physiology of L. cuprina larvae. In addition, CRISPR/Cas9 gene editing was used to obtain a loss-of-function mutation for the L. cuprina odorant coreceptor gene (LcupOrco). The response of mutant larvae and adult females to fresh and rotten meat at different temperatures was evaluated.<br><br>RESULTS: The RNA-Seq analysis suggested that odorant (OR), gustatory, ionotropic, and Pickpocket receptors may not play a central role in the L. cuprina larval sensory signaling and digestive systems. Rather, ATP-binding cassettes (ABCs) were highly enriched in head and gut RNA, and odorant-binding proteins (OBPs) only in the head. To confirm that ORs are not essential for larval detection of rotten beef, diet-choice assays were performed including larvae and adults homozygous for a null mutation in LcupOrco. While the attraction of adult females to rotten beef was disrupted, LcupOrco mutant larvae showed no change in diet preference.<br><br>CONCLUSIONS: The expression pattern of the ABC and OBP gene families suggests a central role in the sensory system of the L. cuprina larva for these receptors. Behavioral assays showed that ORs are essential for the adult female response to rotten beef, but not for larval behavior. These findings are consistent with high levels of expression of LcupOrco in the adult female antenna but very low expression in larvae.},
}
@article {pmid40634958,
year = {2025},
author = {Tian, Y and Bao, X and Lei, S and Huang, Y and Wang, X and Tu, Y and He, Q and Zhang, F and Xu, H and Ashrafizadeh, M and Sethi, G and Wang, F and Zeng, Z},
title = {In vivo CRISPR screening identifies POU3F3 as a novel regulator of ferroptosis resistance in hepatocellular carcinoma via retinoic acid signaling.},
journal = {Cell communication and signaling : CCS},
volume = {23},
number = {1},
pages = {329},
pmid = {40634958},
issn = {1478-811X},
support = {32260169//National Natural Science Foundation of China/ ; 82303903//National Natural Science Foundation of China/ ; BSH-Q-2023-06//the Affiliated Hospital of Guizhou Medical University Postdoctoral fellow Start-up fund/ ; },
mesh = {*Ferroptosis/drug effects/genetics ; Humans ; *Carcinoma, Hepatocellular/genetics/pathology/metabolism/drug therapy ; *Liver Neoplasms/genetics/pathology/metabolism/drug therapy ; Animals ; *Drug Resistance, Neoplasm/genetics/drug effects ; *Signal Transduction/drug effects ; *Tretinoin/metabolism ; Sorafenib/pharmacology ; Mice ; *Octamer Transcription Factor-3/metabolism/genetics ; Cell Line, Tumor ; Mice, Nude ; *CRISPR-Cas Systems/genetics ; },
abstract = {BACKGROUND: Sorafenib, a ferroptosis agonist, is a first-line treatment for advanced hepatocellular carcinoma (HCC). However, its clinical efficacy is limited due to drug resistance, resulting in modest improvements in patient survival. Hence, the present study has been designed to identify critical molecular targets associated with sorafenib resistance and investigate the potential inhibitors in overcoming this therapeutic challenge.<br><br>METHODS: In vivo whole-genome CRISPR/Cas9 library screens were conducted to identify resistance factors to ferroptosis agonists, such as RSL3 and sorafenib, in HCC. The effects and underlying molecular mechanisms of these resistance factors were investigated in HCC cells using ferroptosis detection assays, xenograft tumor models, chromatin immunoprecipitation (ChIP), and dual-luciferase reporter assays. Potential inhibitors targeting these factors were evaluated through computer-aided virtual screening, molecular dynamics simulations, surface plasmon resonance analysis, and functional evaluations.<br><br>RESULTS: A retinoic acid metabolism gene cluster, including ADH4, ALDH1A1, ALDH1A3, FABP5, RBP1, and RDH10, was found demonstrating upregulation in HCC cells treated with ferroptosis agonist, sorafenib. This gene cluster contributes to the ferroptosis resistance by producing the strong reducing agent retinoic acid. The transcription factor POU3F3 was identified as a key regulator for the retinoic acid metabolism gene cluster, which simultaneously binds to their promoters, increasing their transcription and promoting retinoic acid production. Knockdown of POU3F3 significantly enhanced the pro-ferroptotic and inhibitory effects of sorafenib on HCC cells by suppressing retinoic acid metabolism. Furthermore, rosarin was identified as a POU3F3 inhibitor, with an equilibrium dissociation constant of 7.57 &#xb5;M, and demonstrated a synergistic effect with sorafenib against HCC cells both in vitro and in vivo.<br><br>CONCLUSIONS: According to the results, POU3F3 acts as a protective regulator against sorafenib-induced ferroptosis in HCC cells by enhancing the transcription of multiple retinoic acid metabolism genes and promoting retinoic acid production. The POU3F3 inhibitor, rosarin, shows potential as an ideal candidate for overcoming sorafenib resistance in HCC.},
}
@article {pmid40516842,
year = {2025},
author = {Sun, M and Ni, C and Li, A and Liu, J and Guo, H and Xu, F and Li, K and Cao, X and Shi, X and Guo, R},
title = {A biomimetic nanoplatform mediates hypoxia-adenosine axis disruption and PD-L1 knockout for enhanced MRI-guided chemodynamic-immunotherapy.},
journal = {Acta biomaterialia},
volume = {201},
number = {},
pages = {618-632},
doi = {10.1016/j.actbio.2025.06.021},
pmid = {40516842},
issn = {1878-7568},
mesh = {Animals ; *Immunotherapy ; *B7-H1 Antigen/genetics/metabolism/deficiency ; *Nanoparticles/chemistry/therapeutic use ; *Magnetic Resonance Imaging ; Mice ; Cell Line, Tumor ; Manganese Compounds/chemistry/pharmacology ; *Adenosine/metabolism ; Humans ; *Biomimetic Materials/chemistry/pharmacology ; *Gene Knockout Techniques ; CRISPR-Cas Systems ; Oxides/chemistry/pharmacology ; Tumor Microenvironment ; Melanoma, Experimental/therapy/pathology/diagnostic imaging ; Biomimetics ; },
abstract = {Malignant melanoma is an extremely aggressive and fatal form of skin cancer due to the limited efficacy of conventional therapies. While immune checkpoint blockade therapy and chemodynamic therapy (CDT) have emerged as promising strategies for melanoma treatment, their effectiveness is compromised by the immunosuppressive and complex tumor microenvironment (TME). Here, cancer cell membrane-camouflaged nanoplatforms (PPMC@CM) were developed to co-deliver the CRISPR/Cas9-PD-L1 system and manganese dioxide nanoparticles (MnO2 NPs) for magnetic resonance imaging (MRI)-guided CDT and enhanced immunotherapy. The formed PPMC@CM could efficiently accumulate at tumor sites by homologous targeting, generate O2 to relieve hypoxia, and deplete glutathione (GSH) to enhance Mn[2+]-mediated Fenton-like reactions for enhanced CDT. Meanwhile, CRISPR/Cas9-mediated PD-L1 knockout effectively suppressed the PD-L1 expression, while hypoxia relief attenuated the immunosuppressive hypoxia-CD39/CD73-adenosine (ADO) pathway, thereby boosting the PD-L1-mediated immunotherapy. In vivo experimental results demonstrated that PPMC@CM nanoplatform could efficiently inhibit the growth and metastasis of melanoma by enhanced CDT and amplified immunotherapy, and provide targeted MRI of tumors. This work presents a novelty strategy to design biomimetic theranostic nanoplatform for melanoma by the combination of CDT and improved immunotherapy with CRISPR/Cas9-PD-L1 system and hypoxia-ADO axis inhibition. STATEMENT OF SIGNIFICANCE: Malignant melanoma is a highly aggressive and treatment-refractory skin cancer, where conventional therapies exhibit limited efficacy and immune checkpoint blockade (ICB) is often compromised by the immunosuppressive tumor microenvironment (TME). To address these challenges, we developed a biomimetic nanoplatform (PPMC@CM) to codeliver MnO2 nanoparticles and the CRISPR/Cas9-PD-L1 gene-editing system for MRI-guided chemodynamic therapy and enhanced immunotherapy. The PPMC@CM nanoplatform could efficiently accumulate at tumor sites by homologous targeting and relieve hypoxia to suppress the hypoxia-CD39/CD73-adenosine immunosuppressive axis. Additionally, the CRISPR/Cas9-mediated PD-L1 knockout significantly suppresses PD-L1 expression, thereby boosting ICB efficacy. Moreover, PPMC@CM could deplete glutathione in the TME to amplify Mn[2+]-mediated Fenton-like reactions for enhanced chemodynamic therapy. This research represents a promising theranostic nanoplatform for melanoma by combining chemodynamic therapy and immunotherapy.},
}
@article {pmid40499611,
year = {2025},
author = {Wang, C and Wang, Q and Jin, Y and Li, C and Xin, M and Jiang, X and Wan, J},
title = {Lambda exonuclease assisted helicase-dependent amplification CRISPR/Cas12a detection of Listeria monocytogenes.},
journal = {Biochimie},
volume = {235},
number = {},
pages = {106-112},
doi = {10.1016/j.biochi.2025.06.002},
pmid = {40499611},
issn = {1638-6183},
mesh = {*Listeria monocytogenes/genetics/isolation &amp; purification ; *Biosensing Techniques/methods ; *CRISPR-Cas Systems ; *DNA Helicases/metabolism ; DNA, Single-Stranded/genetics ; *Bacterial Proteins/genetics/metabolism ; *Nucleic Acid Amplification Techniques/methods ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {We describe the construction of a protospacer adjacent motif-free CRISPR/Cas12a fluorescent biosensor based on lambda exonuclease (λ-exo) and helicase-dependent amplification (HDA) to detect Listeria monocytogenes(L. monocytogenes). The hlyA gene of L. monocytogenes was amplified by HDA. After λ-exo catalyzed cleavage of 5' phosphorylated single-stranded DNA of amplification product double-stranded DNA, the double-stranded DNA formed single-stranded DNA (ssDNA). The ssDNA as a substrate activated the trans-cleavage capability of CRISPR/Cas12a to cleave the reporter gene to produce fluorescence signals. Under optimized experimental conditions, the lower limit of L. monocytogenes detection by the fluorescent biosensor was 11.5 CFU/mL, with a linear range of detection from 10[1] to 10[7] CFU/mL. The fluorescent biosensor permits simple and sensitive detection of L. monocytogenes and provides a promising analysis platform for clinical diagnosis and biomedical research without protospacer adjacent motif sequence ssDNA.},
}
@article {pmid40634673,
year = {2025},
author = {Asumadu, P and Guo, Z and Qi, S and Liu, C and Li, Y and Shi, Q and Kong, D and Ye, H and Fu, C and Wang, Z},
title = {Programmable DNA aptamer logic gates: from structural design to integrated systems for intelligent nanoscale biosensors.},
journal = {Analytical and bioanalytical chemistry},
volume = {},
number = {},
pages = {},
pmid = {40634673},
issn = {1618-2650},
support = {BZ2022056//Caili Fu/ ; Science//Caili Fu/ ; Technology Project of Jiangsu Province//Caili Fu/ ; 32372417//Hua Ye/ ; National Natural Science Foundation of China//Hua Ye/ ; 1182921902//Dezhao Kong/ ; 32001804//Dezhao Kong/ ; Emerging Science//Dezhao Kong/ ; Technology Innovation Team Fu//Dezhao Kong/ ; National Natural Science Foundation of China//Dezhao Kong/ ; 22406069//Chang Liu/ ; National Natural Science Foundation of China//Chang Liu/ ; 1184882301//Qiaoqiao Shi/ ; Natural Science Foundation of the Jiangsu Higher E//Qiaoqiao Shi/ ; },
abstract = {DNA aptamer-based logic gates represent significant advances in molecular computing, enabling complex biological computations at the nanoscale. These systems leverage the unique programmable properties of DNA aptamers-short, single-stranded oligonucleotides with high specificity and binding affinity for diverse applications across fields such as clinical diagnostics, food/environmental monitoring, and targeted therapeutic delivery, garnering significant research interest in the past few decades. In this review, we first expand on the fundamentals of aptamers, including its SELEX process and post-SELEX modifications. We systematically examine the design principles and operation mechanisms of DNA aptamer-based logic gates, mainly AND, OR, INHIBIT and NOT as reported by researchers. Then, we highlight various logic gates based on different oligonucleotides spanning from intact and split aptamers to DNA origami architectures, DNA nanorobots, and G-quadruplex structural switches, bringing to light their applications across various fields. Recent innovations in multi-input/output gate cascades, CRISPR-Cas-integrated systems and signal amplification approaches are highlighted as key developments in DNA aptamer-based logic gates. Finally, we elucidate challenges relating to DNA aptamer-based systems such as aptamer performance, cross-reactivity in complex multi-input systems and the complexities of merging other systems to amplify output readability, among others, to the end that in addressing these challenges, we will be able to unlock the full potential of this system.},
}
@article {pmid40632881,
year = {2025},
author = {Thi Pham, N and Wang, CH and Chen, CH and Tseng, YT and Guthula, LS and Chau, LK and Yu, CY and Chen, YL},
title = {Integration of CRISPR/Cas12a and a Fiber Optic Particle Plasmon Resonance Sensor for Single Nucleotide Polymorphism Detection in an Aldehyde Dehydrogenase 2 Gene.},
journal = {ACS sensors},
volume = {},
number = {},
pages = {},
doi = {10.1021/acssensors.5c01035},
pmid = {40632881},
issn = {2379-3694},
abstract = {The highly prevalent single nucleotide polymorphism (SNP, rs671) of the aldehyde dehydrogenase (ALDH2) gene in Asian populations instigates various human pathologies and thus accentuates the urgent need for effective diagnostic tools. In this study, we present an ultrasensitive biosensing method by a combination of clustered regularly interspaced short palindromic repeats (CRISPR)/Cas12a with the fiber optic nanogold-linked sorbent assay (FONLISA) for precise SNP identification. This method leverages the sequence-specific recognition capability of the CRISPR/Cas system and the ultrahigh sensitivity via the dual signal enhancement mechanisms by integrating the trans-cleavage mechanism of Cas12a to amplify the signal from an activity reporter and the subsequent waveguide-enhanced nanoplasmonic absorption by a signaling reporter. In this method, Cas12a targets a double-stranded DNA from the ALDH2 SNP and then activates the degradation of the activity reporter, a free biotin-labeled single-stranded DNA probe (ssDNA[b]), by trans-cleavage. An unhybridized complementary single-stranded DNA probe (ssDNA[c]) labeled with a gold nanoparticle (AuNP) as the signaling reporter (AuNP@ssDNA[c]) is subsequently released and captured by the immobilized ssDNA[b] on the fiber core surface, resulting in a detectable nanoplasmonic absorption signal. The method also utilized an indispensable nanoplasmonic signal generator, carboxymethyl dextran-coated AuNP, to improve the preparation and bioconjugation processes. The CRISPR-FONLISA system demonstrates the ability to analyze the ALDH2 rs671 SNP from double-stranded DNA with a limit of detection of 71 aM. Furthermore, both cell lines and unamplified DNA extracted from blood samples were conducted to verify the system accuracy for ALDH2 rs671 SNP detection.},
}
@article {pmid40632864,
year = {2025},
author = {Yi, JY and Choi, H and Kim, M and Jeong, Y and Hahn, JS and Son, B and Park, HH and Sung, C},
title = {High-throughput multiplexed gene and cell doping analysis through CRISPR-Cas12a system integrated with blood direct PCR.},
journal = {Science advances},
volume = {11},
number = {28},
pages = {eadv7234},
doi = {10.1126/sciadv.adv7234},
pmid = {40632864},
issn = {2375-2548},
mesh = {*CRISPR-Cas Systems/genetics ; Humans ; *Doping in Sports ; *Polymerase Chain Reaction/methods ; *High-Throughput Screening Assays ; },
abstract = {Advancements in gene and cell therapies introduce "gene and cell doping," requiring efficient and sensitive detection methods. Here, we report a high-throughput multiplexed gene and cell doping analysis (HiMDA) using CRISPR-Cas12a system integrated with blood direct polymerase chain reaction (PCR). Blood direct PCR enables simultaneous amplification of multiple exogenous genes directly from whole-blood samples. Coupled with sequence-specific DNA recognition and fluorescence reporter system, HiMDA achieves multiplexed, on-target detection of doping genes and cells. Our results demonstrate HiMDA's feasibility with only 5 microliters of blood required for the entire 90-minute process. HiMDA exhibits exceptional sensitivity, detecting as few as 2.5 copies of doping target genes from blood-four times more sensitive than current anti-doping standards-and identifying in vivo doping up to 10 days. These findings highlight HiMDA's robust high-throughput, multiplexed capabilities, satisfying the sensitivity and selectivity demands of anti-doping research. HiMDA offers a flexible solution to meet future doping detection challenges.},
}
@article {pmid40632850,
year = {2025},
author = {Pal, P and Gao, S and Gao, H and Qin, X and Cella, M and Wang, Q and Shan, L},
title = {Establishment of a reverse genetics system for studying human immune functions in mice.},
journal = {Science advances},
volume = {11},
number = {28},
pages = {eadu1561},
doi = {10.1126/sciadv.adu1561},
pmid = {40632850},
issn = {2375-2548},
mesh = {Animals ; Humans ; Mice ; *Reverse Genetics/methods ; Antigens, CD34/metabolism/genetics ; CRISPR-Cas Systems ; Gene Editing ; Fetal Blood/cytology ; Disease Models, Animal ; },
abstract = {Reverse genetics approaches in mice are widely used to understand gene functions and their aberrations in diseases. However, limitations exist in translating findings from animal models to human physiology. Humanized mice provide a powerful bridge to understanding human physiology and mechanisms of disease pathogenesis while maintaining the feasibility of working with small animals. Methods for generating humanized mouse models that allow scientists to probe contributions of particular genes have been rudimentary. Here, we established an efficient method for generating genetically modified human cord blood-derived CD34[+] cells for transplantation, resulting in humanized mice with near-complete loss of specific gene expression by the human immune system. Mice transplanted with Cas9-edited human CD34[+] cells recapitulate functional consequences of specific gene losses in the human immune system. Our approach enables targeted gene knockouts in humanized mice, offering a valuable tool for human gene function studies in vivo.},
}
@article {pmid40632760,
year = {2025},
author = {Chao, A and Wang, J and Xiu, L and Bold, B and Ghonaim, AH and Chen, J and Hu, Q and Yin, K},
title = {CRISPR/Cas-Based Biosensing Strategies for Non-Nucleic Acid Contaminants in Food Safety: Status, Challenges, and Perspectives.},
journal = {Journal of agricultural and food chemistry},
volume = {},
number = {},
pages = {},
doi = {10.1021/acs.jafc.5c04162},
pmid = {40632760},
issn = {1520-5118},
abstract = {Non-nucleic acid targets (non-NATs), such as heavy metals, toxins, and pesticide residues, pose critical threats to food safety. Although CRISPR/Cas systems were initially developed for nucleic acid detection, recent advances have enabled their adaptation to non-NATs analysis by transducing target recognition into nucleic acid signals. Unlike previous reviews categorized by target type, this work establishes a mechanism-centric framework, systematically classifying non-NAT-to-nucleic acid signal conversion methodologies into three paradigms: (1) aptamer-based systems, (2) catalytic nucleic acid-based methods (e.g., DNAzymes), and (3) protein-mediated strategies (e.g., antibodies, transcription factors). When integrated with CRISPR/Cas, these systems achieve rapid, sensitive detection at picomolar (pM) levels without relying on chromatographic or spectroscopic instruments. Furthermore, we critically discuss challenges, including the limited diversity of recognition elements, inefficient signal conversion, and inflexible signal outputs, proposing solutions including synthetic-biology-driven bioreceptor design and artificial-intelligence-based data analysis. By bridging mechanistic principles with applications in complex food matrices, this review provides actionable insights to advance CRISPR-based tools for rapid, on-site, food safety monitoring.},
}
@article {pmid40632661,
year = {2025},
author = {Wang, S and Zheng, J and Zhang, X and Zhao, M and Li, J and Su, M and Qiu, W},
title = {Genome-Wide CRISPR-Cas9 Knockout Screening Identifies Genes Modulating Cisplatin-Induced Cytotoxicity in Renal Proximal Tubule Epithelial Cells.},
journal = {FASEB journal : official publication of the Federation of American Societies for Experimental Biology},
volume = {39},
number = {13},
pages = {e70780},
doi = {10.1096/fj.202402401RR},
pmid = {40632661},
issn = {1530-6860},
support = {5202009//Natural Science Foundation of Beijing Municipality/ ; 5192022//Natural Science Foundation of Beijing Municipality/ ; 81870196//MOST | National Natural Science Foundation of China (NSFC)/ ; },
mesh = {*Cisplatin/toxicity/pharmacology/adverse effects ; *Kidney Tubules, Proximal/drug effects/metabolism/cytology/pathology ; *CRISPR-Cas Systems/genetics ; *Epithelial Cells/drug effects/metabolism/pathology ; Humans ; *Antineoplastic Agents/toxicity ; Animals ; Gene Knockout Techniques ; },
abstract = {Cisplatin is widely used as a first-line chemotherapy drug for various cancers. However, cisplatin-induced nephrotoxicity (CIN) greatly restricts its application. Renal proximal tubular epithelial cells (RPTECs) can be extensively damaged during CIN. However, it still lacks an ideal method to prevent CIN, because the mechanism and therapeutic targets of CIN remain largely unclear. In the present study, we used a genome-scale CRISPR-Cas9 knock-out method to functionally screen key genes of cisplatin-induced RPTEC injury. We found 815 genes significantly enriched (p < 0.05) from positive selection screening strategy, which may synergistically enhance cisplatin cytotoxicity in RPTECs. Importantly, we identified ERAP2 as a novel molecule associated with CIN. We found that the expression of ERAP2 in RPTECs was significantly up-regulated by cisplatin. Data from CCK-8 assay and flow cytometry showed that inhibition of ERAP2 alleviated cisplatin-induced RPTEC injury. Furthermore, RNA-seq and qPCR results revealed that three necroptosis-associated genes, PLA2G4C, HIST1H2AC, and HIST1H2AM, were downregulated following ERAP2 inhibition, suggesting that ERAP2 may be a novel therapeutic target of CIN through the modulation of necroptosis pathway.},
}
@article {pmid40632351,
year = {2025},
author = {Lokya, V and Singh, S and Chaudhary, R and Jangra, A and Tiwari, S},
title = {Emerging trends in transgene-free crop development: insights into genome editing and its regulatory overview.},
journal = {Plant molecular biology},
volume = {115},
number = {4},
pages = {84},
pmid = {40632351},
issn = {1573-5028},
support = {BIRAC/TechTransfer/08/I2/QUT-BBF//Biotechnology Industry Research Assistance Council/ ; Core research grant//BRIC-National Agri-Food and Biomanufacturing Institute (BRIC-NABI)/ ; },
mesh = {*Gene Editing/methods/trends ; *Crops, Agricultural/genetics/growth &amp; development ; CRISPR-Cas Systems ; Plants, Genetically Modified/genetics ; *Genome, Plant ; Transgenes ; },
abstract = {Genome editing tools have revolutionized plant biology research offering unparalleled applications for genome manipulation and trait improvement in crops. Adopting such advanced biotechnological tools is inevitable to meet increasing global food demand and address challenges in food production, including (a)biotic stresses and inadequate nutritional value. Despite reliance on conventional genetic manipulation methods, the CRISPR-Cas-mediated genome editing toolbox allows precise modification of DNA/RNA in a target organism's genome. So far, CRISPR-Cas has been widely used to enhance yield, quality, stress tolerance, and nutritional value in various food crops. However, challenges such as reagent delivery in suitable explants, precise editing with minimal off-target effect, and generating transgene-free plants persist as major bottlenecks in most plant species. Components of CRISPR-Cas construct mainly Cas, guide RNA (gRNA), and selectable marker genes are often integrated into the host genome, which raises regulatory concerns. However, adapting advanced gene-editing strategies, including high-efficiency Cas endonucleases, DNA-independent RNP delivery, morphogenetic regulators, and grafting-mediated editing, are paving the way for transgene-free crop improvement while easing biosafety regulations. Further, regulatory frameworks for genome-edited crops vary globally, with several countries accepting them and others debating their legal status. Hence, the disparity in global regulatory guidelines of genome editing curbs commercialization. The current review highlights the emerging CRISPR-mediated tools or methods and their applications in developing transgene-free designer crops to harness the benefits of advanced genome manipulation.},
}
@article {pmid40632173,
year = {2025},
author = {Thomas, L and Abraham, T},
title = {Disabling iron uptake and pilus assembly in uropathogenic Escherichia coli using CRISPR-Cas9: a step towards antivirulence therapy.},
journal = {Antonie van Leeuwenhoek},
volume = {118},
number = {8},
pages = {110},
pmid = {40632173},
issn = {1572-9699},
support = {363661//University Grants Commission/ ; },
mesh = {*Uropathogenic Escherichia coli/genetics/metabolism/pathogenicity ; *CRISPR-Cas Systems ; *Iron/metabolism ; *Fimbriae, Bacterial/metabolism/genetics ; Escherichia coli Proteins/genetics/metabolism ; Escherichia coli Infections/microbiology ; Virulence Factors/genetics/metabolism ; Virulence/genetics ; Molecular Docking Simulation ; Humans ; Fimbriae Proteins/genetics/metabolism ; Gene Editing ; },
abstract = {Uropathogenic Escherichia coli (UPEC) is the leading cause of urinary tract infections (UTIs), driven by virulence factors such as iron acquisition systems and adhesive pili. In this study, we employed CRISPR-Cas9-mediated genome editing to functionally inactivate two critical virulence genes-iucD, involved in aerobactin-mediated iron uptake, and papC, encoding the outer membrane usher protein essential for P pilus assembly. Using a clinical UPEC isolate, we introduced premature stop codons via homologous repair templates guided by gene-specific single-guide RNAs. Colony PCR and Sanger sequencing confirmed precise site-specific editing, leading to truncated protein variants. In silico analyses using InterPro and Swiss-Model revealed a complete loss of essential domains in both proteins. Molecular docking studies demonstrated a marked reduction in binding affinities of truncated iucD for NAD(P)H and impaired protein-protein interaction between truncated PapC and PapG. This study highlights the utility of CRISPR-Cas9 as a powerful tool for dissecting bacterial pathogenesis and supports the potential of targeting virulence determinants like iucD and papC as part of an antivirulence strategy for managing UPEC infections.},
}
@article {pmid40631558,
year = {2025},
author = {He, C and Zhu, W and Zhang, X and Wu, W and Tan, J and Li, B and Huang, D and Chen, Y and Xiang, Z and Huang, L and Gong, L},
title = {Sensitive and Visualized Detection of Hantavirus Using CRISPR/Cas12a Based on AutoCORDSv2 Design.},
journal = {Journal of medical virology},
volume = {97},
number = {7},
pages = {e70460},
doi = {10.1002/jmv.70460},
pmid = {40631558},
issn = {1096-9071},
support = {//This study is financially supported by The President Foundation of Nanfang Hospital, Southern Medical University (grant number 2023B036); National Science and Technology Infrastructure of China (Project No. National Pathogen Resource Center-NPRC-32); The CAMS Innovation Fund for Medical Sciences (grant number 2021-I2M-1-039); National Key R&amp;D Program of China, grant number (grant number 2022YFC2303400); The Guangdong Basic and Applied Basic Research Foundation (grant number 2023A1515140080); Dongguan Social development technology projects Science and Technology Planning Project (grant number 20231800940792)./ ; },
mesh = {*CRISPR-Cas Systems ; Sensitivity and Specificity ; Humans ; *Molecular Diagnostic Techniques/methods ; *Orthohantavirus/isolation &amp; purification/genetics ; RNA, Viral/genetics ; *Hantavirus Infections/diagnosis/virology ; *Hantaan virus/genetics/isolation &amp; purification ; DNA Primers/genetics ; },
abstract = {In recent years, detection technologies based on the CRISPR/Cas12a method have been extensively utilized in the fields of nucleic acid, enzyme, and macromolecule detection, thereby reinforcing their significant role in the detection landscape. Enhancing the simplicity of design, efficiency, and automation of the CRISPR/Cas12a detection system is essential for advancing its application in diagnostics. Recently, we developed an automated CRISPR/Cas12a design system named AutoCORDSv2. This system can process published genomic sequences of pathogenic bacteria in a high-throughput manner and automatically generate conserved and highly specific crRNA sequences, along with primer sequences for target amplification. This capability facilitates the specific and precise design of the CRISPR/Cas12a detection system. In this study, crRNAs targeting the Hantaan virus (HTNV) and Seoul virus (SEOV), as well as RT-PCR primers and RT-RPA primers, were designed using AutoCORDSv2. The experimental results demonstrated that the CRISPR/Cas12a system, automatically designed by AutoCORDSv2, was specific for the detection of both the HTNV and SEOV, with no cross-reactivity observed with other pathogens. The detection sensitivity reached 6 copies/μL (equivalent to 111 copies per amplification reaction), whether measured by a microplate reader or directly observed with the naked eye. The detection results for 50 samples were consistent with those obtained from commercial RT-qPCR kits, indicating high precision. Furthermore, the CRISPR/Cas12a system designed by AutoCORDSv2 can also be utilized for the development of a single-tube detection system with a sensitivity of 42 copies per reaction. This system combined with a 5-min extraction step and RT-RPA, further underscoring its potential for application.},
}
@article {pmid40630641,
year = {2025},
author = {Pan, K and Zeng, A and Ruan, X and Mo, X and Shen, B and Zhao, J and Zhou, Y},
title = {The apicoplast localized isocitrate dehydrogenase is needed for de novo fatty acid synthesis in the apicoplast of Toxoplasma gondii.},
journal = {Frontiers in cellular and infection microbiology},
volume = {15},
number = {},
pages = {1542122},
pmid = {40630641},
issn = {2235-2988},
mesh = {*Toxoplasma/enzymology/growth &amp; development/genetics/metabolism ; *Isocitrate Dehydrogenase/metabolism/genetics ; Animals ; *Fatty Acids/biosynthesis ; *Apicoplasts/enzymology/metabolism/genetics ; Mice ; Virulence ; CRISPR-Cas Systems ; Gene Deletion ; NADP/metabolism ; Glucose/metabolism ; },
abstract = {Toxoplasma gondii (T. gondii), an apicomplexan parasite, infects a wide range of warm-blooded animals and poses significant risks to human health. The fatty acid synthesis II (FASII) pathway in the apicoplast, which is the major source of fatty acids in parasites, is considered a potential drug target. The apicoplast also harbors some enzymes of central carbon metabolism, which are crucial for its survival, but their biological roles remain unclear. In this study, we focused on apicoplast-localized isocitrate dehydrogenase 1 (ICDH1) and deleted it using CRISPR-Cas9 technology. The Δicdh1 mutant tachyzoites displayed markedly impaired growth kinetics, with further suppression under serum-deprived conditions. However, this deletion did not affect the viability or virulence of the Δicdh1 mutant in mice. NADPH, a product of ICDH1-mediated decarboxylation of isocitrate, is an essential cofactor for fatty acid synthesis. Using [13]C6 glucose as a metabolic carbon source, we showed that the mutant strains had reduced incorporation of glucose-derived carbons into medium-chain length fatty acids (C14:0 and C16:0). Additionally, the growth of the mutant was partially restored by supplementation with exogenous C14:0 and C16:0 fatty acids. These results indicate that ICDH1 deletion affects the FASII pathway and parasite growth. Consistent with previous studies, this study confirms that T. gondii has metabolic flexibility in the apicoplast that allows it to acquire fatty acids through various pathways.},
}
@article {pmid40629383,
year = {2025},
author = {Gou, F and Liu, D and Gong, C and Wang, K and Wang, X and Chen, Y and Liu, Q and Tian, C},
title = {Development of an efficient heterologous protein expression platform in Aspergillus niger through genetic modification of a glucoamylase hyperproducing industrial strain.},
journal = {Microbial cell factories},
volume = {24},
number = {1},
pages = {160},
pmid = {40629383},
issn = {1475-2859},
support = {2023YFC3402300//National Key Research &amp; Developmental Program of China/ ; U22A20441//National Natural Science Foundation of China/ ; XDA0510300//Strategic Priority Research Program of the Chinese Academy of Sciences/ ; },
mesh = {*Aspergillus niger/genetics/metabolism/enzymology ; *Glucan 1,4-alpha-Glucosidase/genetics/biosynthesis/metabolism ; Recombinant Proteins/genetics/biosynthesis/metabolism ; CRISPR-Cas Systems ; Industrial Microbiology ; Genetic Engineering ; },
abstract = {BACKGROUND: Aspergillus niger is widely used in industrial enzyme production due to its strong secretion capacity and the status of generally recognized as safe (GRAS). However, heterologous protein expression in A. niger is frequently constrained by high levels of background endogenous protein secretion, limited access to native high transcription loci, and limitations in the efficiency of the secretory machinery. To address these limitations, this study genetically engineered a chassis strain based on an industrial glucoamylase-producing A. niger strain AnN1 for constructing the improved heterologous protein expression.<br><br>RESULTS: In this study, by using CRISPR/Cas9-assisted marker recycling, we deleted 13 of the 20 copies of the heterologous glucoamylase TeGlaA gene and disrupted the major extracellular protease gene PepA, resulting in the low-background strain AnN2. Compared to the parental strain AnN1, AnN2 exhibited 61% less extracellular protein and significantly reduced glucoamylase activity, while retaining multiple transcriptionally active integration loci. Four diverse proteins were integrated into the high-expression loci originally occupied by the TeGlaA gene in the chassis AnN2. These recombinant protein included a homologous glucose oxidase (AnGoxM), a thermostable pectate lyase A (MtPlyA), a bacterial triose phosphate isomerase (TPI), and a medical protein Lingzhi-8 (LZ8). All target proteins were successfully expressed and secreted within 48-72&#xa0;h, with yields ranging from 110.8 to 416.8&#xa0;mg/L in 50 mL shake-flasks cultivation. The enzyme activities of AnGoxM, MtPlyA and TPI reached&#x2009;~&#x2009;1276&#x2009;-&#x2009;1328 U/mL, ~&#x2009;1627. 43&#x2009;-&#x2009;2105.69 U/mL, and ~&#x2009;1751.02 to 1906.81 U/mg after 48&#xa0;h, respectively. Additionally, Overexpression of Cvc2, a COPI vesicle trafficking component, further enhanced MtPlyA production by 18%, highlighting the benefit of combining transcriptional and secretory pathway engineering.<br><br>CONCLUSIONS: Our results demonstrated that the chassis AnN2 served as a robust, modular, and time-efficient platform for heterologous protein expression in A. niger. Through site-specific integration of target genes into native high-expression loci and strategic modulation of the secretory pathway, we successfully enabled the rapid production of functional enzymes and bioactive proteins from diverse origins. This dual-level optimization strategy, which integrates rational genomic engineering with targeted enhancement of the secretory pathway, enabled high-yield expression while minimizing background interference. Together, these findings offer a practical framework for constructing versatile fungal expression systems and highlight the potential of combining genetic and cellular engineering to improve recombinant protein production in filamentous fungi.},
}
@article {pmid40586242,
year = {2025},
author = {Sreekanth, V and Jan, M and Zhao, KT and Lim, D and Siriwongsup, S and Davis, JR and McConkey, M and Kovalcik, V and Barkal, S and Law, BK and Fife, J and Tian, R and Vinyard, ME and Becerra, B and Kampmann, M and Sherwood, RI and Pinello, L and Liu, DR and Ebert, BL and Choudhary, A},
title = {A Molecular Glue Approach to Control the Half-Life of CRISPR-Based Technologies.},
journal = {Journal of the American Chemical Society},
volume = {147},
number = {27},
pages = {23844-23856},
doi = {10.1021/jacs.5c06230},
pmid = {40586242},
issn = {1520-5126},
mesh = {Half-Life ; Humans ; *Gene Editing/methods ; *CRISPR-Cas Systems ; *Thalidomide/analogs &amp; derivatives/chemistry/pharmacology/metabolism ; Ubiquitination ; },
abstract = {Cas9 is a programmable nuclease that has furnished transformative technologies, including base editors and transcription modulators (e.g., CRISPRi/a), but several applications of these technologies, including therapeutics, mandatorily require precision control of their half-life. For example, such control can help avert any potential immunological and adverse events in clinical trials. Current genome editing technologies to control the half-life of Cas9 are slow, have lower activity, involve fusion of large response elements (>230 amino acids), utilize expensive controllers with poor pharmacological attributes, and cannot be implemented in vivo on several CRISPR-based technologies. We report a general platform for half-life control using the molecular glue, pomalidomide, that binds to a ubiquitin ligase complex and a response-element bearing CRISPR-based technology, thereby causing the latter's rapid ubiquitination and degradation. Using pomalidomide, we were able to control the half-life of large CRISPR-based technologies (e.g., base editors and CRISPRi) and small anti-CRISPRs that inhibit such technologies, allowing us to build the first examples of on-switch for base editors. The ability to switch on, fine-tune, and switch-off CRISPR-based technologies with pomalidomide allowed complete control over their activity, specificity, and genome editing outcome. Importantly, the miniature size of the response element and favorable pharmacological attributes of the drug pomalidomide allowed control of activity of base editor in vivo using AAV as the delivery vehicle. These studies provide methods and reagents to precisely control the dosage and half-life of CRISPR-based technologies, propelling their therapeutic development.},
}
@article {pmid40577080,
year = {2025},
author = {Butti, P and Bellusci, F and Brambilla, E and Branduardi, P},
title = {Genomically integrated cassettes swapping: bringing modularity to the strain level in Saccharomyces cerevisiae.},
journal = {FEMS yeast research},
volume = {25},
number = {},
pages = {},
doi = {10.1093/femsyr/foaf032},
pmid = {40577080},
issn = {1567-1364},
support = {//University of Milano-Bicocca/ ; CN_00000033//Italian Ministry of University and Research/ ; H43C22000530001//Italian Ministry of University and Research/ ; },
mesh = {*Saccharomyces cerevisiae/genetics ; CRISPR-Cas Systems ; *Synthetic Biology/methods ; *Gene Editing/methods ; *Genome, Fungal ; },
abstract = {A large variety of synthetic biology toolkits for the introduction of multiple expression cassettes is available for Saccharomyces cerevisiae. Unfortunately, none of these tools is designed to allow the modification - exchange or removal - of the cassettes already integrated into the genome in a standardized way. The application of the modularity principle therefore ends to the steps preceding the final host engineering, making microbial cell factories construction stiff and strictly sequential. In this work, we describe a system that easily allows CRISPR-mediated swapping or removal of previously integrated cassettes, thus bringing the modularity to the strain level, enhancing the possibility of modifying existing strains with a reduced number of steps. In the system, each cassette is tagged with specific barcodes, which can be used as targets for CRISPR nucleases (Cas9 and Cas12a), allowing the excision of the construct from the genome and its substitution with another expression cassette or the restoration of the wild type locus in one single standardized step. The system has been applied to the previously developed Easy-MISE toolkit and tested by swapping fluorescent protein expression cassettes with an efficiency of ∼90% quantified by PCR and flow cytometry.},
}
@article {pmid40552742,
year = {2025},
author = {Petroulia, S and Hockemeyer, K and Tiwari, S and Berico, P and Shamloo, S and Banijamali, SE and Vega-Saenz de Miera, E and Gong, Y and Thandapani, P and Wang, E and Schloßhauer, JL and Tsirigos, A and Osman, I and Aifantis, I and Imig, J},
title = {Uncovering Novel lncRNAs Linked to Melanoma Growth and Migration with CRISPR Inhibition Screening.},
journal = {Cancer research communications},
volume = {5},
number = {7},
pages = {1102-1118},
doi = {10.1158/2767-9764.CRC-24-0416},
pmid = {40552742},
issn = {2767-9764},
mesh = {Humans ; *Melanoma/genetics/pathology ; *RNA, Long Noncoding/genetics ; Cell Movement/genetics ; Cell Line, Tumor ; Cell Proliferation/genetics ; *Skin Neoplasms/genetics/pathology ; Gene Expression Regulation, Neoplastic ; CRISPR-Cas Systems ; Drug Resistance, Neoplasm/genetics ; Apoptosis/genetics ; },
abstract = {UNLABELLED: Melanoma being one of the most common and deadliest skin cancers has been increasing since the past decade. Patients at advanced stages of the disease have very poor prognoses, as opposed to at the earlier stages. Nowadays, the standard of care of advanced melanoma is resection, followed by immune checkpoint inhibition-based immunotherapy. However, a substantial proportion of patients either do not respond or develop resistance. This underscores a need for novel approaches and therapeutic targets as well as a better understanding of the mechanisms of melanoma pathogenesis. Long noncoding RNAs (lncRNA) comprise a poorly characterized class of functional players and promising targets in promoting malignancy. Certain lncRNAs have been identified to play integral roles in melanoma progression and drug resistance; however, systematic screens to uncover novel functional lncRNAs are scarce. In this study, we profile differentially expressed lncRNAs in patient-derived short-term metastatic cultures and BRAF-MEK inhibition-resistant cells. We conduct a focused growth-related CRISPR inhibition screen of overexpressed lncRNAs, validate, and functionally characterize lncRNA hits with respect to cellular growth, invasive capacities, and apoptosis in vitro as well as the transcriptomic impact of our lead candidate the novel lncRNA XLOC_030781. In sum, we extend the current knowledge of ncRNAs and their potential relevance in melanoma.<br><br>SIGNIFICANCE: LncRNAs have emerged as novel players in regulating many cellular aspects also in melanoma. The number of functional significances of most lncRNAs remains elusive. We provide a comprehensive strategy to identify functionally relevant lncRNAs in melanoma by combining expression profiling with CRISPR inhibition growths screens. Our results broaden the characterized lncRNAs as potential targets for future therapeutic applications.},
}
@article {pmid40482807,
year = {2025},
author = {Geiger, AB and Kennedy, JG and Staker, LG and Wensel, TG and Casson, RJ and Thomas, PQ},
title = {Shining light on CRISPR/Cas9 therapeutics for inherited retinal diseases.},
journal = {Progress in retinal and eye research},
volume = {107},
number = {},
pages = {101376},
doi = {10.1016/j.preteyeres.2025.101376},
pmid = {40482807},
issn = {1873-1635},
mesh = {Humans ; *CRISPR-Cas Systems ; *Gene Editing/methods ; *Genetic Therapy/methods ; *Retinal Diseases/therapy/genetics ; Animals ; },
abstract = {Inherited retinal diseases (IRDs), such as retinitis pigmentosa, are a heterogenous group of genetic eye diseases characterized by degeneration of photoreceptors. They are the leading cause of blindness in the working age population in high-income countries and are an ideal target for the expanding gene editing tool kit, including rapidly evolving CRISPR/Cas9 technology. In this review, we provide a comprehensive analysis of CRISPR/Cas9 technologies currently being explored as therapeutic interventions for IRDs. Given the challenges posed by the growing complexity and size of gene editing systems, the delivery of these therapeutics to the retina has necessitated innovative approaches. We review current delivery methods, including nanoparticles, virus-like particles and traditional viral vectors, highlighting their advantages and limitations. This review underscores the potential transformative impact of gene editing on genetic disease management, emphasising that advancements in these technologies, coupled with improved pre-clinical models, bring clinically safe and effective treatments for IRDs within view.},
}
@article {pmid40434115,
year = {2025},
author = {Wiechert, J and Badia Roigé, B and Dohmen-Olma, D and Hindra, and Zhang, X and Stella, RG and Elliot, MA and Frunzke, J},
title = {CRISPR/dCas-mediated counter-silencing: reprogramming dCas proteins into antagonists of xenogeneic silencers.},
journal = {mBio},
volume = {16},
number = {7},
pages = {e0038225},
doi = {10.1128/mbio.00382-25},
pmid = {40434115},
issn = {2150-7511},
support = {458090666//Deutsche Forschungsgemeinschaft/ ; 162340/CAPMC/CIHR/Canada ; //J&#xfc;rgen Manchot Stiftung/ ; },
mesh = {*CRISPR-Cas Systems ; Promoter Regions, Genetic ; *Bacterial Proteins/genetics/metabolism ; *Streptomyces/genetics ; *Gene Silencing ; *Gene Expression Regulation, Bacterial ; *Corynebacterium glutamicum/genetics/metabolism ; *Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {UNLABELLED: Lsr2-like nucleoid-associated proteins function as xenogeneic silencers (XSs) inhibiting expression of horizontally acquired, adenine-thymine-rich DNA in actinobacteria. Interference by transcription factors can lead to counter-silencing of XS target promoters, but relief of this repression typically requires promoter engineering. In this study, we developed a novel clustered regularly interspaced short palindromic repeats (CRISPR)/dCas-mediated counter-silencing (CRISPRcosi) approach by using nuclease-deficient dCas enzymes to counteract the Lsr2-like XS protein CgpS in Corynebacterium glutamicum or Lsr2 in Streptomyces venezuelae. Systematic in vivo reporter studies with dCas9 and dCas12a and various guide RNAs revealed effective counter-silencing of different CgpS target promoters in response to guide RNA/dCas DNA binding, independent of promoter sequence modifications. The most prominent CRISPRcosi effect was observed when targeting the CgpS nucleation site, an effect that was also seen in S. venezuelae when targeting a known Lsr2 nucleation site within the chloramphenicol biosynthesis gene cluster. Analyzing the system in C. glutamicum strains lacking the XS protein CgpS revealed varying strengths of counteracting CRISPR interference effects based on the target position and strand. Genome-wide transcriptome profiling in single-guide RNA/dCas9 co-expressing C. glutamicum wild-type strains revealed high counter-silencing specificity with minimal off-target effects. Thus, CRISPRcosi provides a promising strategy for the precise upregulation of XS target genes with significant potential for studying gene networks as well as for developing applications in biotechnology and synthetic biology.<br><br>IMPORTANCE: Lsr2-like nucleoid-associated proteins act as xenogeneic silencers (XSs), repressing the expression of horizontally acquired, adenine-thymine-rich DNA in actinobacteria. The targets of Lsr2-like proteins are very diverse, including prophage elements, virulence gene clusters, and biosynthetic gene clusters. Consequently, the targeted activation of XS target genes is of interest for fundamental research and biotechnological applications. Traditional methods for counter-silencing typically require promoter modifications. In this study, we developed a novel clustered regularly interspaced short palindromic repeats (CRISPR)/dCas-mediated counter-silencing (CRISPRcosi) approach, utilizing nuclease-deficient dCas enzymes to counteract repression by Lsr2-like proteins in Corynebacterium glutamicum and Streptomyces venezuelae. The strongest effect was observed when targeting the Lsr2 nucleation site. Genome-wide transcriptome profiling revealed high specificity with minimal off-target effects. Overall, CRISPRcosi emerges as a powerful tool for the precise induction of genes silenced by xenogeneic silencers, offering new opportunities for exploring gene networks and advancing biotechnological applications.},
}
@article {pmid39915620,
year = {2025},
author = {Pagliaro, A and Andreatta, F and Finger, R and Artegiani, B and Hendriks, D},
title = {Generation of human fetal brain organoids and their CRISPR engineering for brain tumor modeling.},
journal = {Nature protocols},
volume = {20},
number = {7},
pages = {1846-1883},
pmid = {39915620},
issn = {1750-2799},
mesh = {Humans ; *Organoids/cytology/metabolism ; *Brain Neoplasms/genetics/pathology ; *Brain/embryology/cytology ; *CRISPR-Cas Systems ; *Gene Editing/methods ; Fetus/cytology ; },
abstract = {The developing human brain displays unique features that are difficult to study in animal models. Current in vitro models based on human brain tissue face several challenges, including the limited cellular heterogeneity in two- or three-dimensional neural stem cell cultures, while tissue slice cultures suffer from short survival. We recently established culture conditions to derive organoid cultures directly from human fetal brain tissue by preserving tissue integrity, which can be long-term expanded and display cellular heterogeneity and complex organization. In this Protocol, we describe detailed procedures to establish human fetal brain organoids (FeBOs) that broadly retain regional characteristics, along with procedures for their passaging and characterization. In addition, we describe genome engineering approaches applied to FeBOs to generate mutant FeBO lines that serve as versatile bottom-up brain cancer models. Lastly, we exemplify various downstream applications applicable to both healthy and mutant FeBOs. Scientists with experience in tissue culture can expect the establishment of human FeBO cultures to take 2-3 weeks, while genome engineering of FeBOs takes 2-4 months.},
}
@article {pmid40627177,
year = {2025},
author = {Azani, A and Sharafi, M and Doachi, R and Akbarzadeh, S and Lorestani, P and Haji Kamanaj Olia, A and Zahed, Z and Gharedaghi, H and Ghasrsaz, H and Foroozand, H and Rahimi, H and Tahmasebi, S and Behfar, Q},
title = {Applications of CRISPR-Cas9 in mitigating cellular senescence and age-related disease progression.},
journal = {Clinical and experimental medicine},
volume = {25},
number = {1},
pages = {237},
pmid = {40627177},
issn = {1591-9528},
mesh = {Humans ; *Cellular Senescence/genetics ; *CRISPR-Cas Systems ; *Aging/genetics ; Epigenesis, Genetic ; *Gene Editing/methods ; Disease Progression ; Animals ; Telomerase/genetics/metabolism ; },
abstract = {Aging is a multifaceted process influenced by many elements. During cell division, the repetitive DNA sequences at the ends of chromosomes called telomeres protect them from degradation. Telomeres shorten alongside each cell division, eventually contributing to cellular senescence and aging. Telomerase as an enzyme has a role in the maintenance of telomere length. Reduced function of telomerase is linked to acceleration of aging and age-related diseases. By affecting cellular function, mutations in particular genes can cause aging. Genes involved in DNA repair, cellular metabolism, and inflammation play the key roles in this process. Accumulated mutations result in cellular dysfunction and age-related diseases over time. Epigenetic changes are the modifications that impact gene expression without altering the DNA sequence. Lifestyle factors (diet, exercise, stress) and environmental influences (toxins, trauma) can cause epigenetic alterations. DNA methylation as well as histone modifications are examples of epigenetic alterations. They influence how cells work and are essential to the aging process. Understanding these molecular mechanisms is essential for developing interventions to promote healthy aging and prevent age-related diseases. This paper explores the potential of CRISPR/Cas9 as a gene-editing tool to target these mechanisms and mitigate age-related conditions, ultimately enhancing longevity and quality of life.},
}
@article {pmid40627029,
year = {2025},
author = {M, KR and C, SD},
title = {Recent insights into actinobacteria research in antimicrobial resistance: a review.},
journal = {Molecular biology reports},
volume = {52},
number = {1},
pages = {683},
pmid = {40627029},
issn = {1573-4978},
mesh = {*Actinobacteria/genetics/metabolism/drug effects ; *Anti-Bacterial Agents/pharmacology ; Multigene Family ; Humans ; *Drug Resistance, Multiple, Bacterial/genetics ; Streptomyces/genetics/metabolism ; *Drug Resistance, Bacterial/genetics ; Biosynthetic Pathways/genetics ; },
abstract = {Antimicrobial resistance (AMR) has emerged as a global health crisis, taking 4.71 million lives in the year 2021 and posing significant challenges to healthcare systems. Actinobacteria, particularly Streptomyces sp., are a well-established source of bioactive secondary metabolites, including antibiotics such as polyketides, aminoglycosides, and macrolides with activity against multidrug-resistant (MDR) bacteria. However, only 10% of the antibiotic genes are expressed, and others are silent in cryptic biosynthetic gene clusters (BGCs) that remain inactive under standard laboratory conditions. Advances in genome mining, bioinformatics tools like antiSMASH, and molecular techniques such as CRISPR-Cas have facilitated the identification of these clusters. Furthermore, innovative strategies such as co-culturing and HDAC inhibitors have shown promise in activating cryptic biosynthetic pathways to combat emerging antimicrobial resistance. Despite these advancements, the rapid evolution of resistance requires continuous research and global collaboration to ensure a sustainable pipeline of effective antibiotics. This review provides insight into actinobacteria-derived antibiotics, resistance mechanisms, and emerging biotechnological interventions to address the AMR crisis, underscoring the urgent need for multidisciplinary antibiotic discovery and stewardship efforts.},
}
@article {pmid40626824,
year = {2025},
author = {Özdemir, BH},
title = {Navigating Immunological Barriers in Xenotransplantation: Recent Advances and Promising Strides.},
journal = {Experimental and clinical transplantation : official journal of the Middle East Society for Organ Transplantation},
volume = {23},
number = {6},
pages = {421-430},
doi = {10.6002/ect.2023.0351},
pmid = {40626824},
issn = {2146-8427},
mesh = {Humans ; Animals ; *Transplantation, Heterologous/adverse effects ; *Graft Rejection/immunology/prevention &amp; control/genetics ; Animals, Genetically Modified ; Swine ; *Immunity, Humoral ; *Graft Survival ; *Antibodies, Heterophile/immunology/genetics/blood ; *Antigens, Heterophile/immunology/genetics ; Immunity, Cellular ; Treatment Outcome ; CRISPR-Cas Systems ; Gene Editing ; },
abstract = {The review introduces the challenges and potential solutions in xenotransplantation, focusing on pig-to-human organ transplant. Xenotransplantation, mainly with the use of pig organs, is a promising solution because of the reproductive capacity, size, and physiological resemblance of pigs to humans. However, immunological barriers, especially humoral and cellular immune responses, pose substantial challenges. The humoral immune response, involving antibodies targeting xenoantigens, is a substantial barrier. Anti-α-galactose antibodies, targeting α-Gal epitopes, are crucial in hyperacute rejection and acute humoral xenograft rejection. Genetic modifications, including CRISPR/Cas9 technology, aim to eliminate xenoantigens like α-Gal, potentially overcoming these challenges. This review discusses the use of genetically modified pigs for xenotransplantation, emphasizing the removal of xenoantigens, expression of human complement regulatory proteins, and transgenic expres-sion of human regulatory factors. Recent advancements, such as the world's first porcine-to-human heart transplant, highlight the potential of genetic manipu-lation in overcoming immune rejection barriers.},
}
@article {pmid40626243,
year = {2025},
author = {Mitousis, L and Musiol-Kroll, E and Wohlleben, W},
title = {CRISPR-Cas in actinomycetes: still a lot to be discovered.},
journal = {microLife},
volume = {6},
number = {},
pages = {uqaf010},
pmid = {40626243},
issn = {2633-6693},
abstract = {Actinomycetes are important producers of valuable natural products that are applied in medicine or industry. The enzymes necessary for the synthesis of those compounds are encoded in biosynthetic gene clusters (BGCs) in the genome. However, the discovery of new natural products or the improvement of production levels can be hindered by difficulties in genetic manipulation, since standard methods often do not or not efficiently work in actinomycetes. One possible explanation for this could be the presence of nucleic acid defense systems such as CRISPR-Cas. Even though there is a lot of research published about CRISPR-Cas systems in general, the knowledge about the function of CRISPR-Cas in actinomycetes is very limited. Based on sequence data it is known that CRISPR-Cas systems occur in around half of all sequenced actinobacterial genomes. Moreover, in silico analyses of those systems have led to the discovery of new subtypes. The few examples of experimental evidence of CRISPR-Cas activity in vivo or in vitro, however, point to some special features, regarding crRNA maturation or life-cycle dependent CRISPR-Cas activity. This short review draws attention to this neglected research area and highlights the available data about CRISPR-Cas in actinomycetes.},
}
@article {pmid40364716,
year = {2025},
author = {Cahn, JKB and Ludwicki, H and Shingler, J and Gulvin, S and Zhang, Y and Kristopeit, A and Ton, C and Winters, MA and Wagner, JM and Moore, J},
title = {CRISPR-Editing of the Vero Cell Line Improves Processability of Live Virus Vaccines by Enabling Targeted Proteolysis of Fibronectin.},
journal = {Biotechnology and bioengineering},
volume = {122},
number = {8},
pages = {2082-2092},
doi = {10.1002/bit.29028},
pmid = {40364716},
issn = {1097-0290},
mesh = {Animals ; Chlorocebus aethiops ; Vero Cells ; *Gene Editing/methods ; *Fibronectins/genetics/metabolism ; *CRISPR-Cas Systems ; *Viral Vaccines/genetics/metabolism ; Proteolysis ; Vaccines, Attenuated/genetics ; },
abstract = {Removal of host cell components is a significant cost driver in the production of live virus vaccines. Filtration processes such as tangential flow filtration can be effective in this capacity by leveraging the relative size difference between viral particles and host proteins; however, filtration membranes can be fouled by larger proteins, particularly those of the extracellular matrix. In this study, we used CRISPR editing to insert the recognition element of the highly-selective TEV protease into various positions of the gene encoding fibronectin in the genome of the Vero cell line, a common platform for viral production. By screening edited cell lines, we identified a promising candidate line in which fibronectin could be effectively removed by treating with the protease during processing, eliminating filter fouling and allowing for viral purification without the need for costly chromatography steps.},
}
@article {pmid40345583,
year = {2025},
author = {Flores-Arenas, C and Malekos, E and Montano, C and Covarrubias, S and Sudek, L and Dempsey, V and Huynh, V and Katzman, S and Carpenter, S},
title = {CRISPRi screen uncovers lncRNA regulators of human monocyte growth.},
journal = {The Journal of biological chemistry},
volume = {301},
number = {6},
pages = {110204},
pmid = {40345583},
issn = {1083-351X},
mesh = {Humans ; *RNA, Long Noncoding/genetics/metabolism ; *Monocytes/cytology/metabolism ; *Cell Proliferation ; *CRISPR-Cas Systems ; THP-1 Cells ; Interferon Regulatory Factors/genetics/metabolism ; Apoptosis ; },
abstract = {Long noncoding RNAs are emerging as critical regulators of biological processes. While there are over 36,000 lncRNAs annotated in the human genome, we do not know the function of the majority. Here, we performed a high-throughput CRISPRi screen to identify those lncRNAs that are important for viability in human monocytes using the cell line THP1. We identified a total of 38 hits from the screen and validated and characterized two of the top intergenic hits. The first is a lncRNA neighboring the macrophage viability transcription factor IRF8 (RP11-542M13.2 hereafter referred to as long noncoding RNA regulator of monocyte proliferation, LNCRMP), and the second is a lncRNA called OLMALINC (oligodendrocyte maturation-associated long intervening non-coding RNA) that was previously found to be important in oligodendrocyte maturation. Transcriptional repression of LNCRMP and OLMALINC from monocytes severely limited their proliferation capabilities. RNA-seq analysis of knockdown lines showed that LNCRMP regulated proapoptotic pathways, while knockdown of OLMALINC impacted genes associated with the cell cycle. Data support both LNCRMP and OLMALINC functioning in cis to regulate their neighboring proteins that are also essential for THP1 cell growth. This research highlights the importance of high-throughput screening as a powerful tool for quickly discovering functional long non-coding RNAs (lncRNAs) that play a vital role in regulating monocyte viability.},
}
@article {pmid40193304,
year = {2025},
author = {Florindi, C and Jang, Y and Shani, K and Moretti, P and Bertarelli, C and Lanzani, G and Parker, KK and Lodola, F and Vurro, V},
title = {A Cardiac Microphysiological System for Studying Ca2+ Propagation via Non-genetic Optical Stimulation.},
journal = {Journal of visualized experiments : JoVE},
volume = {},
number = {217},
pages = {},
doi = {10.3791/67823},
pmid = {40193304},
issn = {1940-087X},
mesh = {*Photic Stimulation ; *Light Signal Transduction ; *Calcium/metabolism ; Induced Pluripotent Stem Cells ; Humans ; *Myocytes, Cardiac/metabolism ; CRISPR-Cas Systems ; CRISPR-Associated Protein 9 ; Cations, Divalent/metabolism ; },
abstract = {In vitro cardiac microphysiological models are highly reliable for scientific research, drug development, and medical applications. Although widely accepted by the scientific community, these systems are still limited in longevity due to the absence of non-invasive stimulation techniques. Phototransducers provide an efficient stimulation method, offering a wireless approach with high temporal and spatial resolution while minimizing invasiveness in stimulation processes. In this manuscript, we present a fully optical method for stimulating and detecting the activity of an in vitro cardiac microphysiological model. Specifically, we fabricated engineered laminar anisotropic tissues by seeding human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) generated in a 3D bioreactor suspension culture. We employed a phototransducer, an amphiphilic azobenzene derivative, named Ziapin2, for stimulation and a Ca[2+] dye (X-Rhod 1) for monitoring the system's response. The results demonstrate that Ziapin2 can photomodulate Ca[2+] responses in the employed system without compromising tissue integrity, viability, or behavior. Furthermore, we showed that the light-based stimulation approach offers a similar resolution compared to electrical stimulation, the current gold standard. Overall, this protocol opens promising perspectives for the application of Ziapin2 and material-based photostimulation in cardiac research.},
}
@article {pmid40624545,
year = {2025},
author = {Ahmed, HMM and Zheng, L and Hunnekuhl, VS},
title = {Transferable approaches to CRISPR-Cas9 induced genome editing in non-model insects: a brief guide.},
journal = {Frontiers in zoology},
volume = {22},
number = {1},
pages = {13},
pmid = {40624545},
issn = {1742-9994},
support = {TE1380/1//Deutsche Forschungsgemeinschaft/ ; },
abstract = {Despite the large variety of insect species with divergent morphological, developmental and physiological features questions on gene function could for a long time only be addressed in few model species. The adaption of the bacterial CRISPR-Cas system for genome editing in eukaryotic cells widened the scope of the field of functional genetics: for the first time the creation of heritable genetic changes had become possible in a very broad range of organisms. Since then, targeted genome editing using the CRISPR-Cas technology has greatly increased the possibilities for genetic manipulation in non-model insects where molecular genetic tools were little established. The technology allows for site-specific mutagenesis and germline transformation. Importantly, it can be used for the generation of gene knock-outs, and for the knock-in of transgenes and generation of gene-reporter fusions. CRISPR-Cas induced genome editing can thus be applied to address questions in basic research in various insect species and other study organisms. Notably, it can also be used in applied insect biotechnology to design new pest and vector control strategies such as gene drives and precision guided Sterile Insect Technique. However, establishing CRISPR in a new model requires several practical considerations that depend on the scientific questions and on the characteristics of the respective study organism. Therefore, this review is intended to give a literature overview on different CRISPR-Cas9 based methods that have already been established in diverse insects. After discussing some required pre-conditions of the study organism, we provide a guide through experimental considerations when planning to conduct CRISPR-Cas9 genome editing, such as the design and delivery of guide RNAs, and of Cas9 endonuclease. We discuss the use of different repair mechanisms including homology directed repair (HDR) for a defined insertion of genetic elements. Furthermore, we describe different molecular methods for genetic screening and the use of visible markers. We focus our review on experimental work in insects, but due to the ubiquitous functionality of the CRISPR-Cas system many considerations are transferable to other non-model organisms.},
}
@article {pmid40623912,
year = {2025},
author = {Wang, YW and Tang, YM},
title = {[Advances in the application strategies of CRISPR/Cas9 technology in chimeric antigen receptor T cell therapy for hematological malignancies].},
journal = {Zhonghua xue ye xue za zhi = Zhonghua xueyexue zazhi},
volume = {46},
number = {5},
pages = {481-488},
doi = {10.3760/cma.j.cn121090-20240911-00343},
pmid = {40623912},
issn = {0253-2727},
mesh = {Humans ; *CRISPR-Cas Systems ; *Hematologic Neoplasms/therapy ; *Receptors, Chimeric Antigen ; *Immunotherapy, Adoptive/methods ; T-Lymphocytes/immunology ; Gene Editing ; },
abstract = {Chimeric antigen receptor (CAR) T-cell therapy has achieved breakthroughs in treating relapsed/refractory B-cell malignancies. However, it still faces challenges, including complex manufacturing processes, limited indications, T-cell exhaustion, and insufficient durability of therapeutic efficacy. CRISPR/Cas9, a highly efficient and relatively simple gene-editing technology, offers new avenues for overcoming these limitations. This review briefly outlines the working mechanism of CRISPR/Cas9 and focuses on its recent applications and clinical practices in developing universal CAR T-cells, enhancing T-cell function, and extending CAR T-cell therapy to T-cell and myeloid leukemias. Furthermore, this review highlights optimization strategies developed over the past two years to enhance the editing precision, delivery efficiency, and safety of the CRISPR/Cas9 system, aiming to provide insights for the optimal design and clinical application of CAR T-cell therapy.},
}
@article {pmid40623181,
year = {2025},
author = {Aumann, RA and Gouvi, G and Gregoriou, ME and Rehling, T and Sollazzo, G and Bourtzis, K and Schetelig, MF},
title = {Decoding and engineering temperature-sensitive lethality in Ceratitis capitata for pest control.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {122},
number = {28},
pages = {e2503604122},
doi = {10.1073/pnas.2503604122},
pmid = {40623181},
issn = {1091-6490},
support = {FF17000//SITplus, Hort Innovation/ ; SCHE 1833/7-1//Deutsche Forschungsgemeinschaft (DFG)/ ; 101059523//Horizon Europe Research and Innovation Action/ ; },
mesh = {Animals ; *Ceratitis capitata/genetics ; *Pest Control, Biological/methods ; Male ; Female ; Temperature ; Mutation ; Phenotype ; },
abstract = {The Sterile Insect Technique (SIT) is a species-specific and environmentally friendly method for effectively controlling pest insect populations based on releasing reared, sterile insects into infested areas. Sex sorting in rearing facilities, enabling male-only releases, is necessary to ensure SIT programs are efficient, cost-effective and, in case of mosquito control, also safe. This can be greatly facilitated by genetic sexing strains (GSS), exhibiting sex-specific phenotypic markers. However, the development of GSS remains challenging. The construction of a temperature-sensitive lethal (tsl)-based GSS in the Mediterranean fruit fly (Ceratitis capitata) over three decades ago was considered a major breakthrough for SIT programs but was never successfully replicated in other pests. After over 30 y of research, we have pinpointed a specific mutation in the C. capitata lysine--tRNA ligase (Lysyl-tRNA synthetase, LysRS) gene responsible for the tsl phenotype. Introducing this specific mutation into a wild-type strain produced full embryonic lethality under heat stress, replicating the original mutant phenotype. The random integration of a LysRS minigene reversed this effect. The high conservation of LysRS among insects suggests that tsl-based GSS could be expanded to multiple pest species and extend applications of SIT programs for disease prevention and the protection of agriculture.},
}
@article {pmid40622878,
year = {2025},
author = {Perry, JK and Schwartzberg, PL and Golec, DP},
title = {Investigating Murine CD4 T Cell Differentiation Using CRISPR-Cas9 Ribonucleoprotein Complex-mediated Gene Ablation.},
journal = {Journal of visualized experiments : JoVE},
volume = {},
number = {220},
pages = {},
doi = {10.3791/67380},
pmid = {40622878},
issn = {1940-087X},
mesh = {Animals ; Mice ; *Ribonucleoproteins/genetics ; *CRISPR-Cas Systems ; *CD4-Positive T-Lymphocytes/cytology/immunology ; Cell Differentiation/genetics ; *Gene Knockout Techniques/methods ; Mice, Knockout ; RNA, Guide, CRISPR-Cas Systems/genetics ; },
abstract = {The widespread accessibility of clustered regularly interspaced short palindromic repeat (CRISPR)-Cas9 technology has made gene targeting in primary cells a routine method for evaluating gene function in T cells. Given the cost and limited availability of knockout (KO) mouse strains, testing preliminary hypotheses involving gene function in T cells can be prohibitive using gene-targeted animal models. However, using commercially available resources, including predesigned guide RNAs (gRNAs), researchers can conveniently generate gene-targeted naïve T cells that can be used for T cell activation and differentiation studies. Here we outline a protocol for using nucleofection-delivered CRISPR-Cas9 ribonucleoprotein complexes (RNPs) to efficiently generate gene KO murine naïve CD4 T cells that can be used to evaluate gene function in CD4 T cell differentiation, in vitro. Isolation of naïve CD4 T cells from mouse secondary lymphoid organs, followed by nucleofection with Cas9-gRNA complexes ensures gene KO is initiated before downstream T cell activation, offering a strategic advantage over retroviral-mediated gRNA delivery, which typically requires preactivation of T cells, preventing the evaluation of effects in naïve T cells. Furthermore, this nucleofection-based method bypasses potential developmental issues associated with gene KO animals. Following Cas9-gRNA delivery, we describe protocols for studying CD4 T cell differentiation into Th1, Th2, Th17, and Treg lineages using in vitro polarization. In addition, this protocol is adaptable to using gene-targeted CD4 or CD8 T cells for numerous downstream applications, including other T cell activation studies in vitro and adoptive transfer studies in vivo. The use of CRISPR-Cas9 methods has streamlined our ability to evaluate gene function in T cells and allows for the routine KO of many genes of interest, freeing researchers from limitations associated with studying gene KO animals.},
}
@article {pmid40622492,
year = {2025},
author = {Parmar, H and Goel, A and Gelaw, TA and Reddy, MK},
title = {Enhancing drought resilience in crops: mechanistic approaches in the face of climate challenge.},
journal = {Plant molecular biology},
volume = {115},
number = {4},
pages = {82},
pmid = {40622492},
issn = {1573-5028},
support = {09/512(0244)/2019-EMR-I//Human Resource Development Centre, Council of Scientific And Industrial Research/ ; },
mesh = {*Crops, Agricultural/genetics/physiology ; *Droughts ; Climate Change ; Quantitative Trait Loci/genetics ; Stress, Physiological ; Plant Breeding ; Gene Editing ; Gene Expression Regulation, Plant ; },
abstract = {Enhancing drought resilience in crops has become a critical challenge in the face of global climate change, which is exacerbating the frequency and severity of drought events. This review explores mechanistic approaches aimed to improve crop drought tolerance, focusing on physiological, biochemical, and molecular mechanisms. We examine the key molecular pathways involved in drought stress responses, including the Mitogen-Activated Protein Kinase (MAPKs) signaling pathway, hormonal regulation, transcriptional control, and post-translational modifications such as ubiquitination-mediated protein degradation, and plant-microbe interaction. The review also delves into the mechanisms of drought stress tolerance, including drought escape, avoidance, and tolerance. It highlights significant traits contributing to drought resilience, such as stomatal regulation and root architecture. Furthermore, we discuss genomics and breeding approaches, including quantitative trait loci (QTL) mapping, marker-assisted selection (MAS), and cutting-edge CRISPR-Cas-based genome editing technologies. These advanced techniques, such as base editing, prime editing, and multiplexing, transform crop improvement strategies by facilitating precise and efficient modifications for enhanced drought resilience, with the success stories in crops such as rice, maize, wheat, and others. Integrating these mechanistic and technological approaches offers promising avenues for developing drought-resilient crops, ensuring food security under increasingly unpredictable climate conditions.},
}
@article {pmid40619763,
year = {2025},
author = {Fritz, B and Lapp, CJ and Gescher, J},
title = {Influence of Different Transposon Families on Genomic Stability of Shewanella oneidensis MR1.},
journal = {Microbial biotechnology},
volume = {18},
number = {7},
pages = {e70188},
doi = {10.1111/1751-7915.70188},
pmid = {40619763},
issn = {1751-7915},
support = {031B0847A//Bundesministerium f&#xfc;r Bildung und Forschung/ ; },
mesh = {*Shewanella/genetics ; *DNA Transposable Elements/genetics ; *Genomic Instability ; Transposases/genetics/metabolism ; Genome, Bacterial ; Plasmids/genetics ; Whole Genome Sequencing ; },
abstract = {Shewanella oneidensis, recognised as an important model organism for exoelectrogenic electron transport, has been extensively studied for its potential applications in bioelectrochemical systems. To date, the activity of transposable elements in this organism has not been conclusively investigated. This study focused on transposases, specifically insertion sequences (IS), which make up approximately 4.7% of the organism's genome, and evaluated their impact on genome stability under stress conditions. Using whole genome sequencing, two IS families, ISSOD1 and ISSOD2, were identified as the most active, both showing similar transposition patterns across all tested stressors. A CRISPR/dCas9 cytosine deaminase system was used to introduce stop codons in the ISSOD2 transposase genes, resulting in a significant reduction of transposition events under stress conditions. Analysis of transposition patterns revealed a high frequency of insertions occurring on the megaplasmid, which predominantly carries non-essential genes. Experiments performed here to delete the megaplasmid resulted in the elimination of approximately 35% of its sequence, including an unexpected complete loss of the ori/repA region. Therefore, it was hypothesised that the megaplasmid either exists in a metastable state, possibly representing a cointegrated intermediate within the ISSOD9 (Tn3 member) transposition mechanism, or consists of two replicons that have been combined in previous assemblies due to long overlapping homologies resulting from the presence of ISSOD9. These findings highlight the dynamics of transposable elements in S. oneidensis and suggest strategies to improve strain stability by inactivating these elements and at least reducing megaplasmid sequences. Such approaches could improve the suitability of the organism for industrial applications.},
}
@article {pmid40384567,
year = {2025},
author = {Schneider, PG and Liu, S and Bullinger, L and Ostendorf, BN},
title = {BEscreen: a versatile toolkit to design base editing libraries.},
journal = {Nucleic acids research},
volume = {53},
number = {W1},
pages = {W68-W72},
doi = {10.1093/nar/gkaf406},
pmid = {40384567},
issn = {1362-4962},
support = {70114327//German Cancer Aid/ ; 10.24.1.010MN//Fritz Thyssen/ ; //Guangzhou/ ; //Charit&#xe9;-BIH Digital Clinician/ ; //German Research Foundation/ ; },
mesh = {*Software ; *Gene Editing/methods ; *Gene Library ; *RNA, Guide, CRISPR-Cas Systems/genetics ; Humans ; Internet ; CRISPR-Cas Systems ; },
abstract = {Base editing enables the high-throughput screening of genetic variants for phenotypic effects. Base editing screens require the design of single guide RNA (sgRNA) libraries to enable either gene- or variant-centric approaches. While computational tools supporting the design of sgRNAs exist, no solution offers versatile and scalable library design enabling all major use cases. Here, we introduce BEscreen, a comprehensive base editing guide design tool provided as a web server (bescreen.ostendorflab.org) and as a command line tool. BEscreen provides variant-, gene-, and region-centric modes to accommodate various screening approaches. The variant mode accepts genomic coordinates, amino acid changes, or rsIDs as input. The gene mode designs near-saturation libraries covering the entire coding sequence of given genes or transcripts, and the region mode designs all possible guides for given genomic regions. BEscreen enables selection of guides by biological consequence, it features comprehensive customization of base editor characteristics, and it offers optional annotation using Ensembl's Variant Effect Predictor. In sum, BEscreen is a highly versatile tool to design base editing screens for a wide range of use cases with seamless scalability from individual variants to large, near-saturation libraries.},
}
@article {pmid40377102,
year = {2025},
author = {Chapdelaine-Trépanier, V and Shenoy, S and Masud, W and Minju-Op, A and Bérubé, MA and Schönherr, S and Forer, L and Fradet-Turcotte, A and Taliun, D and Cuella-Martin, R},
title = {CRISPR-BEasy: a free web-based service for designing sgRNA tiling libraries for CRISPR-dependent base editing screens.},
journal = {Nucleic acids research},
volume = {53},
number = {W1},
pages = {W193-W202},
doi = {10.1093/nar/gkaf382},
pmid = {40377102},
issn = {1362-4962},
support = {FRN:DV2-197674/CAPMC/CIHR/Canada ; //Canada Excellence Chair in Genomic Medicine/ ; //McGill University/ ; //Fonds de Recherche du Qu&#xe9;bec-Sant&#xe9;/ ; //Doctoral Training Scholarships/ ; //Canada Research Chair Tier II in Molecular Biology and Genomic Instability/ ; FRN:DV2-197674/CAPMC/CIHR/Canada ; },
mesh = {Internet ; *Gene Editing/methods ; *Software ; *CRISPR-Cas Systems ; *RNA, Guide, CRISPR-Cas Systems/genetics ; Gene Library ; Humans ; },
abstract = {CRISPR-dependent base editing (BE) enables the modeling and correction of genetic mutations at single-base resolution. Base editing screens, where point mutations are queried en masse, are powerful tools to systematically draw genotype-phenotype associations and characterise the function of genes and other genomic elements. However, the lack of user-friendly web-based tools for designing base editing screens can hinder broad technology adoption. Here, we introduce CRISPR-BEasy (https://crispr-beasy.cerc-genomic-medicine.ca), a free, automated web-based server that streamlines the creation of single guide (sg)RNA tiling libraries for base editing screens. Researchers can provide their genes or genomic features of interest, their base editors of choice, and target sequences to act as positive and negative controls. The server designs and annotates sgRNA libraries by integrating custom code with publicly available tools such as crisprVerse and Ensembl's Variant Effect Predictor. CRISPR-BEasy provides downloadable results, including sgRNA on/off-target scores, predicted mutational outcomes per base editor, and intuitive interactive visualizations for data quality assessment. CRISPR-BEasy also provides a separate tool that assembles sgRNA libraries into oligonucleotides for cloning following the detailed protocol documented in the searchable web server manual. Together, CRISPR-BEasy ensures the seamless design of cloning-ready sgRNA libraries, seeking to democratise access to base editing screening technologies.},
}
@article {pmid40337925,
year = {2025},
author = {Mekonnen, AM and Seong, K and Kim, H and Park, J},
title = {Variant-aware Cas-OFFinder: web-based in silico variant-aware potential off-target site identification for genome editing applications.},
journal = {Nucleic acids research},
volume = {53},
number = {W1},
pages = {W118-W124},
doi = {10.1093/nar/gkaf389},
pmid = {40337925},
issn = {1362-4962},
support = {RS-2024-00322053//Rural Development Administration/ ; //National Research Foundation of Korea/ ; //Korean government/ ; RS-2023-00278658//MSIT/ ; //Bio &amp; Medical Technology Development/ ; RS-2024-00439078//Korean government (MSIT)/ ; BK21 Four//Korean government (MSIT)/ ; },
mesh = {*Gene Editing/methods ; *Software ; Humans ; Internet ; *CRISPR-Cas Systems ; Genome, Human ; Genetic Variation ; Haplotypes ; Computer Simulation ; },
abstract = {Genome editing based on CRISPR systems has been widely used in the vast areas of biomedical and agricultural applications. However, identifying the potential off-target sites remains challenging, particularly in individuals with diverse genetic variations. Several in silico tools have been developed to predict potential off-target sites, but they have limitations on their performance and scalability. In this paper, we present "Variant-aware Cas-OFFinder," a novel pipeline based on Cas-OFFinder for identifying potential off-target sites by accounting for individual genetic variants. We benchmarked the pipeline's improved scalability and performance with the human genome and pepper cultivars, having unique potential off-target sites on each allele at the haplotype level. The web tool is open to all users without a login requirement and is freely available online at https://rgetoolkit.com/var-cas-offinder.},
}
@article {pmid40334276,
year = {2025},
author = {Liu, H and Dong, J and Wu, R and Dai, J and Lou, X and Xia, F and Willner, I and Huang, F},
title = {Light-Triggered CRISPR/Cas12a for Genomic Editing and Tumor Regression.},
journal = {Angewandte Chemie (International ed. in English)},
volume = {64},
number = {28},
pages = {e202502892},
doi = {10.1002/anie.202502892},
pmid = {40334276},
issn = {1521-3773},
support = {22377112//National Natural Science Foundation of China/ ; 22090050//National Natural Science Foundation of China/ ; 2021YFA1200400//National Key R&amp;D Program of China/ ; 22161142020//Joint NSFC-ISF Research Grant Program/ ; },
mesh = {*Gene Editing/methods ; Humans ; *CRISPR-Cas Systems/genetics ; *Light ; Animals ; Hep G2 Cells ; Mice ; Hepatocyte Growth Factor/genetics ; Cell Proliferation ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {A photo-triggered CRISPR/Cas12a machinery for in vitro and in vivo gene editing is introduced. The system consists of a caged, inactive ortho-nitrobenzyl phosphate ester photo-responsive crRNA, which, upon light-induced deprotection, yields the active CRISPR/Cas12a gene editing machinery (LAC12aGE). The LAC12aGE system induces specific thymidine-rich (TTTN) protospacer-adjacent motif (PAM)-guided double-stranded breaks in genomic DNA, which upon non-homologous end-joining lead to gene repair. The LAC12aGE machinery is applied for gene editing of an exogenous dual fluorescent reporter gene in living cells, as well as the endogenous gene encoding DNA methyltransferase 1. In addition, the LAC12aGE is applied for in vitro gene editing and disruption of the hepatocyte growth factor (HGF) gene in HepG2 cells, where knockout of the HGF gene results in inhibited cell proliferation and migration, as well as enhanced apoptosis. Moreover, the in vivo knockout and disruption of the HGF gene in HepG2 tumors by the LAC12aGE machinery is demonstrated. The cyclic temporal development of the LAC12aGE system in tumors shows effective inhibition of tumor growth and enhanced apoptosis/necrosis of tumor tissues compared to control systems.},
}
@article {pmid40308216,
year = {2025},
author = {Marino, GB and Evangelista, JE and Clarke, DJB and Ma'ayan, A},
title = {L2S2: chemical perturbation and CRISPR KO LINCS L1000 signature search engine.},
journal = {Nucleic acids research},
volume = {53},
number = {W1},
pages = {W338-W350},
doi = {10.1093/nar/gkaf373},
pmid = {40308216},
issn = {1362-4962},
support = {OT2OD036435/GF/NIH HHS/United States ; OT2OD030160/GF/NIH HHS/United States ; U24CA264250/GF/NIH HHS/United States ; U24CA271114/GF/NIH HHS/United States ; R01DK131525/GF/NIH HHS/United States ; RC2DK131995/GF/NIH HHS/United States ; },
mesh = {Humans ; *CRISPR-Cas Systems ; Gene Knockout Techniques ; *Software ; Internet ; *Search Engine ; Gene Expression Profiling ; Small Molecule Libraries/pharmacology ; Cell Line ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Transcriptome ; },
abstract = {As part of the Library of Integrated Network-Based Cellular Signatures (LINCS) NIH initiative, 248 human cell lines were profiled with the L1000 assay to measure the effect of 33 621 small molecules and 7508 single-gene CRISPR knockouts. From this massive dataset, we computed 1.678 million sets of up- and down-regulated genes. These gene sets are served for search by the LINCS L1000 Signature Search (L2S2) web server application. With L2S2, users can identify small molecules and single gene CRISPR KOs that produce gene expression profiles similar or opposite to their submitted single or up/down gene sets. L2S2 also includes a consensus search feature that ranks perturbations across all cellular contexts, time points, and concentrations. To demonstrate the utility of L2S2, we crossed the L2S2 gene sets with gene sets collected for the RummaGEO resource. The analysis identified clusters of differentially expressed genes that match drug classes, tissues, and diseases, pointing to many opportunities for drug repurposing and drug discovery. Overall, the L2S2 web server application can be used to further the development of personalized therapeutics while expanding our understanding of complex human diseases. The L2S2 web server application is available at https://l2s2.maayanlab.cloud.},
}
@article {pmid39729054,
year = {2025},
author = {Ballantine, J and Tisdale, JF},
title = {Gene therapy for sickle cell disease: recent advances, clinical trials and future directions.},
journal = {Cytotherapy},
volume = {27},
number = {7},
pages = {826-834},
pmid = {39729054},
issn = {1477-2566},
support = {T32 CA060441/CA/NCI NIH HHS/United States ; ZIA HL006008/ImNIH/Intramural NIH HHS/United States ; },
mesh = {Humans ; *Anemia, Sickle Cell/therapy/genetics ; *Genetic Therapy/methods ; Gene Editing ; Clinical Trials as Topic ; Hematopoietic Stem Cell Transplantation ; CRISPR-Cas Systems/genetics ; Genetic Vectors/genetics ; Animals ; beta-Thalassemia/therapy/genetics ; },
abstract = {Sickle cell disease (SCD) is the most common inherited blood disorder worldwide, impacting millions and imposing severe healthcare challenges, particularly in resource-limited regions. Current treatments have variable efficacy and require lifelong adherence. Allogeneic Hematopoietic Stem Cell Transplantation can be curative but comes with significant side effects and limited donor availability limits its widespread applicability. Gene therapy, by addressing the root genetic causes, offers a revolutionary alternative. This article discusses the molecular mechanisms of SCD and β-thalassemia and highlights advancements in gene therapy, such as gene addition via lentiviral vectors and gene editing with CRISPR/Cas9 technology. Clinical trials have brought about significant progress but challenges remain, including leukemogenesis, delivery efficiency and cost. Future efforts must focus on enhancing efficiency, reducing costs, developing nongenotoxic conditioning regimens and methods for in vivo application.},
}
@article {pmid40619482,
year = {2025},
author = {Bodmer, N and Uth, K and Mehmeti, R and Demir, CS and Stroka, D and Ghaffari-Tabrizi-Wizsy, N and Lugli, A and de Wever, O and Zlobec, I and Tschan, MP},
title = {CDX2 loss in colorectal cancer cells is associated with invasive properties and tumor budding.},
journal = {Scientific reports},
volume = {15},
number = {1},
pages = {24113},
pmid = {40619482},
issn = {2045-2322},
support = {ESKAS, nr. 2021.0019 / Kosovo / OP//Swiss Government Excellence Scholarship/ ; 31003A_166578/1//Schweizerischer Nationalfonds zur F&#xf6;rderung der Wissenschaftlichen Forschung/ ; 31003A_166578/1//Schweizerischer Nationalfonds zur F&#xf6;rderung der Wissenschaftlichen Forschung/ ; },
mesh = {*CDX2 Transcription Factor/genetics/metabolism ; *Colorectal Neoplasms/pathology/genetics/metabolism ; Humans ; Animals ; Cell Movement/genetics ; Cell Line, Tumor ; Neoplasm Invasiveness ; Mice ; Cell Proliferation ; Epithelial-Mesenchymal Transition/genetics ; Cadherins/metabolism ; Gene Expression Regulation, Neoplastic ; CRISPR-Cas Systems ; },
abstract = {In colorectal cancer (CRC), tumor buds (TB) are observed histologically as single tumor cell or small tumor cell clusters located mainly at the advancing tumor edge. TB are a marker of poor prognosis and correlate with metastatic disease in CRC patients. They often lack expression of CDX2 and overexpress markers involved in epithelial-mesenchymal transition (EMT). We evaluated the function of CDX2 in CRC proliferation and migration using CRISPR/Cas9 technology and demonstrated a possible link to tumor dissociation and tumor budding. Knocking out CDX2 in CRC cell lines significantly increased migration. Importantly, the observed phenotypes could be rescued by re-expressing CDX2 and by specific CRISPR synergistic activation mediator (SAM) of endogenous CDX2 in CDX2 low expressing CRC cell lines. Multiplex immunofluorescence (mIF) analysis of primary tumor regions compared to TB in a CDX2-positive CRC patient sample as well as patient derived xenografts (PDX) revealed significantly lower CDX2 expression and correlating E-cadherin levels in TB compared to primary tumor regions, in both models. Accordingly, increased invasiveness of CRC CDX2 knockout cells was seen in ex ovo xenografts. Taken together, our results provide further insight into the function of CDX2 in preventing CRC cell migration, tumor budding and tumor aggressiveness.},
}
@article {pmid40617436,
year = {2025},
author = {Marpaung, DSS and Chen, YY and Singuru, MMR and Chuang, MC},
title = {Structure-transformable poly (thymine) activators of CRISPR/Cas12a for highly sensitive detection of mercury (II) ions.},
journal = {International journal of biological macromolecules},
volume = {319},
number = {Pt 4},
pages = {145748},
doi = {10.1016/j.ijbiomac.2025.145748},
pmid = {40617436},
issn = {1879-0003},
abstract = {Off-target effects of the CRISPR/Cas system refer to suboptimal activity triggered by activators containing unintended modifications beyond the intended target, which can lead to unwanted genetic changes-particularly problematic in therapeutic applications. In this study, the off-target effects of Cas12a are strategically repurposed for advanced Hg[2+] detection by leveraging a structure-transformable activator. The proposed approach integrates a two-segment poly-T DNA activator with the CRISPR/Cas12a system, where the activator forms hairpin structures through thymine-Hg[2+]-thymine base pairing in response to mercury concentrations. Increasing Hg[2+] concentrations promote the formation and stability of hairpin DNA, significantly suppressing Cas12a activity and resulting in an ON-OFF fluorescence signal modulated by trans-cleavage activity. The effect of varying thymine segment lengths in the poly-T activator was investigated to increase the hybridization burden for crRNA, ultimately enhancing detection sensitivity. The Hg[2+]-dependent structural transformation of the activator was characterized using circular dichroism (CD), while urea and native PAGE analyses confirmed the catalytic behavior of both cis- and trans-cleavage activities, respectively, as a function of Hg[2+] concentration. The trans-cleavage efficiency (kcat/KM) decreased by 3.03-fold as Hg[2+] concentration increased from 0 to 40 nM. Under optimized conditions, the biosensor demonstrated a linear detection range of 0-30 nM, a detection limit as low as 0.372 nM, and high selectivity for Hg[2+] over other metal ions. Furthermore, the biosensor was successfully applied for Hg[2+] detection in field water samples. These findings establish a robust foundation for exploiting the off-target effects of the CRISPR/Cas12a system for the efficient detection of heavy metals in environmental monitoring.},
}
@article {pmid40617152,
year = {2025},
author = {Rangu, SS and Misra, CS and Shaikh, S and Singh, M and Bindal, G and Singh, S and Purageri, S and Shettigar, S and Warke, R and Cherian, S and Rath, D},
title = {RNA extraction-free CRISPR-based SARS-CoV-2 detection in viral transport medium and dry swab-a comparative analysis: Short title: Extraction-free CRISPR-based Covid detection.},
journal = {Diagnostic microbiology and infectious disease},
volume = {113},
number = {3},
pages = {116982},
doi = {10.1016/j.diagmicrobio.2025.116982},
pmid = {40617152},
issn = {1879-0070},
abstract = {CRISPR-Cas-based methods have shown high efficacy in detecting SARS-CoV-2. Amidst the Covid-19 pandemic, numerous studies have explored SARS-CoV-2 detection methods without the need for RNA extraction, aiming to reduce cost and processing time. Here, we assessed a CRISPR-based SARS-CoV-2 detection method's ability to detect the virus in viral transport medium (VTM). Swabs obtained from Covid-19 positive patients and stored in two Indian brands of VTM were examined alongside dry swab samples. The samples underwent proteinase -K treatment followed by heat incubation. The released nucleic acids were tested by RT-LAMP and CRISPR-based detection. We conclude that SARS-CoV-2 can be detected in VTM of two commercial preparations as well as dry swab samples without RNA extraction and purification. COVID detection was found to be more efficient for dry swab samples compared to VTM samples. The work flow described in this paper can be extended to other respiratory diseases.},
}
@article {pmid40057303,
year = {2025},
author = {Wang, L and Hu, Y and Qiu, Y and Lin, H and Li, X and Fu, S and Zeng, YY and Ghouse, M and Long, C and Liu, Y and Fei, JF},
title = {Establishing a semi-homology-directed recombination method for precision gene integration in axolotls.},
journal = {Journal of genetics and genomics = Yi chuan xue bao},
volume = {52},
number = {7},
pages = {942-953},
doi = {10.1016/j.jgg.2025.03.001},
pmid = {40057303},
issn = {1673-8527},
mesh = {Animals ; CRISPR-Cas Systems/genetics ; *Gene Knock-In Techniques/methods ; *Ambystoma mexicanum/genetics ; Gene Editing/methods ; SOXB1 Transcription Factors/genetics ; *Homologous Recombination ; },
abstract = {The axolotl is broadly used in regenerative, developmental, and evolutionary biology research. Targeted gene knock-in is crucial for precision transgenesis, enabling disease modeling, visualization, tracking, and functional manipulation of specific cells or genes of interest (GOIs). Existing CRISPR/Cas9-mediated homology-independent method for gene knock-in often causes "scars/indels" at integration junctions. Here, we develop a CRISPR/Cas9-mediated semi-homology-directed recombination (HDR) knock-in method using a donor construct containing a single homology arm for the precise integration of GOIs. This semi-HDR approach achieves seamless single-end integration of the Cherry reporter gene and a large inducible Cre cassette into intronless genes like Sox2 and Neurod6 in axolotls, which are challenging to modify with the homology-independent method. Additionally, we integrate the inducible Cre cassette into intron-containing loci (e.g., Nkx2.2 and FoxA2) without introducing indels via semi-HDR. GOIs are properly expressed in F0 founders, with approximately 5%-10% showing precise integration confirmed by genotyping. Furthermore, using the Nkx2.2:CreER[T2] line, we fate-map spinal cord p3 neural progenitor cells, revealing that Nkx2.2[+] cells adopt different lineages in development and regeneration, preferentially generating motoneurons over oligodendrocytes during regeneration. Overall, this semi-HDR method balances efficiency and precision in the integration of GOIs, providing a valuable tool for generating knock-in axolotls and potentially extending to other species.},
}
@article {pmid40616579,
year = {2025},
author = {Urnov, F and Kassim, S and Musunuru, K and Liu, D and Lee, A and Barrera, L and Stetkiewicz, P and Bruno, J and Hewitt, M and Lister, T and Malech, H and Gasch, L and Diver, M and Gertler, N and Grignon, F and Le, A and Lehmicke, M and Almendro-Navarro, V and Lembong, J},
title = {Advancing gene-editing platforms to improve the viability of rare-disease therapeutics: key insights from a 2024 Scientific Exchange hosted by ARM, ISCT, and Danaher.},
journal = {Cytotherapy},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.jcyt.2025.06.010},
pmid = {40616579},
issn = {1477-2566},
abstract = {Rare-disease therapeutics face viability challenges due to small patient populations and drug-development and regulatory frameworks that were not developed to address rapidly progressive or quickly fatal conditions. Because the majority of rare diseases are genetic in nature, gene-editing modalities offer substantial promise. This Scientific Exchange, co-hosted by the Alliance for Regenerative Medicine, the International Society for Cell and Gene Therapy, and Danaher Corporation in November 2024, set out to address the challenge of realizing the full promise of gene editing for rare-disease therapies by advancing platforms that leverage stable and reusable processes or components to develop multiple therapies. Through multi-stakeholder engagement and discussions of case studies in CRISPR/Cas nuclease, base, and prime editing, 4 key opportunities emerged that deliver value by holding platform elements constant and/or streamlining development steps: (1) consistent delivery vehicle; (2) consistent manufacturing; (3) benefit-risk appropriate quality requirements; and (4) expansive clinical trial designs. Together, these opportunities could yield up to 5-fold efficiency gains and result in substantial value creation for patients, regulators, and developers, potentially decreasing the time required to dose patients with a new gene-editing therapy from years down to 6 months.},
}
@article {pmid40615444,
year = {2025},
author = {Novotná, M and Tinti, M and Faria, JRC and Horn, D},
title = {Precision-edited histone tails disrupt polycistronic gene expression controls in trypanosomes.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {6194},
pmid = {40615444},
issn = {2041-1723},
support = {217105/Z/19/Z//Wellcome Trust (Wellcome)/ ; },
mesh = {*Histones/genetics/metabolism ; *Trypanosoma brucei brucei/genetics/metabolism ; Protozoan Proteins/genetics/metabolism ; RNA Polymerase II/metabolism/genetics ; Promoter Regions, Genetic/genetics ; CRISPR-Cas Systems ; *Gene Expression Regulation ; Gene Editing ; Mutation ; Acetylation ; Lysine/metabolism ; Protein Processing, Post-Translational ; Methylation ; Gene Expression Profiling ; Chromatin/metabolism ; },
abstract = {Transcription of protein coding genes in trypanosomatids is atypical and almost exclusively polycistronic. In Trypanosoma brucei, for example, approximately 150 polycistrons, and 8000 genes, are constitutively transcribed by RNA polymerase II. The RNA pol-II promoters are also unconventional and characterised by regions of chromatin enriched for histones with specific patterns of post-translational modification on their divergent N-terminal tails. To investigate the roles of histone tail-residues in gene expression control in T. brucei, we engineered strains exclusively expressing mutant histones. We used an inducible CRISPR-Cas9 system to delete >40 histone H4 genes, complementing the defect with a single ectopic H4 gene. The resulting "hist[one]H4" strains were validated using whole-genome sequencing and transcriptome analysis. We then performed saturation mutagenesis of six histone H4 N-terminal tail lysine residues, that are either acetylated or methylated, and profiled relative fitness of 384 distinct precision-edited mutants. H4[lys10] mutations were not tolerated, but we derived nineteen strains exclusively expressing distinct H4[lys4] or H4[lys14] mutants. Proteomic and transcriptomic analysis of H4[lys4] glutamine mutants revealed significantly reduced expression of genes adjacent to RNA pol-II promoters, where glutamine mimics abnormally elevated acetylation. Thus, we present direct evidence for polycistronic expression control by modified histone H4 N-terminal tail residues in trypanosomes.},
}
@article {pmid40615387,
year = {2025},
author = {Lou, S and DJiake Tihagam, R and Wasko, UN and Equbal, Z and Venkatesan, S and Braczyk, K and Przanowski, P and Il Koo, B and Saltani, I and Singh, AT and Likhite, S and Powers, S and Souza, GMPR and Maxwell, RA and Yu, J and Zhu, LJ and Beenhakker, M and Abbott, SBG and Lu, Z and Green, MR and Meyer, KC and Tushir-Singh, J and Bhatnagar, S},
title = {Targeting microRNA-dependent control of X chromosome inactivation improves the Rett Syndrome phenotype.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {6169},
pmid = {40615387},
issn = {2041-1723},
mesh = {*Rett Syndrome/genetics/therapy ; *MicroRNAs/genetics/metabolism ; *X Chromosome Inactivation/genetics ; Animals ; Female ; Methyl-CpG-Binding Protein 2/genetics/metabolism ; Humans ; Mice ; RNA, Long Noncoding/genetics/metabolism ; Phenotype ; Fibroblasts/metabolism ; CRISPR-Cas Systems ; Disease Models, Animal ; Male ; },
abstract = {X chromosome inactivation (XCI) is induced by Xist long non-coding RNA and protein-coding genes. However, the role of small non-coding RNA function in XCI remains unidentified. Our genome-wide, loss-of-function CRISPR/Cas9 screen in female fibroblasts identified microRNAs (miRNAs) as regulators of XCI. A striking finding is the identification of miR106a among the top candidates from the screen. Loss of miR106a is accompanied by altered Xist interactome, leading to dissociation and destabilization of Xist. XCI interference via miR106a inhibition has therapeutic implications for Rett syndrome (RTT) girls with a defective X-linked MECP2 gene. Here, we discovered that the inhibition of miR106a significantly improves several facets of RTT pathology: it increases the life span, enhances locomotor activity and exploratory behavior, and diminishes breathing variabilities. Our results suggest that miR106a targeting offers a feasible therapeutic strategy for RTT and other monogenic X-linked neurodevelopmental disorders.},
}
@article {pmid40613815,
year = {2025},
author = {Pinto, D and Mendes, I and Cunha, MV},
title = {Genomic Survey Reveals no Detectable Bacteriophage Activity in Mycobacterium bovis Across a Large Population.},
journal = {FEMS microbiology ecology},
volume = {},
number = {},
pages = {},
doi = {10.1093/femsec/fiaf072},
pmid = {40613815},
issn = {1574-6941},
abstract = {Phages are major drivers of bacterial evolution, yet their ecological and evolutionary interactions with Mycobacterium bovis, a key member of the Mycobacterium tuberculosis complex (MTBC), remain understudied. In this work, we investigate the elusive phage-bacterium interface in M. bovis by integrating comparative genomics of 200 isolates from infected animals with molecular analyses of M. bovis-positive environmental samples. Despite employing diverse and complementary approaches, we found no evidence of active or recent phage infections: no novel prophages beyond the conserved phiRv1, no expansion of CRISPR arrays, and no co-occurrence of M. bovis and mycobacteriophages in host tissues or environmental matrices. Intriguingly, we identified multiple independent excision events of phiRv1 across closely related lineages, suggesting recent prophage mobilization driven by unidentified ecological or genomic triggers. These findings echo previous observations in M. tuberculosis and point toward a stable, phage-scarce landscape across MTBC members. Our results raise compelling questions about the barriers to phage predation in M. bovis, the functionality of its CRISPR-Cas system, and the selective pressures underlying prophage retention and loss. By shedding light on these underexplored dynamics, our study reveals critical gaps in the ecological understanding of M. bovis and highlights opportunities for phage-based innovation in TB control.},
}
@article {pmid40613714,
year = {2025},
author = {Wu, D and Snead, S and Ganguly, C and Babu, K and Li, Y and Kathiresan, V and Shen, R and Zhang, X and Rajan, R and Qin, PZ},
title = {Structural integrity and side-chain interaction at the loop region of the bridge helix modulate Cas9 substrate discrimination.},
journal = {Nucleic acids research},
volume = {53},
number = {12},
pages = {},
doi = {10.1093/nar/gkaf557},
pmid = {40613714},
issn = {1362-4962},
support = {MCB-1716744//National Science Foundation/ ; MCB-1818107//National Science Foundation/ ; MCB-1716423//National Science Foundation/ ; MCB-2424888//National Science Foundation/ ; R35-GM145341/NH/NIH HHS/United States ; //Arnold and Mabel Beckman Foundation/ ; },
mesh = {*CRISPR-Associated Protein 9/chemistry/genetics/metabolism ; RNA, Guide, CRISPR-Cas Systems/metabolism/chemistry/genetics ; DNA/metabolism/chemistry/genetics ; Substrate Specificity ; CRISPR-Cas Systems ; Models, Molecular ; Protein Binding ; DNA Cleavage ; Protein Conformation ; },
abstract = {CRISPR-Cas9 (clustered regularly interspaced short palindromic repeats-CRISPR-associated protein 9) has been revolutionizing genome engineering, and in-depth understanding of mechanisms governing its DNA discrimination is critical for continuing technology advances. An arginine-rich bridge helix (BH) connecting the nuclease lobe and the recognition lobe, which is conserved across the Cas9 family, exists in a helix-loop-helix conformation in the apo wild-type protein but converts to a long contiguous helix in the Cas9/RNA binary complex. In this work, distances measured with spin labels were utilized to investigate BH's conformational transitions in the solution state upon single-guide RNA (sgRNA) binding, which is a critical early event preceding DNA binding and cleavage. Analyses show that sgRNA binding drives BH conformational changes in the wild-type SpyCas9 (SpyCas9WT) as well as in two BH-loop variants, SpyCas92Pro and SpyCas92Ala. Each Cas9-sgRNA binary complex, however, exhibits distinct BH features that reveal mutation-specific effects on helical integrity versus side-chain interactions. In addition, the BH conformational variations can be correlated to the observed changes in the mismatch cleavage profiles of the Cas9 variants. The work represents the first use of distances measured by site-directed spin labeling to investigate Cas9 protein conformational changes in the solution state and advances our understanding on the structure-dynamic-function relationship governing DNA target discrimination by Cas9.},
}
@article {pmid40613710,
year = {2025},
author = {Hibshman, GN and Taylor, DW},
title = {Structural basis of a dual-function type II-B CRISPR-Cas9.},
journal = {Nucleic acids research},
volume = {53},
number = {12},
pages = {},
doi = {10.1093/nar/gkaf585},
pmid = {40613710},
issn = {1362-4962},
support = {R35GM138348/NH/NIH HHS/United States ; F-1938//Welch Foundation/ ; },
mesh = {*CRISPR-Cas Systems ; *CRISPR-Associated Protein 9/chemistry/genetics/metabolism ; *Francisella/genetics/enzymology ; Gene Editing ; *Bacterial Proteins/chemistry/genetics/metabolism ; RNA, Guide, CRISPR-Cas Systems/genetics/metabolism ; Streptococcus pyogenes/genetics/enzymology ; DNA/genetics/chemistry/metabolism ; DNA Cleavage ; },
abstract = {Cas9 from Streptococcus pyogenes (SpCas9) revolutionized genome editing by enabling programmable DNA cleavage guided by an RNA. However, SpCas9 tolerates mismatches in the DNA-RNA duplex, which can lead to deleterious off-target editing. Here, we reveal that Cas9 from Francisella novicida (FnCas9) possesses a unique structural feature-the REC3 clamp-that underlies its intrinsic high-fidelity DNA targeting. Through kinetic and structural analyses, we show that the REC3 clamp forms critical contacts with the PAM-distal region of the R-loop, thereby imposing a novel checkpoint during enzyme activation. Notably, F. novicida encodes a noncanonical small CRISPR-associated RNA (scaRNA) that enables FnCas9 to repress an endogenous bacterial lipoprotein gene, subverting host immune detection. Structures of FnCas9 with scaRNA illustrate how partial R-loop complementarity hinders REC3 clamp docking and prevents cleavage in favor of transcriptional repression. The REC3 clamp is conserved across type II-B CRISPR-Cas9 systems, pointing to a potential path for engineering precise genome editors or developing novel antibacterial strategies. These findings reveal the molecular basis of heightened specificity and virulence enabled by FnCas9, with broad implications for biotechnology and therapeutic development.},
}
@article {pmid40613705,
year = {2025},
author = {Schwartz, CI and Abell, NS and Li, A and Aradhana, and Tycko, J and Truong, A and Montgomery, SB and Hess, GT},
title = {Toward optimizing diversifying base editors for high-throughput mutational scanning studies.},
journal = {Nucleic acids research},
volume = {53},
number = {12},
pages = {},
doi = {10.1093/nar/gkaf620},
pmid = {40613705},
issn = {1362-4962},
support = {R35GM150462/NH/NIH HHS/United States ; NIH-1F99DK126120-01/NH/NIH HHS/United States ; NIH-4K00DK126120-03/NH/NIH HHS/United States ; S10RR025518-01/NH/NIH HHS/United States ; },
mesh = {*Gene Editing/methods ; Humans ; CRISPR-Cas Systems ; Cytidine Deaminase/genetics/metabolism ; HEK293 Cells ; CRISPR-Associated Protein 9/genetics/metabolism ; Point Mutation ; RNA, Guide, CRISPR-Cas Systems/genetics ; Mutation ; DNA Mutational Analysis/methods ; AICDA (Activation-Induced Cytidine Deaminase) ; },
abstract = {Base editors, including diversifying base editors that create C>N mutations, are potent tools for systematically installing point mutations in mammalian genomes and studying their effect on cellular function. Numerous base editor options are available for such studies, but little information exists on how the composition of the editor (deaminase, recruitment method, and fusion architecture) affects editing. To address this knowledge gap, the effect of various design features, such as deaminase recruitment and delivery method (electroporation or lentiviral transduction), on editing was assessed across ∼200 synthetic target sites. The direct fusion of a hyperactive variant of activation-induced cytidine deaminase to the N-terminus of dCas9 (DivA-BE) produced the highest editing efficiency, ∼4-fold better than the previous CRISPR-X method. Additionally, DivA-BE mutagenized the DNA strand that anneals to the targeting sgRNA (target strand) to create complementary C>N mutations, which were absent when the deaminase was fused to the C-terminus of dCas9. Based on these studies that comprehensively analyze the editing patterns of several popular base editors, DivA-BE editors efficiently diversified their target sites, albeit with increased indel frequencies. Overall, the improved editing efficiency makes the DivA-BE editors ideal for discovering functional variants in mutational scanning assays.},
}
@article {pmid40613203,
year = {2025},
author = {Criollo Delgado, L and Zamalutdinov, A and Potokina, E},
title = {Identification of Soybean E1-E4 Gene Orthologs in the Guar Genome Using Comprehensive Transcriptome Assembly and Annotation.},
journal = {Frontiers in bioscience (Scholar edition)},
volume = {17},
number = {2},
pages = {26548},
doi = {10.31083/FBS26548},
pmid = {40613203},
issn = {1945-0524},
support = {24-26-00073//Russian Science Foundation/ ; },
mesh = {*Glycine max/genetics ; *Cyamopsis/genetics ; *Transcriptome/genetics ; *Genome, Plant ; Molecular Sequence Annotation ; Photoperiod ; Gene Expression Regulation, Plant ; Plant Proteins/genetics ; *Genes, Plant ; Gene Expression Profiling ; Galactans ; Mannans ; Plant Gums ; },
abstract = {BACKGROUND: We publish the first available transcriptome assembly of guar (Cyamopsis tetragonoloba (L.) Taub.), a well-known source of guar gum (food additive E 412). At high latitudes, e.g., in Russia, the main challenge for guar cultivation is the long photoperiod during summer, which delays flowering and maturation of guar plants. Meanwhile, identifying of genes affecting the photoperiod sensitivity of guar would have a major impact on the development of marker-assisted breeding of this valuable food crop.<br><br>METHODS: RNA isolated from leaves of early and late flowering guar plants grown under long-day conditions were used to generate de novo transcriptome assembly. A similarity search was conducted using BLASTN 2.2.31+ with default settings to identify homologous sequences of soybean maturity genes E1-E4 in guar transcriptome and genome assembly. Gene prediction tools such as AUGUSTUS and FGENESH+ were used to predict the exon-intron structure of the candidate genes. Functional annotation of the amino acid sequence was performed using InterProScan v. 5.68-100.<br><br>RESULTS: The transcriptome assembly contained sequences of 96,447 clustered transcript isoforms in the leaves of guar plants grown under long-day conditions. The transcriptome assembly was annotated using BLAST against the Glycine max genome, and 42,615 guar transcripts (44.2%) were found to be similar to soybean genes. We used the developed transcriptome assembly to discover orthologs of the E1-E4 soybean loci in the guar genome that have the greatest impact on the flowering and maturation of this closely related, short-day legume crop. A high level of identity was detected between peptide sequences encoding by orthologous genes E1 and CtE1 (80%), E2 and CtE2 (93%), E3 and CtE3 (83%), and E4 and CtE4 (91%). The sequences and the intron-exon structure of the genes in soybean and guar were similar, suggesting that the genetic pathways underlying basic flowering mechanisms are conserved between these two legume crops.<br><br>CONCLUSIONS: The revealed intron-exon structure of the guar genes CtE1-CtE4 creates possibilities for their targeted mutagenesis, e.g., using CRISPR-Cas and developing new guar germplasm with low sensitivity to photoperiod.},
}
@article {pmid40611477,
year = {2025},
author = {Bulle, M and Abbagani, S and Raza, A},
title = {Genome blaze: engineering chilli pepper chloroplasts for sustainable production of capsaicinoids through organellar genome editing.},
journal = {Plant biology (Stuttgart, Germany)},
volume = {},
number = {},
pages = {},
doi = {10.1111/plb.70067},
pmid = {40611477},
issn = {1438-8677},
support = {PDF/2018/001226//Science and Engineering Research Board/ ; },
abstract = {The quest for superior parental lines of chilli pepper, enriched with heightened levels of phytochemicals, such as capsinoids (CATs) and capsaicinoids (CAPs), has recently gained momentum. Plant scientists now pay more attention to generating pepper cultivars that offer both increased yield and richness in these crucial ingredients. Indeed, the average pepper yield per hectare, as well as richness of these ingredients, are affected by multiple environmental stimuli. However, expression of specific genes is crucial to stimulate the CAT and CAP levels in response to environmental conditions. Recently, new technologies, like genome editing using CRISPR/Cas and engineering of chloroplasts, have been tested in chilli pepper. The advances in genome editing tools, such as the prime editor and base editor methods, have tremendous potential for plant organelle genome engineering. In this article, we highlight recent advancements in CRISPR/Cas and plastid engineering within Capsicum, coupled with application of base editing approaches for editing plant organelle DNA. We also assess the challenges and opportunities in the pursuit of commercial and sustainable production of bioactive compounds specific to Capsicum species.},
}
@article {pmid40611420,
year = {2025},
author = {Rezaei, M and Jalali, A and Sadah Al-Azzawi, DH},
title = {Engineered Bacteriophages: Advances in Phage Genome Redesign Strategies for Therapeutic and Environmental Applications.},
journal = {Protein and peptide letters},
volume = {},
number = {},
pages = {},
doi = {10.2174/0109298665372719250616085616},
pmid = {40611420},
issn = {1875-5305},
abstract = {Bacteriophages, or phages, have emerged as powerful platforms in synthetic biology, offering innovative solutions for therapeutic and environmental challenges through advanced genome redesign strategies. This review explores a wide range of phage engineering techniques, including CRISPR (clustered regularly-interspaced short palindromic repeats)-Cas systems, phage display, random and site-directed mutagenesis, retrons, and rebooting approaches, highlighting their potential to create phages with tailored functionalities. CRISPR-Cas systems enable precise genome editing, allowing the development of phages with expanded host ranges, biofilm degradation capabilities, and targeted antimicrobial activity. Phage display facilitates the presentation of peptides on phage surfaces, enabling applications in targeted drug delivery, tumor imaging, and bioremediation. Beyond these, techniques like retron-mediated recombination and homologous recombination offer additional avenues for precise phage genome modification. In the therapeutic realm, engineered phages show promise in combating drug-resistant infections, modulating the microbiome, and delivering targeted therapies for cancer and other diseases. Environmentally, phage-based strategies, such as the use of phage-displayed metal-binding peptides, provide innovative solutions for bioremediation and reducing exposure to toxic heavy metals. This review also addresses challenges, such as phage resistance, immune responses, and the limitations of current engineering methods, while exploring future directions, including the development of improved CRISPR systems, phage-based biosensors, and high-throughput screening platforms. By integrating cutting-edge genome redesign strategies with diverse applications, this review underscores the transformative potential of engineered bacteriophages in addressing global healthcare and environmental sustainability challenges.},
}
@article {pmid40611351,
year = {2025},
author = {Smidler, AL and Akbari, OS},
title = {CRISPR technologies for the control and study of malaria-transmitting anopheline mosquitoes.},
journal = {Parasites &amp; vectors},
volume = {18},
number = {1},
pages = {252},
pmid = {40611351},
issn = {1756-3305},
support = {R01AI151004/GF/NIH HHS/United States ; R01AI151004/GF/NIH HHS/United States ; },
mesh = {*Anopheles/genetics/parasitology/physiology ; Animals ; *Mosquito Control/methods ; *Mosquito Vectors/genetics/parasitology/physiology ; *CRISPR-Cas Systems ; *Malaria/transmission/prevention &amp; control ; Gene Editing ; Clustered Regularly Interspaced Short Palindromic Repeats ; Humans ; Female ; Animals, Genetically Modified ; },
abstract = {Malaria is one of the deadliest diseases on the planet, killing approximately 600,000 people annually, and is transmitted by the bite of an anopheline mosquito. Anophelines, and the diseases they transmit, have changed the course of history and the fate of nations, and their successful control promises to end the transmission of malaria. With the advent of clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) technologies, the study and control of these deadly pests have been revolutionized. As the release of genetically modified anophelines is being considered, here we outline the advances in CRISPR/Cas9 technologies and how they have revolutionized the study of anopheline basic biology and the development of innovative vector control strategies. We outline the major findings of CRISPR-based basic biological research into traits relevant for vector control including, but not limited to, olfaction, chemosensation, neurobiology, and reproduction. Further, we summarize the advancements in CRISPR-based innovative vector control strategies, such as the precision-guided sterile insect technique (pgSIT), inherited female elimination by genetically encoded nucleases to interrupt alleles (IFEGENIA), X-shredder, Y-linked editors, and gene drives. All in all, this review summarizes the basic biological and vector control research undertaken using CRISPR since its advent approximately a decade ago.},
}
@article {pmid40516528,
year = {2025},
author = {Matthews, RE and Danac, JMC and Naden, EL and Farleigh Smith, LE and Lestari, S and Gungi, A and Appert, A and Buttress, T and Verma, A and Sinclair, O and Chong, F and Suberu, J and Antrobus, R and Bonev, B and Dawson, MA and Reid, AJ and Timms, RT and Ahringer, J and Tchasovnikarova, IA},
title = {CRAMP1 drives linker histone expression to enable Polycomb repression.},
journal = {Molecular cell},
volume = {85},
number = {13},
pages = {2503-2516.e8},
doi = {10.1016/j.molcel.2025.05.031},
pmid = {40516528},
issn = {1097-4164},
mesh = {*Histones/genetics/metabolism ; *Polycomb Repressive Complex 2/genetics/metabolism ; Animals ; Humans ; Mice ; Promoter Regions, Genetic ; Heterochromatin/genetics/metabolism ; CRISPR-Cas Systems ; *Epigenetic Repression ; },
abstract = {In contrast to the well-understood role of core histones in DNA packaging, the function of the linker histone (H1) remains enigmatic. Challenging the prevailing view that linker histones are a general feature of heterochromatin, here we show a critical requirement for H1 in Polycomb repressive complex 2 (PRC2) function. A CRISPR-Cas9 genetic screen using a fluorescent PRC2 reporter identified an essential role for the poorly characterized gene CRAMP1 in PRC2-mediated repression. CRAMP1 localizes to the promoters of expressed H1 genes and positively regulates their transcription. CRAMP1 ablation simultaneously depletes all linker histones, which results in selective decompaction of H3K27me3-marked loci and derepression of PRC2 target genes without concomitant loss of PRC2 occupancy or enzymatic activity. Strikingly, we find that linker histones preferentially localize to genomic loci marked by H3K27me3 across diverse cell types and organisms. Altogether, these data demonstrate a prominent role for linker histones in epigenetic repression by PRC2.},
}
@article {pmid40359943,
year = {2025},
author = {Wang, Y and Wu, J and Yang, S and Li, X and Wang, J and Lv, Q and Zhu, X and Lu, G and Zhang, J and Shen, WH and Liu, B and Lin, J and Dong, A},
title = {Structural and functional interrelationships of histone H2A with its variants H2A.Z and H2A.W in Arabidopsis.},
journal = {Structure (London, England : 1993)},
volume = {33},
number = {7},
pages = {1240-1249.e5},
doi = {10.1016/j.str.2025.04.015},
pmid = {40359943},
issn = {1878-4186},
mesh = {*Histones/chemistry/genetics/metabolism ; *Arabidopsis/genetics/metabolism ; *Arabidopsis Proteins/chemistry/metabolism/genetics ; Nucleosomes/metabolism/chemistry ; Cryoelectron Microscopy ; CRISPR-Cas Systems ; Mutation ; Protein Isoforms/chemistry/metabolism/genetics ; },
abstract = {Multiple histone H2A variants are known in eukaryotes. However, the functional relationship between H2A and its variants in plants remains largely obscure. Using CRISPR-Cas9 editing, we generated a mutant lacking four H2A isoforms in Arabidopsis and analyzed the functional and structural relationships between H2A, H2A.Z, and H2A.W. RNA sequencing and phenotype analyses revealed mild changes in gene transcription and plant development in mutants lacking H2A, H2A.Z, or H2A.W compared with the wild-type plants. Chromatin immunoprecipitation sequencing analysis showed that H2A can substitute for both H2A.Z and H2A.W across the genome, including in euchromatin and heterochromatin regions. However, H2A.Z replaced both H2A and H2A.W primarily within the euchromatin regions. By using DNA and histones from Arabidopsis, we constructed nucleosomes containing H2A, H2A.Z, or H2A.W and resolved their cryogenic electron microscopy (cryo-EM) structures at near-atomic resolution. Collectively, the results reveal the structural similarity and functional redundancy of H2A and its variants in Arabidopsis.},
}
@article {pmid38944889,
year = {2025},
author = {Kang, ES and Kim, NH and Lim, HK and Jeon, H and Han, K and No, YH and Kim, K and Khaleel, ZH and Shin, D and Eom, K and Nam, J and Lee, BS and Kim, HJ and Suh, M and Lee, J and Thach, TT and Hyun, J and Kim, YH},
title = {Structure-Guided Engineering of Thermodynamically Enhanced SaCas9 for Improved Gene Suppression.},
journal = {Advanced materials (Deerfield Beach, Fla.)},
volume = {37},
number = {26},
pages = {e2404680},
doi = {10.1002/adma.202404680},
pmid = {38944889},
issn = {1521-4095},
support = {//National Research Foundation of Korea/ ; NRF-2023R1A2C3005731//Korean government/ ; RS-2023-00302458)//Korean government/ ; IBS-R015-D1//Institute for Basic Science/ ; },
mesh = {Animals ; Mice ; *Protein Engineering/methods ; Thermodynamics ; Humans ; Molecular Dynamics Simulation ; *CRISPR-Associated Protein 9/chemistry/genetics/metabolism ; CRISPR-Cas Systems ; HEK293 Cells ; DNA/metabolism ; },
abstract = {Proteins with multiple domains play pivotal roles in various biological processes, necessitating a thorough understanding of their structural stability and functional interplay. Here, a structure-guided protein engineering approach is proposed to develop thermostable Cas9 (CRISPR-associated protein 9) variant for CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) interference applications. By employing thermodynamic analysis, combining distance mapping and molecular dynamics simulations, deletable domains are identified to enhance stability while preserving the DNA recognition function of Cas9. The resulting engineered Cas9, termed small and dead form Cas9, exhibits improved thermostability and maintains target DNA recognition function. Cryo-electron microscopy analysis reveals structural integrity with reduced atomic density in the deleted domain. Fusion with functional elements enables intracellular delivery and nuclear localization, demonstrating efficient gene suppression in diverse cell types. Direct delivery in the mouse brain shows enhanced knockdown efficiency, highlighting the potential of structure-guided engineering to develop functional CRISPR systems tailored for specific applications. This study underscores the significance of integrating computational and experimental approaches for protein engineering, offering insights into designing tailored molecular tools for precise biological interventions.},
}
@article {pmid40612691,
year = {2024},
author = {Lin, J and Yang, J},
title = {CRISPR-Cas systems: A revolution in genome editing and its diverse applications.},
journal = {Journal of biomed research},
volume = {5},
number = {1},
pages = {108-114},
pmid = {40612691},
issn = {2693-5910},
abstract = {The clustered regularly interspaced short palindromic repeats (CRISPR) Cas (CRISPR6 associated protein) system is an advanced adaptive immune system found in prokaryotes. First discovered in1987, CRISPR Cas has revolutionized genetic research in the past two decades. CRISPR-Cas9 the most widespread system enables precise gene editing by creating double strand breaks. Its ease of use and cost-effectiveness has lowered the barrier to entry for genetic research. CRISPR holds immense potential in many fields from agriculture to medicine. In agriculture, CRISPR has accelerated crop improvement by enabling precise gene edits for desirable traits. In medicine, CRISPR holds promise in xenotransplant, cancers and infectious diseases (HIV) treatment. This review traces the historical development of CRISPR-Cas systems, explores their unique applications, and discusses future advancements aimed at enhancing CRISPR's precision and expanding its applications through technologies like prime and base editing.},
}
@article {pmid40610423,
year = {2025},
author = {Fuhrmeister, ER and Kim, S and Mairal, SA and McCormack, C and Chieng, B and Swarthout, JM and Paulos, AH and Njenga, SM and Pickering, AJ},
title = {Context-Seq: CRISPR-Cas9 targeted nanopore sequencing for transmission dynamics of antimicrobial resistance.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {5898},
pmid = {40610423},
issn = {2041-1723},
support = {OPP1129535//Bill and Melinda Gates Foundation (Bill &amp; Melinda Gates Foundation)/ ; UL1TR002544//U.S. Department of Health &amp; Human Services | National Institutes of Health (NIH)/ ; 1906957//National Science Foundation (NSF)/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; Animals ; Humans ; *Nanopore Sequencing/methods ; *Drug Resistance, Bacterial/genetics ; Kenya ; Anti-Bacterial Agents/pharmacology ; Dogs ; *Bacteria/genetics/drug effects ; Klebsiella pneumoniae/genetics/drug effects ; Child ; Adult ; Escherichia coli/genetics/drug effects ; Poultry/microbiology ; beta-Lactamases/genetics ; Genome, Bacterial ; Haemophilus influenzae/genetics/drug effects ; },
abstract = {Precisely understanding how and to what extent antimicrobial resistance (AMR) is exchanged between animals and humans is needed to inform control strategies. Metagenomic sequencing has low detection for rare targets such as antibiotic resistance genes, while whole genome sequencing of isolates misses exchange between uncultured bacterial species. We introduce Context-Seq, CRISPR-Cas9 targeted sequencing of ARGs and their genomic context with long-reads. Using Context-Seq, we investigate genetically similar AMR elements containing the ARGs blaCTX-M and blaTEM between adults, children, poultry, and dogs in Nairobi, Kenya. We identify genetically distinct clusters containing blaTEM and blaCTX-M that are shared between animals and humans within and between households. We also uncover potentially pathogenic hosts of ARGs including Escherichia coli, Klebsiella pneumoniae, and Haemophilus influenzae in this study context. Context-Seq complements conventional methods to obtain an additional view of bacterial and mammalian hosts in the proliferation of AMR.},
}
@article {pmid40610421,
year = {2025},
author = {Sek, K and Chen, AXY and Cole, T and Armitage, JD and Tong, J and Yap, KM and Munoz, I and Dunbar, PA and Wu, S and van Elsas, MJ and Hidajat, O and Scheffler, C and Giuffrida, L and Henderson, MA and Meyran, D and Souza-Fonseca-Guimaraes, F and Nguyen, D and Huang, YK and de Menezes, MN and Derrick, EB and Chan, CW and Todd, KL and Chan, JD and Li, J and Lai, J and Petley, EV and Mardiana, S and Bosco, A and Waithman, J and Parish, IA and Mølck, C and Stewart, GD and Kats, L and House, IG and Darcy, PK and Beavis, PA},
title = {Tumor site-directed A1R expression enhances CAR T cell function and improves efficacy against solid tumors.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {6123},
pmid = {40610421},
issn = {2041-1723},
support = {836379//Cancer Council Victoria/ ; },
mesh = {Animals ; Humans ; Mice ; *Receptors, Chimeric Antigen/genetics/metabolism/immunology ; *Neoplasms/therapy/immunology/genetics ; *Immunotherapy, Adoptive/methods ; *T-Lymphocytes/immunology/metabolism ; CRISPR-Cas Systems ; Tumor Microenvironment/immunology ; Cell Line, Tumor ; Female ; Receptor, Adenosine A2A/genetics/metabolism ; Mice, Inbred C57BL ; },
abstract = {The efficacy of Chimeric Antigen Receptor T cells against solid tumors is limited by immunosuppressive factors in the tumor microenvironment including adenosine, which suppresses Chimeric Antigen Receptor T cells through activation of the A2A receptor. To overcome this, Chimeric Antigen Receptor T cells are engineered to express A1 receptor, a receptor that signals inversely to A2A receptor. Using murine and human Chimeric Antigen Receptor T cells, constitutive A1 receptor overexpression significantly enhances Chimeric Antigen Receptor T cell effector function albeit at the expense of Chimeric Antigen Receptor T cell persistence. Through a CRISPR/Cas9 homology directed repair "knock-in" approach we demonstrate that Chimeric Antigen Receptor T cells engineered to express A1 receptor in a tumor-localized manner, enhances anti-tumor therapeutic efficacy. This is dependent on the transcription factor IRF8 and is transcriptionally unique when compared to A2A receptor deletion. This data provides a novel approach for enhancing Chimeric Antigen Receptor T cell efficacy in solid tumors and provides proof of principle for site-directed expression of factors that promote effector T cell differentiation.},
}
@article {pmid40610153,
year = {2025},
author = {Ren, W and Li, M and Liu, X and You, W and You, Q and Li, B and Ye, H and Zhang, R},
title = {Specific detection of DNA and RNA by the CRISPR-Cas12a system containing spacer split crRNA.},
journal = {Analytica chimica acta},
volume = {1367},
number = {},
pages = {344204},
doi = {10.1016/j.aca.2025.344204},
pmid = {40610153},
issn = {1873-4324},
mesh = {*CRISPR-Cas Systems/genetics ; Humans ; *DNA/analysis/genetics ; *RNA/analysis/genetics ; MicroRNAs/analysis/genetics ; *CRISPR-Associated Proteins/genetics ; Bacterial Proteins ; Endodeoxyribonucleases ; },
abstract = {BACKGROUND: The CRISPR/Cas12a system has emerged as a versatile molecular diagnostic tool due to its dual cis- and trans-cleavage activities. However, two key limitations hinder its broad application: high tolerance to single-base mismatches in DNA targets and strict reliance on DNA activators. To address these challenges, we hypothesized that structural reengineering of crRNA could enhance specificity and functional versatility. This study aimed to develop a modified Cas12a system capable of detecting DNA and RNA targets with improved single-base resolution, thereby expanding its utility in molecular diagnostics and clinical subclassification.<br><br>RESULTS: We engineered split crRNAs by introducing a split site within the spacer region, creating a spacer-split crRNA-activated Cas12a system (SPCas12a). This system exhibited three key advantages: First, SPCas12a demonstrated significantly enhanced specificity in discriminating single-base mutations compared to conventional full-sized crRNA systems. Second, it bypassed the DNA activator requirement, enabling direct detection of miRNA targets without reverse transcription. In addition, AlphaFold Server predictive structural modeling analysis showed that the split site selected by SPCas12a gives the Cas12a complex an open structural domain, which is conducive to the stable function of Cas12a. Third, integration with isothermal amplification enabled constructing an "AND" logic gate detection platform that processes multiple inputs within 40&#xa0;min. As a proof-of-concept, SPCas12a successfully distinguished triple-negative breast cancer (TNBC) subtype cell lines by analyzing miRNA-210 and miRNA-21 biomarkers in different cell lines.<br><br>SIGNIFICANCE: SPCas12a overcomes fundamental limitations of current CRISPR diagnostics by unifying high-specificity DNA mutation detection and direct RNA sensing in a single platform. The split-crRNA design principle provides a universally adaptable strategy to enhance CRISPR-Cas systems, with immediate applications in precision oncology and infectious disease stratification where base-level discrimination and multi-target detection are critical.},
}
@article {pmid40609997,
year = {2025},
author = {Kalinina, NO and Spechenkova, NA and Taliansky, ME},
title = {Biotechnological Approaches to Plant Antiviral Resistance: CRISPR-Cas or RNA Interference?.},
journal = {Biochemistry. Biokhimiia},
volume = {90},
number = {6},
pages = {804-817},
doi = {10.1134/S0006297925600139},
pmid = {40609997},
issn = {1608-3040},
mesh = {*RNA Interference ; *CRISPR-Cas Systems ; *Plant Diseases/virology/genetics/prevention &amp; control ; Gene Editing/methods ; *Plants/virology/genetics ; *Disease Resistance/genetics ; *Biotechnology/methods ; Plant Viruses/genetics ; },
abstract = {Established genome editing technologies, such as CRISPR-Cas and RNA interference (RNAi), have significantly advanced research studies in nearly all fields of life sciences, including biotechnology and medicine, and have become increasingly in demand in plant biology. In the review, we present the main principles of the CRISPR-Cas and RNAi technologies and their application in model plants and crops for the control of viral diseases. The review explores the antiviral effects they provide, including direct suppression of genomes of DNA- and RNA-containing viruses and inhibition of activity of host genes that increase plant susceptibility to viruses. We also provide a detailed comparison of the effectiveness of CRISPR-Cas and RNAi methods in plant protection, as well as discuss their advantages and disadvantages, factors limiting their application in practice, and possible approaches to overcome such limitations.},
}
@article {pmid40609996,
year = {2025},
author = {Kazakova, AA and Leonova, EI and Sopova, JV and Chirinskaite, AV and Minskaya, ES and Kukushkin, IS and Ivanov, RA and Reshetnikov, VV},
title = {Progress in CRISPR/CAS13-Mediated Suppression of Influenza A and SARS-CoV-2 Virus Infection in in vitro and in vivo Models.},
journal = {Biochemistry. Biokhimiia},
volume = {90},
number = {6},
pages = {786-803},
doi = {10.1134/S0006297925601212},
pmid = {40609996},
issn = {1608-3040},
mesh = {*CRISPR-Cas Systems ; Animals ; *SARS-CoV-2/genetics ; Humans ; *COVID-19/therapy/virology ; *Influenza A virus/genetics ; *Influenza, Human/therapy/virology/genetics ; Disease Models, Animal ; RNA, Viral/genetics/metabolism ; Antiviral Agents ; },
abstract = {The worldwide number of deaths from complications caused by severe influenza and COVID-19 is about 1 million cases annually. Development of the effective antiviral therapy strategies for the disease treatment is one of the most important tasks. Use of the CRISPR/Cas13 system, which specifically degrades viral RNA and significantly reduces titer of the virus, could be a solution of this problem. Despite the fact that Cas13 nucleases have been discovered only recently, they already have shown high efficiency in suppressing viral transcripts in cell cultures. The recent advances in mRNA technology and improvements in non-viral delivery systems have made it possible to effectively use CRISPR/Cas13 in animal models as well. In this review, we analyzed experimental in vitro and in vivo studies on the use of CRISPR/Cas13 systems as an antiviral agent in cell cultures and animal models and discussed main directions for improving the CRISPR/Cas13 system. These data allow us to understand prospects and limitations of the further use of CRISPR/Cas13 in the treatment of viral diseases.},
}
@article {pmid40609995,
year = {2025},
author = {Volodina, OV and Demchenko, AG and Anuchina, AA and Ryzhkova, OP and Kovalskaya, VA and Kondrateva, EV and Tabakov, VY and Lavrov, AV and Smirnikhina, SA},
title = {Selection of Optimal pegRNAs to Enhance Efficiency of Prime Editing in AT-Rich Genome Regions.},
journal = {Biochemistry. Biokhimiia},
volume = {90},
number = {6},
pages = {773-785},
doi = {10.1134/S0006297924604672},
pmid = {40609995},
issn = {1608-3040},
mesh = {*Gene Editing/methods ; Humans ; *Cystic Fibrosis Transmembrane Conductance Regulator/genetics ; *Cystic Fibrosis/genetics/therapy ; CRISPR-Cas Systems ; },
abstract = {Prime editing is a highly promising strategy for treating hereditary disorders due to its superior efficiency and safety profile compared to the conventional CRISPR-Cas9 systems. This study is dedicated to development of a causal therapy for cystic fibrosis by targeting the F508del variant of the CFTR gene using prime editing, as this specific deletion accounts for a substantial proportion of cystic fibrosis cases. While prime editing has shown remarkable precision in introducing targeted genetic modifications, its application in AT-rich genomic regions, such as the one containing the F508del variant, remains challenging. To overcome this limitation, we systematically evaluated 24 pegRNAs designed for two distinct prime editing systems, PEmax and PE2-NG. Efficiency of the F508del variant correction reached 2.81% (without normalization for transfection efficiency) in the airway basal cells from the patients with homozygous F508del mutation. However, the average transfection efficiency was only 11.9%, emphasizing critical need for the advancements in delivery methodologies. These findings highlight potential of prime editing as an approach for treating cystic fibrosis, while also underscoring necessity for further optimization of both editing constructs and delivery vectors to achieve clinically relevant correction levels.},
}
@article {pmid40609268,
year = {2025},
author = {Allan, AC and Scott, B and Tate, W and Jameson, PE and MacRae, E and Conner, AJ and Drummond, R and Foster, A and Martin, C and Caradus, J},
title = {Human-mediated outdoor genome editing is not possible so therefore poses no risk to the environment.},
journal = {Ecotoxicology and environmental safety},
volume = {302},
number = {},
pages = {118609},
doi = {10.1016/j.ecoenv.2025.118609},
pmid = {40609268},
issn = {1090-2414},
abstract = {There is a world-wide re-examination of the regulations that surround genetic technologies, including gene edited organisms. In many countries, crop plants with small gene edits and where no foreign DNA is introduced are exempt from detailed regulatory assessment. This will allow these types of plants to be released, after assessment for benefit and risk, by plant breeders or plant scientists. The full regulatory risk assessment and risk management of novel (including transgenic) plants is well established and focusses on five key criteria (weediness, gene flow, plant pests, non-target impact, biodiversity). However, plants produced by "traditional" plant breeding technologies, many of which have been subject to random mutagenesis or wide crosses that may introduce enormous numbers of DNA changes, are almost never considered novel so are not assessed for risk through a regulatory system. The most targeted, versatile and widely used gene editing technique involves the enzyme-RNA complex, CRISPR-Cas. This method can produce far more precise and targeted changes than "traditional" mutagenesis techniques. Getting the CRISPR-Cas machinery into plant or animal cells requires highly sterile tissue culture and sophisticated delivery tools. Therefore, gene editing, in the open environment by "field spraying", is not currently possible. While other uses of nucleotide chemistry - such as double stranded RNA - have been applied to plants to knock down gene expression, this is not gene editing and produces no DNA change. Suggestions that gene editing using CRISPR-Cas can occur through spraying directly on to plants in the outside environment is fanciful, incorrect and misleading.},
}
@article {pmid40608406,
year = {2025},
author = {Ito, K and Ito, Y},
title = {Comparative genomic analysis of Latilactobacillus sakei strains provides new insights into their association with different niche adaptations.},
journal = {Microbiology (Reading, England)},
volume = {171},
number = {7},
pages = {},
doi = {10.1099/mic.0.001578},
pmid = {40608406},
issn = {1465-2080},
mesh = {*Genome, Bacterial ; Phylogeny ; *Adaptation, Physiological/genetics ; Plasmids/genetics ; *Latilactobacillus sakei/genetics/classification/physiology/growth &amp; development/metabolism ; Genomics ; Carbohydrate Metabolism/genetics ; Fermentation ; Genetic Variation ; },
abstract = {Latilactobacillus sakei, a lactic acid bacterium in diverse environments such as fermented foods, meat and the human gastrointestinal tract, exhibits significant genetic diversity and niche-specific adaptations. This study conducts a comprehensive comparative genomic analysis of 29 complete L. sakei genomes to uncover the genetic mechanisms underlying these adaptations. Phylogenetic analysis divided the species into three distinct clades that did not correlate with the source of isolation and did not suggest any niche-specific evolutionary direction. The pan-genome analysis revealed a substantial core genome alongside a diverse genetic repertoire, indicating both high genetic conservation and adaptability. Predicted growth rates based on codon use bias analysis suggest that L. sakei strains have an overall faster growth rate and may be able to efficiently dominate in competitive environments. Plasmid analysis revealed a variety of plasmids carrying genes essential for carbohydrate metabolism, enhancing L. sakei's ability to thrive in various fermentation substrates. It was also found that the number of genes belonging to the GH1 family amongst sugar metabolism-related genes present on chromosomes and plasmids varies between strains and that AA1, which is involved in alcohol oxidation, has been acquired from plasmids. blast analysis revealed that some strains have environmental adaptation gene clusters of cell surface polysaccharides that may mediate attachment to food and mucosa. The knowledge gleaned from this study lays a solid foundation for future research aimed at harnessing the genetic traits of L. sakei strains for industrial and health-related applications.},
}
@article {pmid40608358,
year = {2025},
author = {Li, YG and Haeusser, D and Margolin, W and Christie, PJ},
title = {Conjugative delivery of toxin genes ccdB and kil confers synergistic killing of bacterial recipients.},
journal = {Journal of bacteriology},
volume = {},
number = {},
pages = {e0016825},
doi = {10.1128/jb.00168-25},
pmid = {40608358},
issn = {1098-5530},
abstract = {The bacterial type IV secretion systems (T4SS) are medically problematic for their roles in the dissemination of mobile genetic elements or effector proteins, but they also have great potential for new antimicrobial therapies. Recent studies have deployed the T4SS subfamily of conjugation systems to deliver gene editing CRISPR/Cas systems to disrupt drug resistance genes or kill targeted bacterial recipients. However, the therapeutic potential of conjugative CRISPR/Cas delivery is compromised by mutations or host repair systems that diminish the efficiency with which CRISPR/Cas induces double-strand breaks in new transconjugants. Here, we compared the efficiencies of conjugation-based killing systems based on the delivery of CRISPR-Cas9 elements or toxin genes encoding the bacteriophage lambda Kil peptide or the F plasmid-encoded CcdB. Escherichia coli equipped with one of two efficient conjugation systems, pKM101 (IncN) or F (IncF), served as donors to mobilize plasmids carrying the cognate oriT sequence and one or more toxic elements. Overall, toxin gene delivery proved significantly more effective than CRISPR-Cas9 in killing of transconjugant population, but the highest levels of growth suppression of both E. coli and Klebsiella pneumoniae recipients were achieved by a combination of CRISPR-Cas9 plus one or two toxin genes. By contrast, capsule production conferred no or very slight protective effects on plasmid acquisition and killing of either species. We propose that the conjugative co-transfer of two or more toxic elements with distinct mechanisms of action has strong potential for growth suppression of targeted species in environmental or clinical settings.IMPORTANCEThe prevalence of antibiotic resistance emphasizes the need for alternative antimicrobial intervention strategies. We engineered Escherichia coli for conjugative transmission of plasmids encoding CRISPR-Cas9 elements or genes encoding the cell division inhibitor Kil or gyrase poisoner CcdB. Delivery of toxin genes more effectively suppressed the growth of E. coli recipients than CRISPR-Cas9, but the combinatorial delivery of CRISPR-Cas9 and a toxin gene or two toxin genes elicited the strongest killing effects. Capsule production by E. coli or Klebsiella pneumoniae recipient cells had no or little protective effect on plasmid acquisition or growth suppression. Our findings suggest that probiotic donor strains equipped for conjugative delivery of two or more toxic elements may prove effective as an alternative or adjunct to traditional antimicrobials.},
}
@article {pmid40607431,
year = {2025},
author = {Wang, JW and Liu, JH and Xun, JJ},
title = {CCR5 gene editing and HIV immunotherapy: current understandings, challenges, and future directions.},
journal = {Frontiers in immunology},
volume = {16},
number = {},
pages = {1590690},
pmid = {40607431},
issn = {1664-3224},
mesh = {*Receptors, CCR5/genetics/immunology ; Humans ; *Gene Editing/methods ; *HIV Infections/therapy/immunology/genetics ; *Immunotherapy/methods ; *HIV-1/immunology ; CRISPR-Cas Systems ; Genetic Therapy/methods ; },
abstract = {Human immunodeficiency virus (HIV) infection remains a major global public health challenge. Although highly active antiretroviral therapy (HAART or ART) can effectively control viral replication, it fails to eradicate latent viral reservoirs and poses limitations such as lifelong medication and cumulative drug toxicity. This study focuses on the pivotal role of C-C chemokine receptor 5 (CCR5) gene editing in HIV immunotherapy, particularly highlighting the natural resistance to R5-tropic HIV strains observed in the "Berlin" and "London" patients carrying the homozygous CCR5-Δ32 mutation. We further explore the synergistic potential of multiplex gene editing strategies-including CCR5, CXCR4, and HIV LTR loci-and the combinatorial mechanisms between gene editing technologies and immunotherapy. A personalized treatment framework is proposed to address the clinical heterogeneity among people living with HIV. In addition, we assess the balance between long-term safety and global accessibility of gene-editing approaches such as CRISPR/Cas9, emphasizing strategies to enhance therapeutic efficacy while reducing cost and off-target effects. Our findings suggest that the integration of CCR5-targeted gene editing with immune-based interventions holds great promise for overcoming current therapeutic limitations and achieving functional HIV cure. However, key challenges-such as immune rejection, viral tropism switching, and economic feasibility-must be resolved. This integrative approach provides a robust theoretical and technical foundation for the next generation of HIV treatment paradigms.},
}
@article {pmid40605043,
year = {2025},
author = {Salazar-García, LM and Damas-Ramos, LC and Trejo-Alarcón, LM and Rago, D and Ahonen, L and Cruz-Morales, P and Ponce-Noyola, P and Licona-Cassani, C},
title = {CRISPR-driven enhanced hydrocarbon emulsification in an environmental Pseudomonas aeruginosa strain.},
journal = {Microbial cell factories},
volume = {24},
number = {1},
pages = {151},
pmid = {40605043},
issn = {1475-2859},
support = {NNF20CC0035580//Novo Nordisk Fonden/ ; },
mesh = {*Pseudomonas aeruginosa/metabolism/genetics/isolation &amp; purification ; Glycolipids/metabolism/biosynthesis ; *CRISPR-Cas Systems ; *Hydrocarbons/metabolism/chemistry ; Bacterial Proteins/genetics/metabolism ; Pyocyanine/metabolism/biosynthesis ; Emulsions ; Gene Editing ; Sigma Factor/genetics ; Petroleum Pollution ; },
abstract = {BACKGROUND: Oil spills are a major concern due to the economic impact and severe effects on the ecosystem. To mitigate oil spills, hydrocarbon dispersion through emulsification is a promising approach, as it makes oil more susceptible to degradation by microorganisms. Environmental strains of Pseudomonas aeruginosa have demonstrated significant potential for producing rhamnolipids (RMLs) and pyocyanin (PYO), secondary metabolites associated to hydrocarbon emulsification. In this study, we isolated and characterized an environmental strain from an oil-contaminated site in the Gulf of Mexico. Upon genome sequencing and taxonomic classification, we developed genetic engineering tools and assessed their capacity to produce PYO and RMLs, molecules relevant for hydrocarbon emulsification.<br><br>RESULTS: Using the CRISPR/Cas9-APOBEC1-UGI system, we generated a targeted cytosine to thymine transition in the rpoS gene to generate a premature STOP codon. The resulting mutant exhibited increased production of PYO and RMLs, along with enhanced gasoline emulsification in cell-free supernatants, demonstrating successful modulation of a key regulatory gene. While the strain IGLPR01 retains certain virulence-associated features, this study contributes to the exploration of environmental isolates as future candidate chassis for biosurfactant production, emphasizing the need for further safety evaluation and rational attenuation strategies.<br><br>CONCLUSION: This study provides a successful example of implementing CRISPR-based editing in an environmental P. aeruginosa strain. Despite the technical challenges, a genetic editing system was established and validated through a proof of concept to increase production of relevant metabolites. Our work demonstrates the applicability of genetic engineering tools in non-model environmental isolates, facilitating further developments. Importantly, the presence of virulence-associated features highlights the need for in-depth evaluation of pathogenicity and containment strategies before considering any future biotechnological applications.},
}
@article {pmid40605002,
year = {2025},
author = {Chou, J and Esmaeili Anvar, N and Elghaish, R and Chen, J and Hart, T},
title = {Z-scores outperform similar methods for analyzing CRISPR paralog synthetic lethality screens.},
journal = {Genome biology},
volume = {26},
number = {1},
pages = {188},
pmid = {40605002},
issn = {1474-760X},
support = {R35GM130119/GM/NIGMS NIH HHS/United States ; R35GM130119/GM/NIGMS NIH HHS/United States ; R35GM130119/GM/NIGMS NIH HHS/United States ; P30CA16672/CA/NCI NIH HHS/United States ; P30CA16672/CA/NCI NIH HHS/United States ; R35CA274234/CA/NCI NIH HHS/United States ; P30CA16672/CA/NCI NIH HHS/United States ; },
mesh = {Humans ; *CRISPR-Cas Systems ; *Synthetic Lethal Mutations ; Cell Line, Tumor ; },
abstract = {Genetic screens offer a promising strategy for identifying tumor-specific therapeutic targets, but single-gene knockout screens often miss functionally redundant paralogs. Multiplex Cas9 and Cas12a CRISPR systems have been deployed to assay genetic interactions, but analysis pipelines vary considerably. Here we evaluate data from four in4mer CRISPR/Cas12a screens in cancer cell lines, using delta log fold change, Z-transformed dLFC, and rescaled dLFC approaches to identify synthetic lethal interactions. Both ZdLFC and RdLFC provide more consistent identification of synthetic lethal pairs across cell lines compared to the unscaled dLFC method, while ZdLFC benefits from not requiring a training set of known interactors.},
}
@article {pmid40577485,
year = {2025},
author = {Rissone, A and La Spina, M and Bresciani, E and Syed, ZA and Combs, CA and Kirby, M and Elkahloun, A and Chen, V and Sood, R and Burgess, SM and Puertollano, R},
title = {The transcription factors Tfeb and Tfe3 are required for survival and embryonic development of pancreas and liver in zebrafish.},
journal = {PLoS genetics},
volume = {21},
number = {6},
pages = {e1011754},
doi = {10.1371/journal.pgen.1011754},
pmid = {40577485},
issn = {1553-7404},
mesh = {Animals ; *Zebrafish/genetics/embryology/growth &amp; development ; *Basic Helix-Loop-Helix Leucine Zipper Transcription Factors/genetics/metabolism ; *Zebrafish Proteins/genetics/metabolism ; *Pancreas/embryology/metabolism/growth &amp; development ; *Liver/embryology/metabolism/growth &amp; development ; *Embryonic Development/genetics ; Autophagy/genetics ; Gene Expression Regulation, Developmental ; Apoptosis/genetics ; Oxidative Stress/genetics ; CRISPR-Cas Systems ; },
abstract = {The transcription factors TFEB and TFE3 modulate expression of lysosomal, autophagic, and metabolic genes to restore energy and cellular homeostasis in response to a variety of stress conditions. Since their role during vertebrate development is less characterized, we used CRISPR/Cas9 to deplete tfeb, tfe3a, and tfe3b in zebrafish. The simultaneous lack of these genes compromised embryo survival during early development, with an almost complete lethality of the larvae by 8-10 dpf. The knockout animals showed apoptosis in brain and retina and alterations in pancreas, liver, and gut. Exocrine pancreas presented the most severe defects, with accumulation of abnormal zymogen granules leading to acinar atrophy in embryos and pancreatitis-like phenotypes in adults; likely due to a block of the autophagy machinery implicated in removal of damaged granules. Knockout animals displayed increased susceptibility to oxidative and heat-shock stress. Our work reveals an essential role of Tfeb and Tfe3 in maintaining cellular and tissue homeostasis during development.},
}
@article {pmid40399675,
year = {2025},
author = {Han, M and Fu, ML and Zhu, Y and Choi, AA and Li, E and Bezney, J and Cai, S and Miles, L and Ma, Y and Qi, LS},
title = {Programmable control of spatial transcriptome in live cells and neurons.},
journal = {Nature},
volume = {643},
number = {8070},
pages = {241-251},
pmid = {40399675},
issn = {1476-4687},
mesh = {Animals ; *Neurons/metabolism/cytology ; *Transcriptome/genetics ; *CRISPR-Cas Systems/genetics ; RNA, Messenger/metabolism/genetics ; Mice ; Neurites/metabolism ; Humans ; RNA Transport/genetics ; Ribosomes/metabolism ; Neuronal Outgrowth/genetics ; Actins/genetics/metabolism ; Rats ; Protein Biosynthesis ; Cells, Cultured ; Microtubules/metabolism ; Female ; Axons/metabolism ; },
abstract = {Spatial RNA organization has a pivotal role in diverse cellular processes and diseases[1-4]. However, functional implications of the spatial transcriptome remain largely unexplored due to limited technologies for perturbing endogenous RNA within specific subcellular regions[1,5]. Here we present CRISPR-mediated transcriptome organization (CRISPR-TO), a system that harnesses RNA-guided, nuclease-dead dCas13 for programmable control of endogenous RNA localization in live cells. CRISPR-TO enables targeted localization of endogenous RNAs to diverse subcellular compartments, including the outer mitochondrial membrane, p-bodies, stress granules, telomeres and nuclear stress bodies, across various cell types. It allows for inducible and reversible bidirectional RNA transport along microtubules via motor proteins, facilitating real-time manipulation and monitoring of RNA localization dynamics in living cells. In primary cortical neurons, we demonstrate that repositioned mRNAs undergo local translation along neurites and at neurite tips, and co-transport with ribosomes, with β-actin mRNA localization enhancing the formation of dynamic filopodial protrusions and inhibiting axonal regeneration. CRISPR-TO-enabled screening in primary neurons identifies Stmn2 mRNA localization as a driver of neurite outgrowth. By enabling large-scale perturbation of the spatial transcriptome, CRISPR-TO bridges a critical gap left by sequencing and imaging technologies, offering a versatile platform for high-throughput functional interrogation of RNA localization in living cells and organisms.},
}
@article {pmid40344384,
year = {2025},
author = {Yao, Z and Li, W and He, K and Wang, H and Xu, Y and Wu, Q and Wang, L and Nie, Y},
title = {Facilitating crRNA Design by Integrating DNA Interaction Features of CRISPR-Cas12a System.},
journal = {Advanced science (Weinheim, Baden-Wurttemberg, Germany)},
volume = {12},
number = {25},
pages = {e2501269},
doi = {10.1002/advs.202501269},
pmid = {40344384},
issn = {2198-3844},
support = {32172175//National Natural Science Foundation of China/ ; 32172307//National Natural Science Foundation of China/ ; 2023C02006//Key R&amp;D Program of Zhejiang/ ; KYCX24_2592//Postgraduate Research &amp; Practice Innovation Program of Jiangsu Province/ ; NO.111-2-06//Priority Academic Program Development of Jiangsu Higher Education Institutions, the 111 Project/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; *DNA/genetics/metabolism ; Molecular Dynamics Simulation ; *CRISPR-Associated Proteins/genetics/metabolism ; RNA, Guide, CRISPR-Cas Systems/genetics ; Bacterial Proteins ; Endodeoxyribonucleases ; },
abstract = {The CRISPR-Cas12a system has gained significant attention as a rapid nucleic acid diagnostic tool due to its crRNA-guided trans-cleavage activity. Accurately predicting the activity of different targets is significant to facilitate the crRNA availability but remains challenging. In this study, a novel approach is presented that combines molecular dynamics simulations and neural network modeling to predict the trans-cleavage activity. Unlike conventional tools that rely solely on the base sequences, our method integrated sequence features and molecular interaction features of DNA in the CRISPR-Cas12a system, significantly improving prediction accuracy. Through feature importance analysis, key sequence features that influence Cas12a trans-cleavage activity are identified. Additionally, a crRNA-DNA library with over 23 456 feature sequences from representative viruses and bacteria is established, and validated the high predictive accuracy of the model (Pearson's r = 0.9328) by screening crRNAs from reference targets. This study offers new insights into the molecular interactions of Cas12a/crRNA-DNA and provides a reliable framework for optimizing crRNA design, facilitating the application of the CRISPR-Cas12a in rapid nucleic acid diagnostics.},
}
@article {pmid40302201,
year = {2025},
author = {Hao, JH and Kang, X and Zhang, L and Chen, J and Wang, D and Dong, S and Li, X and Gao, L and Yang, G and Yuan, X and Chu, X and Wang, JG},
title = {CRISPR/Cas9-Mediated SiEPF2 Mutagenesis Attenuates Drought Tolerance and Yield in Foxtail Millet (Setaria italica).},
journal = {Plant, cell &amp; environment},
volume = {48},
number = {8},
pages = {6043-6046},
doi = {10.1111/pce.15597},
pmid = {40302201},
issn = {1365-3040},
support = {//This study was supported by the National Key Research and Development Programme of China (2023YFD1202702), National Natural Science Foundation (32400217 and 32200222) and Start-up Fund of Shanxi Agricultural University (2021BQ84)./ ; },
mesh = {*Setaria Plant/genetics/physiology/growth &amp; development ; Droughts ; *Plant Proteins/genetics/metabolism/physiology ; *CRISPR-Cas Systems/genetics ; Mutagenesis ; Plant Stomata/physiology ; Gene Expression Regulation, Plant ; Drought Resistance ; },
abstract = {Plants employ peptide ligands to coordinate development and integrate environmental signals via dedicated cascades (#ref-0013). Epidermal patterning factor (EPF), plays a significant role in regulating stomatal density, seed germination and panicle development (#ref-0008). EPF/EPFL enhance drought tolerance by reducing stomatal density have been reported in multiple species, including Hordeum vulgare, Arabidopsis thaliana, Populus spp., Vitis vinifera, Sorghum bicolor and Brassica napus (#ref-0001). Our previous study has shown that EPF can mediate drought resistance in foxtail millet by regulating stomatal density (#ref-0004). Furthermore, evidence suggests that plants can regulate photosynthesis through stomatal modification, ultimately enhancing yield (#ref-0005). Beyond stomatal density regulation, the EPF/EPFL gene family modulates seed germination through phytohormone signalling and regulates inflorescence development via ligand-receptor interactions (#ref-0007). OsEPFLs act as upstream ligands for the OsER1 receptor, activating the MAPK signalling cascade to regulate panicle morphogenesis (#ref-0002). In this study, we sought to elucidate how SiEPF2 balances drought resistance and yield in foxtail millet by modulating stomatal density and panicle morphology. Our findings not only provide novel insights into SiEPF2's role in abiotic stress responses but also contribute valuable genetic resources for high-yield breeding programmes in millet crops.},
}
@article {pmid40280813,
year = {2025},
author = {Hu, N and Tian, H and Li, Y and Li, X and Li, D and Li, L and Wang, S and Zhang, Y and Shi, X and Huang, B and Lu, Q and Wang, T and Pan, X and Tu, L and Dai, D and Zhang, B and Peng, R and Yan, F},
title = {pHNRhCas9NG, single expression cassette-based dual-component dual-transcription unit CRISPR/Cas9 system for plant genome editing.},
journal = {Trends in biotechnology},
volume = {43},
number = {7},
pages = {1788-1808},
doi = {10.1016/j.tibtech.2025.03.016},
pmid = {40280813},
issn = {1879-3096},
mesh = {*CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; *Genome, Plant/genetics ; Nicotiana/genetics ; Plants, Genetically Modified/genetics ; Solanum lycopersicum/genetics ; Arabidopsis/genetics ; DNA, Bacterial/genetics ; Promoter Regions, Genetic ; },
abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 genome-editing (GEd) technology has revolutionized plant science, facilitating gene function studies and crop improvement. Despite its success, plant-specific CRISPR/Cas9 systems require further optimization. This study aims to boost plant GEd efficiency by revamping the CRISPR/Cas9 system. We addressed large fragment deletions in T-DNA (transfer DNA) postgenomic insertion by developing a binary expression vector, pHNR, which maintains T-DNA integrity using protective sequences. We discovered an artificial promoter, P35SIC47, effective in tobacco, Arabidopsis, and tomato transformation, and designed a dual-component dual-transcription unit CRISPR/Cas9 system (DDS) with optimal gene expression at a poly(A) length of ~150 base pairs. Enhancing the poly(A) tail length of Cas9 mRNA significantly boosted plant GEd efficiency. We also identified compatible hCas9 versions through transitory expression in tobacco leaves. Utilizing pHNRhCas9NG, we efficiently knocked out ten genes in tomato, achieving almost 100% gene-editing efficiency. Our system offers a novel, scalable tool for plant GEd, advancing CRISPR/Cas9 capabilities.},
}
@article {pmid40263591,
year = {2025},
author = {Rohde, T and Demirtas, TY and Süsser, S and Shaw, AH and Kaulich, M and Billmann, M},
title = {BaCoN (Balanced Correlation Network) improves prediction of gene buffering.},
journal = {Molecular systems biology},
volume = {21},
number = {7},
pages = {807-824},
pmid = {40263591},
issn = {1744-4292},
support = {397484323 - TRR259//Deutsche Forschungsgemeinschaft (DFG)/ ; },
mesh = {Humans ; CRISPR-Cas Systems ; *Gene Regulatory Networks ; Cell Line, Tumor ; Algorithms ; Transcriptome ; *Computational Biology/methods ; Neoplasms/genetics ; },
abstract = {Buffering between genes, where one gene can compensate for the loss of another gene, is fundamental for robust cellular functions. While experimentally testing all possible gene pairs is infeasible, gene buffering can be predicted genome-wide under the assumption that a gene's buffering capacity depends on its expression level and its absence primes a severe fitness phenotype of the buffered gene. We developed BaCoN (Balanced Correlation Network), a post hoc unsupervised correction method that amplifies specific signals in expression-vs-fitness correlation networks. We quantified 147 million potential buffering relationships by associating CRISPR-Cas9-screening fitness effects with transcriptomic data across 1019 Cancer Dependency Map (DepMap) cell lines. BaCoN outperformed state-of-the-art methods, including multiple linear regression based on our compiled gene buffering prediction metrics. Combining BaCoN with batch correction or Cholesky data whitening further boosts predictive performance. We characterized 808 high-confidence buffering predictions and found that in contrast to buffering gene pairs overall, buffering paralogs were on different chromosomes. BaCoN performance increases with more screens and genes considered, making it a valuable tool for gene buffering predictions from the growing DepMap.},
}
@article {pmid40205048,
year = {2025},
author = {Fell, CW and Villiger, L and Lim, J and Hiraizumi, M and Tagliaferri, D and Yarnall, MTN and Lee, A and Jiang, K and Kayabolen, A and Krajeski, RN and Schmitt-Ulms, C and Ramani, H and Yousef, SM and Roberts, N and Vakulskas, CA and Nishimasu, H and Abudayyeh, OO and Gootenberg, JS},
title = {Reprogramming site-specific retrotransposon activity to new DNA sites.},
journal = {Nature},
volume = {642},
number = {8069},
pages = {1080-1089},
pmid = {40205048},
issn = {1476-4687},
mesh = {*Retroelements/genetics ; Animals ; Humans ; CRISPR-Cas Systems/genetics ; Finches/genetics ; *Gene Editing/methods ; *DNA/genetics ; Genome/genetics ; *Mutagenesis, Insertional/genetics ; CRISPR-Associated Protein 9/metabolism/genetics ; Mice ; Reverse Transcription ; },
abstract = {Retroelements have a critical role in shaping eukaryotic genomes. For instance, site-specific non-long terminal repeat retrotransposons have spread widely through preferential integration into repetitive genomic sequences, such as microsatellite regions and ribosomal DNA genes[1-6]. Despite the widespread occurrence of these systems, their targeting constraints remain unclear. Here we use a computational pipeline to discover multiple new site-specific retrotransposon families, profile members both biochemically and in mammalian cells, find previously undescribed insertion preferences and chart potential evolutionary paths for retrotransposon retargeting. We identify R2Tg, an R2 retrotransposon from the zebra finch, Taeniopygia guttata, as an orthologue that can be retargeted by payload engineering for target cleavage, reverse transcription and scarless insertion of heterologous payloads at new genomic sites. We enhance this activity by fusing R2Tg to CRISPR-Cas9 nickases for efficient insertion at new genomic sites. Through further screening of R2 orthologues, we select an orthologue, R2Tocc, with natural reprogrammability and minimal insertion at its natural 28S site, to engineer SpCas9[H840A]-R2Tocc, a system we name site-specific target-primed insertion through targeted CRISPR homing of retroelements (STITCHR). STITCHR enables the scarless, efficient installation of edits, ranging from a single base to 12.7 kilobases, gene replacement and use of in vitro transcribed or synthetic RNA templates. Inspired by the prevalence of nLTR retrotransposons across eukaryotic genomes, we anticipate that STITCHR will serve as a platform for scarless programmable integration in dividing and non-dividing cells, with both research and therapeutic applications.},
}
@article {pmid40199626,
year = {2025},
author = {Lu, Y and Wang, J and Xu, Y and Xu, M and Li, B and Fan, Z and Liu, J and Li, X and Cai, Z and Zheng, Y and Wang, W and Yang, J and Zhang, Z and Liu, Z},
title = {Long-offset paired nicking-based efficient and precise strategy for in vivo targeted insertion.},
journal = {Trends in biotechnology},
volume = {43},
number = {7},
pages = {1743-1764},
doi = {10.1016/j.tibtech.2025.02.020},
pmid = {40199626},
issn = {1879-3096},
mesh = {Animals ; Mice ; *CRISPR-Cas Systems/genetics ; Humans ; *Gene Editing/methods ; Genetic Therapy/methods ; Gene Knock-In Techniques/methods ; *Gene Targeting/methods ; DNA Repair ; Hepatocytes/metabolism ; Deoxyribonuclease I/metabolism/genetics ; },
abstract = {Clustered regularly interspaced short palindromic repeat (CRISPR)-based targeted insertion of DNA fragments holds great promise for gene therapy. However, designing highly efficient and precise integration of large DNA segments in somatic cells while avoiding unpredictable products remains challenging. Here, we devised a novel long-offset paired nicking target integration (LOTI) strategy, which enhances the capacity of Cas9 nickase (Cas9n) in targeted gene integration in somatic cells, yielding higher knock-in (KI) efficiency compared with classical nickase-based approaches. The underlying repair mechanism involves the DNA repair proteins Rad51 and Rad52, and Ligase I/III. Moreover, we achieved efficient KI of at least 1.5-kb gene fragments in hepatocytes and recovery 55% FIX activity in a hemophilia B mouse model using only one-dose plasmid DNA delivery. Compared with the Cas9-based strategy, LOTI reduces off-target activity and restricts the formulation of unwanted insertions and deletions (indels) at the target site. Thus, LOTI provides a precise and efficient strategy for gene integration in somatic cells in vivo.},
}
@article {pmid40192038,
year = {2025},
author = {Shi, K and Huang, W and Zhu, M and Teng, S and Zhang, J and Duan, Z and Zhu, C and Hu, T and Wang, K and Wang, Z},
title = {Efficient genetic transformation and genome editing via an Agrobacterium-mediated in commercial oat (Avena sativa L.) cultivars.},
journal = {Journal of integrative plant biology},
volume = {67},
number = {7},
pages = {1697-1699},
doi = {10.1111/jipb.13915},
pmid = {40192038},
issn = {1744-7909},
support = {2005DKA21003//the National Center for Forestry and Grassland Genetic Resources/ ; },
mesh = {*Avena/genetics/microbiology ; *Gene Editing/methods ; *Transformation, Genetic ; *Agrobacterium/genetics ; Plants, Genetically Modified ; CRISPR-Cas Systems/genetics ; Genome, Plant/genetics ; },
abstract = {An optimized Agrobacterium-mediated transformation protocol for immature and mature oat embryos increased transformation efficiencies and the number of transformable cultivars and enabled highly efficient CRISPR/Cas9 and CRISPR/Cas12i genome editing to accelerate oat biotechnology breeding.},
}
@article {pmid40187931,
year = {2025},
author = {Jiang, Y and Xiao, Z and Luo, Z and Zhou, S and Tong, C and Jin, S and Liu, X and Qin, R and Xu, R and Pan, L and Li, J and Wei, P},
title = {Improving plant C-to-G base editors with a cold-adapted glycosylase and TadA-8e variants.},
journal = {Trends in biotechnology},
volume = {43},
number = {7},
pages = {1765-1787},
doi = {10.1016/j.tibtech.2025.03.001},
pmid = {40187931},
issn = {1879-3096},
mesh = {*Gene Editing/methods ; Oryza/genetics ; Plants, Genetically Modified/genetics ; *Cytidine Deaminase/genetics/metabolism ; *Uracil-DNA Glycosidase/genetics/metabolism ; Cytosine/metabolism ; Guanine/metabolism ; Humans ; CRISPR-Cas Systems ; Animals ; Cold Temperature ; },
abstract = {Plant cytosine (C)-to-guanine (G) base editors (CGBEs) have been established but suffer from limited editing efficiencies and low outcome purities. This study engineered a cod uracil DNA glycosylase (cod UNG, coUNG) from the cold-adapted fish Gadus morhua for plant CGBE, demonstrating 1.71- to 2.54-fold increases in C-to-G editing efficiency compared with the CGBE using human UNG (hUNG). Further engineering took advantage of TadA-8e-derived cytidine deaminases (TadA-CDs). These variants induced C substitutions with efficiencies ranging from 26.28% to 30.82% in rice cells, whereas adenine (A) conversion was negligible. By integrating coUNG and TadA-CDc elements with SpCas9 nickase, the resulting CDc-CGBEco achieved pure C-to-G editing without byproducts in up to 52.08% of transgenic lines. Whole-genome sequencing (WGS) analysis revealed no significant off-target effects of the CDc-BEs in rice. In addition, CDc-CGBEco enabled precise C-to-G editing in soybean and tobacco. These engineered CGBEs enhanced editing efficiency, purity, and specificity, suggesting their broad potential for applications in scientific research and crop breeding.},
}
@article {pmid40119518,
year = {2025},
author = {He, X and Yan, T and Song, Z and Xiang, L and Xiang, J and Yang, Y and Ren, K and Bu, J and Xu, X and Li, Z and Guo, X and Lin, B and Zhou, Q and Lin, G and Gu, F},
title = {Correcting a patient-specific Rhodopsin mutation with adenine base editor in a mouse model.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {33},
number = {7},
pages = {3101-3113},
doi = {10.1016/j.ymthe.2025.03.021},
pmid = {40119518},
issn = {1525-0024},
mesh = {Animals ; Disease Models, Animal ; Mice ; *Gene Editing/methods ; *Adenine/metabolism ; Humans ; *Retinitis Pigmentosa/genetics/therapy ; *Mutation ; *Rhodopsin/genetics ; Gene Knock-In Techniques ; Dependovirus/genetics ; Genetic Vectors/genetics ; Electroretinography ; RNA, Guide, CRISPR-Cas Systems/genetics ; CRISPR-Cas Systems ; Retina/metabolism/pathology ; Genetic Therapy/methods ; },
abstract = {Genome editing offers a great promise to treating human genetic diseases. To assess genome-editing-mediated therapeutic effects in vivo, an animal model is indispensable. The genomic disparities between mice and humans often impede the direct clinical application of genome-editing-mediated treatments using conventional mouse models. Thus, the generation of a mouse model with a humanized genomic segment containing a patient-specific mutation is highly sought after for translational research. In this study, we successfully developed a knockin mouse model for autosomal-dominant retinitis pigmentosa (adRP), designated as hT17M knockin, which incorporates a 75-nucleotide DNA segment with the T17M mutation (Rhodopsin-c.C50T; p.T17M). This model demonstrated significant reductions in electroretinogram amplitudes and exhibited disruptions in retinal structure. Subsequently, we administered an adeno-associated virus vectors carrying an adenine base editor (ABE) and a single-guide RNA specifically targeting the T17M mutation, achieving a peak correction rate of 39.7% at the RNA level and significantly improving retinal function in ABE-injected mice. These findings underscore that the hT17M knockin mouse model recapitulates the clinical features of adRP patients and exhibits therapeutic effects with ABE-mediated treatments. It offers a promising avenue for the development of gene-editing therapies for RP.},
}
@article {pmid40603034,
year = {2025},
author = {Sugo, T and Shirasago, Y and Yoshimoto, S and Kitajima, M},
title = {[GenAhead Bio: your partner for extensive support of genome editing and co-‍development of nucleic acid delivery].},
journal = {Nihon yakurigaku zasshi. Folia pharmacologica Japonica},
volume = {160},
number = {4},
pages = {274-278},
doi = {10.1254/fpj.25004},
pmid = {40603034},
issn = {0015-5691},
mesh = {*Gene Editing ; Humans ; *Nucleic Acids/administration &amp; dosage ; CRISPR-Cas Systems ; Animals ; RNA, Small Interfering ; },
abstract = {Inspired by my experiences working in research at an overseas biotech venture, I founded GenAhead Bio Inc. in 2018. GenAhead Bio adopts a unique dual-business structure, providing contract services for generating genetically modified cells using highly efficient CRISPR/Cas9 genome editing technology for researchers, while simultaneously pursuing a nucleic acid drug business aiming to develop nucleic acid drugs such as antisense oligonucleotides and siRNAs. Based on the emerging delivery system called Antibody-Nucleic acid Conjugate, where an antibody is covalently linked to a nucleic acid as a targeting ligand, we are conducting drug developmental research by delivering nucleic acids to the organs where antibodies accumulate. Our ultimate goal is to apply this technology to genome editing for gene modification in specific cell types. In this review, we will introduce some case studies of genome editing, including single nucleotide substitutions, as well as the delivery of siRNA to the skeletal muscle using anti-transferrin receptor (CD71) antibody and its therapeutic effects on muscular diseases.},
}
@article {pmid40601773,
year = {2025},
author = {Chafe, SC and Zhai, K and Aghaei, N and Miletic, P and Huang, Z and Brown, KR and Mobilio, D and Young, D and Suk, Y and Grewal, S and McKenna, D and Alizada, Z and Kieliszek, AM and Lam, FC and Escudero, L and Huang, Q and Huebner, A and Lu, J and Ang, P and Anand, A and Custers, S and Apel, E and Slassi, S and Brakel, B and Kim, J and Liu, JKC and Bassey-Archibong, BI and Abdo, R and Shargall, Y and Lu, JQ and Cutz, JC and Zhang, Q and Li, SS and Venugopal, C and Hynds, RE and Dufour, A and Moffat, J and Swanton, C and Bao, S and Singh, SK},
title = {A genome-wide in vivo CRISPR activation screen identifies BACE1 as a therapeutic vulnerability of lung cancer brain metastasis.},
journal = {Science translational medicine},
volume = {17},
number = {805},
pages = {eadu2459},
doi = {10.1126/scitranslmed.adu2459},
pmid = {40601773},
issn = {1946-6242},
mesh = {Humans ; *Brain Neoplasms/secondary/genetics ; *Lung Neoplasms/pathology/genetics ; *Amyloid Precursor Protein Secretases/metabolism/genetics ; *Aspartic Acid Endopeptidases/metabolism/genetics ; Animals ; *Carcinoma, Non-Small-Cell Lung/genetics/pathology ; Cell Line, Tumor ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Mice ; ErbB Receptors/metabolism ; *CRISPR-Cas Systems/genetics ; Xenograft Model Antitumor Assays ; Gene Expression Regulation, Neoplastic ; },
abstract = {Brain metastasis occurs in up to 40% of patients with non-small cell lung cancer (NSCLC). Considerable genomic heterogeneity exists between the primary lung tumor and respective brain metastasis; however, the identity of the genes capable of driving brain metastasis is incompletely understood. Here, we carried out an in vivo genome-wide CRISPR activation screen to identify molecular drivers of brain metastasis from an orthotopic xenograft model derived from a patient with NSCLC. We found that activating expression of the Alzheimer's disease-associated beta-secretase 1 (BACE1) led to a substantial increase in brain metastases. Furthermore, genetic and pharmacological inhibition of BACE1 blocked NSCLC brain metastasis. Mechanistically, we identified that BACE1 acts through epidermal growth factor receptor to drive this metastatic phenotype. Together, our data highlight the power of in vivo CRISPR activation screening to unveil molecular drivers and potential therapeutic targets of NSCLC brain metastasis.},
}
@article {pmid40599263,
year = {2025},
author = {Yang, R and Tang, T and Wulae, and Liu, P and Wang, X and Jiang, Y and Zhang, S and Wang, M and Yang, L and Wang, C},
title = {[One-Step Detection of Human Influenza B Virus Through Recombinase Polymerase Amplification and CRISPR/Cas12a Protein].},
journal = {Sichuan da xue xue bao. Yi xue ban = Journal of Sichuan University. Medical science edition},
volume = {56},
number = {2},
pages = {549-555},
pmid = {40599263},
issn = {1672-173X},
mesh = {*Influenza B virus/isolation &amp; purification/genetics ; Humans ; *Nucleic Acid Amplification Techniques/methods ; *CRISPR-Cas Systems ; *Recombinases/genetics/metabolism ; Sensitivity and Specificity ; *Influenza, Human/diagnosis/virology ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {OBJECTIVE: To establish a one-step detection method based on recombinase polymerase amplification (RPA) and CRISPR/Cas12a protein for the rapid and sensitive detection of human influenza B virus.<br><br>METHODS: RPA amplification primers were designed according to the conserved gene (NS1 gene) of human influenza B virus (Victoria lineage). The reaction system was established using the standard plasmid as the template. First of all, the reaction system was incubated at 37 &#x2103; for 15 minutes for RPA amplification. Then, the CRISPR/Cas12a system on the tube cap was thoroughly mixed with the RPA amplification product at the bottom of the tube through fast centrifugation, and real-time fluorescence detection was carried out at 37 &#x2103;. The reaction conditions were optimized to establish a one-step RPA-CRISPR/Cas12a detection method for human influenza B virus. The sensitivity of the testing method was evaluated using standard plasmids and pseudoviruses, and the specificity was evaluated using other viruses that may cause febrile respiratory syndrome. The consistency between the results of the one-step detection method and those of RT-qPCR detection was evaluated by testing real samples.<br><br>RESULTS: A one-step detection method based on RPA-CRISPR/Cas12a was successfully established. The optimal reaction conditions included a reaction temperature of 37 &#x2103;, a Cas12a/crRNA concentation ratio of 1&#x2236;1, a Cas12a concentration of 120 nmol/L, a single-stranded DNA (ssDNA) probe concentration of 300 nmol/L, and a primer concentration of 480 nmol/L. The method could detect standard plasmid DNA as low as 2.8 copies/&#x3bc;L within 25 minutes and pseudoviruses as low as 2.77 copies/&#x3bc;L within 30 minutes. The testing method showed high specificity, and no cross-reaction was observed with the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), influenza A (H1N1) virus, or respiratory syncytial virus subgroup A. When testing clinical samples, the sensitivity and the specificity for examining clinical samples were 93.33% and 100%, respectively, and consistency with RT-qPCR results was 97.14%.<br><br>CONCLUSION: With the one-step detection method based on RPA-CRISPR/Cas12a established in this study, the whole sample detection process, including nucleic acid release, reverse transcription, isothermal amplification, CRISPR/Cas12a system cleavage, and fluorescence signal output, can be completed within 30 minutes. Its high sensitivity, specificity, and successful application in clinical samples highlight its potential for rapid point-of-care testing in clinical settings.},
}
@article {pmid40598996,
year = {2025},
author = {Han, HG and Nandre, R and Eom, H and Choi, YJ and Ro, HS},
title = {Development of a CRISPR/Cas9 RNP-mediated genetic engineering system in Paecilomyces variotii.},
journal = {Journal of microbiology (Seoul, Korea)},
volume = {63},
number = {6},
pages = {e2502011},
doi = {10.71150/jm.2502011},
pmid = {40598996},
issn = {1976-3794},
support = {2023R1A2C1007213//National Research Foundation of Korea/ ; //Ministry of Science and ICT/ ; RS2024-00322425//Rural Development Administration/ ; },
mesh = {*CRISPR-Cas Systems ; *Paecilomyces/genetics/metabolism ; Promoter Regions, Genetic ; *Gene Editing/methods ; *Ribonucleoproteins/genetics/metabolism ; *Genetic Engineering/methods ; Green Fluorescent Proteins/genetics/metabolism ; Fungal Proteins/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; },
abstract = {A thermophilic strain of Paecilomyces variotii (MR1), capable of surviving temperatures above 40°C, was isolated from a paper mill and investigated as a host for heterologous protein production. To prevent environmental dissemination of spores, UV mutagenesis was employed to create a conidia-deficient strain, UM7. This strain underwent gene editing using Cas9-gRNA ribonucleoprotein (RNP) with HR donor DNA fragments, incorporating promoter sequences amplified from the genomic DNA of P. variotii (PH4, PP2, PS8, Ptub, Ptef1, and PgpdA), along with a signal sequence-tagged eGFP, flanked by 5'-upstream (336 bp) and 3'-downstream (363 bp) regions of pyrG. Co-transformation of HR donor DNA with RNP into protoplasts yielded 48 mutant pyrG transformants capable of surviving in the presence of 5-fluoroorotic acid (5-FOA). Sequence analysis identified 16 of the 48 pyrG-disrupted mutants carrying complete HR donor DNAs with the six different promoter sequences, indicating successful homology-directed repair (HDR). Evaluation of promoter strength revealed that PgpdA was the most effective for intracellular GFP production; however, it resulted in negligible extracellular GFP signal under all promoter conditions. A newly edited strain with an HDR integration module connecting PgpdA directly to eGFP, without the signal sequence, exhibited enhanced GFP expression in both mycelial cells and culture broth, suggesting that the signal peptide negatively affect protein expression and secretion. This work represents the first successful RNP-mediated gene editing in P. variotii, contributing to the application of this thermophilic fungus in protein production.},
}
@article {pmid40598890,
year = {2025},
author = {Wei, W and Gao, CH and Jiang, X and Qiao, J and Zhang, L and Yan, Y and Zhao, G and Yang, K and Yan, J and Yang, M},
title = {CARF-dependent preferential RNA cleavage by Csm6 increases drug susceptibility of mycobacteria.},
journal = {Nucleic acids research},
volume = {53},
number = {12},
pages = {},
doi = {10.1093/nar/gkaf622},
pmid = {40598890},
issn = {1362-4962},
support = {32070079//National Natural Science Foundation of China/ ; 32370127//National Natural Science Foundation of China/ ; 2662025ZHPY001//Central Universities/ ; 2020kfyXJJS119//Central Universities/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; *Mycobacterium smegmatis/genetics/drug effects/metabolism ; *RNA Cleavage ; *Mycobacterium tuberculosis/genetics/drug effects ; *Bacterial Proteins/genetics/metabolism/chemistry ; Drug Resistance, Bacterial/genetics ; Mycolic Acids/metabolism ; Gene Expression Regulation, Bacterial ; },
abstract = {CRISPR-Cas systems are prokaryotic adaptive immune systems that defend against invading mobile genetic elements. The type III-A CRISPR-Cas system has been studied in the evolutionary and epidemiological context of Mycobacterium tuberculosis, the causative agent of tuberculosis. However, its biological function remains poorly understood. Here, we demonstrate that heterologous expression of csm6, a single-stranded RNA ribonuclease of the CRISPR-Cas system, exhibits preferential RNA cleavage activity targeting host transcripts. This activity significantly downregulates ribosomal and mycolic acid biosynthesis pathway genes, leading to a global reduction in translation levels and an increased drug susceptibility of Mycobacterium smegmatis. Furthermore, mutagenesis analysis revealed that Csm6's biological function critically depends on its CARF domain rather than its HEPN domain. In conclusion, our study elucidates the biological role of the Csm6 protein in the CRISPR-Cas system, both in vitro and in vivo, highlighting how preferential RNA cleavage impacts multiple mycobacterial processes. These findings provide novel insights into the functional diversity of CRISPR-Cas systems in mycobacteria.},
}
@article {pmid40598087,
year = {2025},
author = {Garimella, SS and Minami, SA and Khanchandani, AN and Abad Santos, JC and Schaffer, SR and Shah, PS},
title = {A simplified two-plasmid system for orthogonal control of mammalian gene expression using light-activated CRISPR effector.},
journal = {BMC biotechnology},
volume = {25},
number = {1},
pages = {58},
pmid = {40598087},
issn = {1472-6750},
support = {n/a//AMPAC Fine Chemicals Summer Research Internship/ ; n/a//Hellman Family Foundation/ ; n/a//Good Food Institute/ ; },
mesh = {*Plasmids/genetics ; HEK293 Cells ; Humans ; *Optogenetics/methods ; Green Fluorescent Proteins/genetics/metabolism ; Light ; *CRISPR-Cas Systems/genetics ; Animals ; Clustered Regularly Interspaced Short Palindromic Repeats ; *Gene Expression Regulation ; Gene Expression ; },
abstract = {BACKGROUND: Optogenetic systems use light-responsive proteins to control gene expression, ion channels, protein localization, and signaling with the "flip of a switch". One such tool is the light activated CRISPR effector (LACE) system. Its ability to regulate gene expression in a tunable, reversible, and spatially resolved manner makes it attractive for many applications. However, LACE relies on delivery of four separate components on individual plasmids, which can limit its use. Here, we optimize LACE to reduce the number of plasmids needed to deliver all four components.<br><br>RESULTS: The two-plasmid LACE (2pLACE) system combines the four components of the original LACE system into two plasmids. Following construction, the behavior of 2pLACE was rigorously tested using optogenetic control of enhanced green fluorescent protein (eGFP) expression as a reporter. Using human HEK293T cells, we optimized the ratio of the two plasmids, measured activation as a function of light intensity, and determined the frequency of the light to activate the maximum fluorescence. Overall, the 2pLACE system showed a similar dynamic range, tunability, and activation kinetics as the original four plasmid LACE (4pLACE) system. Interestingly, 2pLACE also had less variability in activation signal compared to 4pLACE. We also demonstrate the optimal LACE system also depends on cell type. In mouse myoblast C2C12 cells, 2pLACE displayed less variability compared to 4pLACE, similar to HEK293T cells. However, 2pLACE also had a smaller dynamic range in C2C12 cells compared to 4pLACE.<br><br>CONCLUSIONS: This simplified system for optogenetics will be more amenable to biotechnology applications where variability needs to be minimized. By optimizing the LACE system to use fewer plasmids, 2pLACE becomes a flexible tool in multiple research applications. However, the optimal system may depend on cell type and application.},
}
@article {pmid40598062,
year = {2025},
author = {Ajdanian, L and Torkamaneh, D},
title = {Mother transformer: A High-Throughput, Cost-Effective in Planta Hairy Root Transformation Method for Cannabis.},
journal = {BMC biotechnology},
volume = {25},
number = {1},
pages = {60},
pmid = {40598062},
issn = {1472-6750},
mesh = {*Plant Roots/genetics/microbiology ; *Plants, Genetically Modified/genetics ; *Agrobacterium/genetics ; *Transformation, Genetic ; *Cannabis/genetics ; Cost-Benefit Analysis ; *High-Throughput Screening Assays/methods ; },
abstract = {BACKGROUND: Hairy root (HR) transformation assays mediated by Agrobacterium rhizogenes, both in vitro and ex vitro, are essential tools in plant biotechnology and functional genomics. These assays can be significantly influenced by various factors, which ultimately can enhance the efficiency. In this study, we optimized a two-step ex vitro HR transformation method using the actual mother plant combined with the RUBY system and compared with existing methods.<br><br>RESULTS: The two-step ex vitro method proved more efficient than both the one-step ex vitro and in vitro methods, with the highest transformation efficiency of 90% observed in the actual plant. This technique also demonstrated a faster and less complicated approach, reducing time to achieve massive transgenic HR formation by 9-29 days compared to other methods.<br><br>CONCLUSIONS: A novel, quicker, less complicated, and more efficient two-step transformation method for cannabis has been established, presenting a significantly lower risk of contamination. This protocol is particularly interesting to produce secondary metabolites using the CRISPR/Cas system in cannabis. We anticipate that this method will facilitate substantial time savings by rapidly producing hundreds of transformed samples.},
}
@article {pmid40597657,
year = {2025},
author = {Malekos, E and Montano, C and Carpenter, S},
title = {CRISPRware: a software package for contextual gRNA library design.},
journal = {BMC genomics},
volume = {26},
number = {1},
pages = {607},
pmid = {40597657},
issn = {1471-2164},
support = {F31AI179201//National Institute of Allergy and Infectious Diseases/ ; R35GM137801/GM/NIGMS NIH HHS/United States ; },
mesh = {*Software ; *RNA, Guide, CRISPR-Cas Systems/genetics ; *Gene Library ; *CRISPR-Cas Systems ; High-Throughput Nucleotide Sequencing ; Humans ; Animals ; },
abstract = {We present CRISPRware, an efficient method for generating guide RNA (gRNA) libraries against transcribed, translated, and noncoding regions. CRISPRware leverages next-generation sequencing data to design context-specific gRNAs and can account for genetic variation, which allows allele-specific guide design on a genome-wide scale. As a demonstration of use and to create a publicly available resource, we use CRISPRware to identify and score gRNAs against coding sequences in six model organisms for Cas9 and Cas12A and host these in a publicly available session on the UCSC Genome Browser.},
}
@article {pmid40596971,
year = {2025},
author = {Martí-Díaz, R and Sánchez-Del-Campo, L and Montenegro, MF and Hernández-Caselles, T and Piñero-Madrona, A and Cabezas-Herrera, J and Rodríguez-López, JN},
title = {Ex vivo engineering of phagocytic signals in breast cancer cells for a whole tumor cell-based vaccine.},
journal = {BMC cancer},
volume = {25},
number = {1},
pages = {1029},
pmid = {40596971},
issn = {1471-2407},
support = {21407/FPI/20//Fundaci&#xf3;n S&#xe9;neca/ ; FSRM/10.13039/100007801(22544/PI/24)//Fundaci&#xf3;n S&#xe9;neca/ ; FSRM/10.13039/100007801(22544/PI/24)//Fundaci&#xf3;n S&#xe9;neca/ ; FSRM/10.13039/100007801(22544/PI/24)//Fundaci&#xf3;n S&#xe9;neca/ ; FSRM/10.13039/100007801(22544/PI/24)//Fundaci&#xf3;n S&#xe9;neca/ ; FSRM/10.13039/100007801(22544/PI/24)//Fundaci&#xf3;n S&#xe9;neca/ ; PID2023-149281OB-I00//Ministerio de Ciencia, Innovaci&#xf3;n y Universidades/ ; CPP2023-010510//Ministerio de Ciencia, Innovaci&#xf3;n y Universidades/ ; PID2023-149281OB-I00//Ministerio de Ciencia, Innovaci&#xf3;n y Universidades/ ; CPP2023-010510//Ministerio de Ciencia, Innovaci&#xf3;n y Universidades/ ; PID2023-149281OB-I00//Ministerio de Ciencia, Innovaci&#xf3;n y Universidades/ ; PID2023-149281OB-I00//Ministerio de Ciencia, Innovaci&#xf3;n y Universidades/ ; },
mesh = {Animals ; Female ; Mice ; *Phagocytosis/immunology ; *Cancer Vaccines/immunology ; *Breast Neoplasms/immunology/therapy/pathology ; Humans ; CD47 Antigen/genetics ; Cell Line, Tumor ; CRISPR-Cas Systems ; Macrophages/immunology ; Mice, Inbred BALB C ; },
abstract = {BACKGROUND: Today, cell therapies are constantly evolving and providing new options for cancer patients. These therapies are mostly based on the inoculation of immune cells extracted from a person's own tumor; however, some studies using whole tumor cell-based vaccines are approaching the level of maturity required for clinical use. Although these latest therapies will have to be developed further and adapted to overcome many ethical barriers, there is no doubt that therapeutic cancer vaccines are the next frontier of immunotherapy.<br><br>METHODS: Ionizing radiation and CD47 knockout via CRISPR-Cas9 genome editing were used to optimize the macrophage-mediated phagocytosis of breast cancer cells. These cells were subsequently used in several mouse models to determine their potential as novel whole-cell-based vaccines to drive antitumor immunity. To improve the recognition of tumor cells by activated immune cells, this cellular therapy was combined with anti-PD-1 antibody treatments.<br><br>RESULTS: Here, we showed that irradiation of 4T1 breast cancer cells increases their immunogenicity and, when injected into the blood of immunocompetent mice, elicits a complete antitumor immune response mediated, in part, by the adaptive immune system. Next, to improve the macrophage-mediated phagocytosis of breast cancer cells, we knocked out CD47 in 4T1 cells. When injected in the bloodstream, irradiated CD47 knockout cells activated both the adaptive and the innate immune systems. Therefore, we used these ex vivo engineered cells as a whole tumor cell-based vaccine to treat breast tumors in immunocompetent mice. A better response was obtained when these cells were combined with an anti-PD-1 antibody.<br><br>CONCLUSION: These results suggest that tumor cells obtained from surgical samples of a breast cancer patient could be engineered ex vivo and used as a novel cell therapy to drive antitumor immunity.},
}
@article {pmid40596718,
year = {2025},
author = {Schuster, B and Dobiášovská, I and Ćurčić, J and Pajer, P and Borna, &#x160; and Bartůněk, P},
title = {SWITCHER, a CRISPR-inducible floxed wild-type Cre regulating CRISPR activity.},
journal = {Communications biology},
volume = {8},
number = {1},
pages = {982},
pmid = {40596718},
issn = {2399-3642},
mesh = {*CRISPR-Cas Systems/genetics ; *Integrases/genetics/metabolism ; *Clustered Regularly Interspaced Short Palindromic Repeats ; *Gene Editing/methods ; Humans ; CRISPR-Associated Proteins/genetics/metabolism ; Endodeoxyribonucleases/genetics/metabolism ; HEK293 Cells ; RNA, Guide, CRISPR-Cas Systems/genetics ; Bacterial Proteins ; },
abstract = {Although several Cre-regulated CRISPR/Cas platforms exist, a CRISPR/Cas-controlled Cre-system remains a challenge. Here, we present a genetic switch we term SWITCHER based on a floxed wild-type Cre-construct representing a CRISPR-inducible and self-limiting kill switch. By leveraging CRISPR/Cas12a-mediated crRNA-array maturation, we showcase SWITCHER's dual role-not just as a recombinase but as a CRISPR switch, capable of orchestrating distinct Cas12a/crRNA-encoded programs.},
}
@article {pmid40595661,
year = {2025},
author = {Boswell, CW and Hoppe, C and Sherrard, A and Miao, L and Kojima, ML and Martino, P and Zhao, N and Stasevich, TJ and Nicoli, S and Giraldez, AJ},
title = {Genetically encoded affinity reagents are a toolkit for visualizing and manipulating endogenous protein function in vivo.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {5503},
pmid = {40595661},
issn = {2041-1723},
support = {R01 HD100035/HD/NICHD NIH HHS/United States ; R35 GM122580/GM/NIGMS NIH HHS/United States ; },
mesh = {Animals ; Zebrafish/genetics/embryology/metabolism ; CRISPR-Cas Systems/genetics ; Zebrafish Proteins/metabolism/genetics ; Single-Domain Antibodies/genetics/metabolism ; Epitopes/genetics/metabolism ; Gene Knock-In Techniques/methods ; Humans ; Single-Chain Antibodies/genetics/metabolism ; },
abstract = {Probing endogenous protein localization and function in vivo remains challenging due to laborious gene targeting and monofunctional alleles. Here, we develop a multifunctional and adaptable toolkit based on genetically encoded affinity reagents (GEARs). GEARs use small epitopes recognized by nanobodies and single chain variable fragments to enable fluorescent visualization, manipulation and degradation of protein targets in vivo. Furthermore, we outline a CRISPR/Cas9-based epitope tagging pipeline to demonstrate its utility for producing knock-in alleles that have broad applications. We use GEARs to examine the native behavior of the pioneer transcription factor Nanog and the planar cell polarity protein Vangl2 during early zebrafish development. Together, this toolkit provides a versatile system for probing and perturbing endogenous protein function while circumventing challenges associated with conventional gene targeting and is broadly available to the model organism community.},
}
@article {pmid40595633,
year = {2025},
author = {Fu, W and Ma, J and Wang, Z and Tang, N and Pan, D and Su, M and Wu, Z and Gan, J and Ji, Q},
title = {Mechanisms and engineering of a miniature type V-N CRISPR-Cas12 effector enzyme.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {5667},
pmid = {40595633},
issn = {2041-1723},
support = {22277078//National Natural Science Foundation of China (National Science Foundation of China)/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; RNA, Guide, CRISPR-Cas Systems/metabolism/genetics ; Cryoelectron Microscopy ; Gene Editing/methods ; *CRISPR-Associated Proteins/metabolism/genetics/chemistry/ultrastructure ; Humans ; DNA/metabolism/genetics ; *Bacterial Proteins/metabolism/genetics/chemistry/ultrastructure ; Protein Engineering ; },
abstract = {Type V CRISPR-Cas12 systems are highly diverse in their functionality and molecular compositions, including miniature Cas12f1 and Cas12n genome editors that provide advantages for efficient in vivo therapeutic delivery due to their small size. In contrast to Cas12f1 nucleases that utilize a homodimer structure for DNA targeting and cleavage with a preference for T- or C-rich PAMs, Cas12n nucleases are likely monomeric proteins and uniquely recognize rare A-rich PAMs. However, the molecular mechanisms behind RNA-guided genome targeting and cleavage by Cas12n remain unclear. Here, we present the cryo-electron microscopy (cryo-EM) structure of Rothia dentocariosa Cas12n (RdCas12n) bound to a single guide RNA (sgRNA) and target DNA, illuminating the intricate molecular architecture of Cas12n and its sgRNA, as well as PAM recognition and nucleic-acid binding mechanisms. Through structural comparisons with other Cas12 nucleases and the ancestral precursor TnpB, we provide insights into the evolutionary significance of Cas12n in the progression from TnpB to various Cas12 nucleases. Additionally, we extensively modify the sgRNA and convert RdCas12n into an effective genome editor in human cells. Our findings enhance the understanding of the evolutionary mechanisms of type V CRISPR-Cas12 systems and offer a molecular foundation for engineering Cas12n genome editors.},
}
@article {pmid40595632,
year = {2025},
author = {Zhang, Y and Zhang, T and Xiao, X and Wang, Y and Kawalek, A and Ou, J and Ren, A and Sun, W and de Bakker, V and Liu, Y and Li, Y and Yang, L and Ye, L and Jia, N and Veening, JW and Liu, X},
title = {CRISPRi screen identifies FprB as a synergistic target for gallium therapy in Pseudomonas aeruginosa.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {5870},
pmid = {40595632},
issn = {2041-1723},
support = {82270012//National Natural Science Foundation of China (National Science Foundation of China)/ ; 82200047//National Natural Science Foundation of China (National Science Foundation of China)/ ; },
mesh = {*Pseudomonas aeruginosa/drug effects/genetics ; *Gallium/pharmacology/therapeutic use ; Animals ; *Pseudomonas Infections/drug therapy/microbiology ; Mice ; Biofilms/drug effects/growth &amp; development ; *Anti-Bacterial Agents/pharmacology ; Humans ; Microbial Sensitivity Tests ; Reactive Oxygen Species/metabolism ; *Bacterial Proteins/genetics/metabolism ; Oxidative Stress/drug effects ; CRISPR-Cas Systems ; Female ; Iron/metabolism ; },
abstract = {With the rise of antibiotic-resistant bacteria, non-antibiotic therapies like gallium gain increasing attention. Intravenous gallium nitrate is under Phase II clinical trials to treat chronic Pseudomonas aeruginosa infections in cystic fibrosis patients. However, its clinical efficacy is constrained by the achievable peak concentration in human tissue. To address this limitation, we apply a genome-wide CRISPR interference approach (CRISPRi-seq) to identify potential synergistic targets with gallium. We classify the essential genes by response time and growth reduction, pinpointing the most vulnerable therapeutic targets in this species. In addition, we identify a highly conserved gene, fprB, encoding a ferredoxin-NADP[+] reductase, whose deletion sensitizes P. aeruginosa to gallium, lowering its MIC by 32-fold and shifting mode of action from bacteriostatic to bactericidal. Further investigation reveals that FprB plays a critical role in modulating oxidative stress induced by gallium, via control of iron homeostasis and reactive oxygen species accumulation. Deleting fprB enhances gallium's efficacy against biofilm formation and improves outcomes in a murine lung infection model of P. aeruginosa, suggesting FprB is a promising drug target in combination with gallium. Overall, our data show CRISPRi-seq as a powerful tool for systematic genetic analysis of P. aeruginosa, advancing the identification of novel therapeutic targets.},
}
@article {pmid40595611,
year = {2025},
author = {Irie, N and Takeda, N and Satou, Y and Araki, K and Ono, M},
title = {Machine learning-assisted decoding of temporal transcriptional dynamics via fluorescent timer.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {5720},
pmid = {40595611},
issn = {2041-1723},
support = {DCRPGF\100007//Cancer Research UK (CRUK)/ ; JP21K07082, JP21H00433, JP24K10259, JPJSCCA2020008//MEXT | Japan Society for the Promotion of Science (JSPS)/ ; JPJSCCA2020008//MEXT | Japan Society for the Promotion of Science (JSPS)/ ; JP16H0627601, JP22H04922//MEXT | Japan Society for the Promotion of Science (JSPS)/ ; 24gm1810001s0103//Japan Agency for Medical Research and Development (AMED)/ ; },
mesh = {Animals ; *Machine Learning ; *Forkhead Transcription Factors/genetics/metabolism ; Mice ; *Transcription, Genetic ; Flow Cytometry/methods ; Gene Expression Regulation ; Mice, Inbred C57BL ; Single-Cell Analysis/methods ; Enhancer Elements, Genetic ; T-Lymphocytes, Regulatory/metabolism ; Genes, Reporter ; Neural Networks, Computer ; Spleen/metabolism/cytology ; CRISPR-Cas Systems ; },
abstract = {Investigating the temporal dynamics of gene expression is crucial for understanding gene regulation across various biological processes. Using the Fluorescent Timer protein, the Timer-of-cell-kinetics-and-activity system enables analysis of transcriptional dynamics at the single-cell level. However, the complexity of Timer fluorescence data has limited its broader application. Here, we introduce an integrative approach combining molecular biology and machine learning to elucidate Foxp3 transcriptional dynamics through flow cytometric Timer analysis. We have developed a convolutional neural network-based method that incorporates image conversion and class-specific feature visualisation for class-specific feature identification at the single-cell level. Biologically, we developed a novel CRISPR mutant of Foxp3 fluorescent Timer reporter mice lacking the enhancer Conserved Non-coding Sequence 2, which revealed new roles of this enhancer in regulating Foxp3 transcription frequency under specific conditions. Furthermore, analysis of wild-type Foxp3 fluorescent Timer reporter mice at different ages uncovered distinct patterns of Foxp3 expression from neonatal to aged mice, highlighting prominent thymus-like features of neonatal splenic Foxp3[+] T cells. In conclusion, our study uncovers previously unrecognised Foxp3 transcriptional dynamics, establishing a proof-of-concept for integrating CRISPR, single-cell dynamics analysis, and machine learning methods as advanced techniques to understand transcriptional dynamics in vivo.},
}
@article {pmid40595537,
year = {2025},
author = {Hofmann, R and Herman, C and Mo, CY and Mathai, J and Marraffini, LA},
title = {Deep mutational scanning identifies Cas1 and Cas2 variants that enhance type II-A CRISPR-Cas spacer acquisition.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {5730},
pmid = {40595537},
issn = {2041-1723},
mesh = {*CRISPR-Cas Systems/genetics ; *Streptococcus pyogenes/genetics/virology ; *CRISPR-Associated Proteins/genetics/metabolism ; Mutation ; *Bacterial Proteins/genetics/metabolism ; Bacteriophages/genetics ; *Endonucleases/genetics/metabolism ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; },
abstract = {A remarkable feature of CRISPR-Cas systems is their ability to acquire short sequences from invading viruses to create a molecular record of infection. These sequences, called spacers, are inserted into the CRISPR locus and mediate sequence-specific immunity in prokaryotes. In type II-A CRISPR systems, Cas1, Cas2 and Csn2 form a supercomplex with Cas9 to integrate viral sequences. While the structure of the integrase complex has been described, a detailed functional analysis of the spacer acquisition machinery is lacking. We developed a genetic system that combines deep mutational scanning (DMS) of Streptococcus pyogenes cas genes with a method to select bacteria that acquire new spacers. Here, we show that this procedure reveals key interactions at the Cas1-Cas2 interface critical for spacer integration, identifies Cas variants with enhanced spacer acquisition and immunity against phage infection, and provides insights into the molecular determinants of spacer acquisition, offering a platform to improve CRISPR-Cas-based applications.},
}
@article {pmid40595093,
year = {2025},
author = {Wei, T and Yan, Y and Niu, M and Dong, X and Li, H and Sun, Y and Fa, Y},
title = {A rapid LASV detection method based on CRISPR-Cas13a and recombinase aided amplification with special lateral-flow test strips.},
journal = {Scientific reports},
volume = {15},
number = {1},
pages = {20640},
pmid = {40595093},
issn = {2045-2322},
support = {2023YFC2605100//National Key Research and Development Program of China/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; *Nucleic Acid Amplification Techniques/methods ; *Recombinases/metabolism ; *Lassa virus/isolation &amp; purification/genetics ; Humans ; *Molecular Diagnostic Techniques/methods ; Sensitivity and Specificity ; *Lassa Fever/diagnosis/virology ; Reagent Strips ; Animals ; },
abstract = {Lassa virus (LASV) is a high-risk pathogen associated with severe viral hemorrhagic fever in both humans and animals. Owing to its significant treatment challenges and high infectivity, LASV is classified as a biosafety level 4 (BSL-4) pathogen. It is essential to establish a rapid LASV detection method to prevent and control the disease. To address the biosecurity threats caused by LASV, in this study, we developed a new test method for LASV detection by combining the recombinase-mediated isothermal amplification (RAA) and CRISPR-Cas13a detection technology. The detection efficiency of this method was evaluated and compared with existing methods. The results demonstrate that this new detection maintains relatively high sensitivity and specificity, while having excellent simplicity and rapidity. The sensitivity of the method for detecting the LASV can achieve a threshold of 10[1] copies/µL using fluorescence detection in 90 min and 10[2] copies/µL with lateral flow strip detection in just an hour, which only needs a simple constant temperature equipment to achieve. The application of this detection method holds substantial biosecurity significance for underdeveloped regions (e.g., West Africa), as well as for countries like China, which have a vast territory and uneven development of medical testing levels in various regions.},
}
@article {pmid40594904,
year = {2025},
author = {Sayem, M and Rafi, MA and Mishu, ID and Mahmud, Z},
title = {Comprehensive genomic analysis reveals virulence and antibiotic resistance genes in a multidrug-resistant Bacillus cereus isolated from hospital wastewater in Bangladesh.},
journal = {Scientific reports},
volume = {15},
number = {1},
pages = {22915},
pmid = {40594904},
issn = {2045-2322},
mesh = {*Bacillus cereus/genetics/pathogenicity/isolation &amp; purification/drug effects ; *Wastewater/microbiology ; Bangladesh ; *Drug Resistance, Multiple, Bacterial/genetics ; Phylogeny ; Hospitals ; Virulence/genetics ; Genome, Bacterial ; Whole Genome Sequencing ; Genomics/methods ; Anti-Bacterial Agents/pharmacology ; Virulence Factors/genetics ; Humans ; },
abstract = {Hospital wastewater represents a significant reservoir for antimicrobial-resistant bacteria, including multidrug-resistant (MDR) Bacillus cereus, a pathogen of growing concern due to its potential impact on public health and environmental safety. This study characterizes the genomic features, antimicrobial resistance (AMR) mechanisms, and virulence potential of Bacillus cereus MBC, isolated from hospital wastewater in Dhaka, Bangladesh. Using whole-genome sequencing (WGS) and advanced bioinformatics, we analyzed the isolate's taxonomy, phylogenetics, functional annotation, and biosynthetic potential. The genome, spanning 5.6 Mb with a GC content of 34.84%, contained 5,881 protein-coding sequences, including 1,424 hypothetical proteins, and 28 genes associated with AMR. Phylogenetic analysis revealed a close genetic relationship with Bacillus cereus ATCC 14579, sharing virulence factors such as hemolysin BL (HBL), non-hemolytic enterotoxin (NHE), and cytotoxin K (CytK), all contributing to its pathogenicity. The ability to form biofilms further enhances the strain's persistence and resistance in hospital environments. AMR profiling identified genes conferring resistance to beta-lactams (e.g., BcI, BcII, BcIII), tetracyclines (tetB(P)), glycopeptides (vanY), and fosfomycin, highlighting the bacterium's capacity to resist a wide array of antibiotics. Functional annotation revealed metabolic pathways involved in iron acquisition and the biosynthesis of siderophores such as petrobactin and bacillibactin, reinforcing the bacterium's adaptability in nutrient-limited environments. Mobile genetic elements, including prophages, CRISPR-Cas systems, and transposable elements, suggest significant horizontal gene transfer (HGT), enhancing genetic plasticity and resistance spread. Pangenomic analysis, involving 125 B. cereus strains, revealed a high degree of genetic diversity and close relationships with strains from clinical, food, and agricultural environments, emphasizing the overlap between clinical and environmental reservoirs of resistance. The strain's isolation from hospital wastewater underscores the complex interplay between environmental contaminants and bacterial evolution, which fosters MDR traits. Our findings underscore the urgent need for enhanced genomic surveillance and wastewater management strategies to mitigate the spread of MDR B. cereus and AMR genes in hospital environments.},
}
@article {pmid40594722,
year = {2025},
author = {Ren, X and Yao, XR and Chen, K and Xiao, WT and He, JY},
title = {CRISPR-Cas9 screening identifies a gene signature predictive of prognosis in glioblastoma.},
journal = {Scientific reports},
volume = {15},
number = {1},
pages = {21077},
pmid = {40594722},
issn = {2045-2322},
support = {82304083//National Natural Science Foundation of China/ ; 2023JJ40584//Natural Science Foundation of Hunan Province/ ; },
mesh = {Humans ; *Glioblastoma/genetics/pathology/mortality ; Prognosis ; *CRISPR-Cas Systems/genetics ; *Brain Neoplasms/genetics/pathology/mortality ; Gene Expression Regulation, Neoplastic ; Biomarkers, Tumor/genetics ; Cell Proliferation/genetics ; Gene Expression Profiling ; Gene Regulatory Networks ; *Transcriptome ; },
abstract = {Glioblastoma (GBM) is the most aggressive primary brain malignancy, characterized by a poor prognosis and limited therapeutic options. Identifying essential genes and pathways involved in GBM proliferation is important for developing prognostic biomarkers and potential therapeutic targets. In this study, genome-wide CRISPR-Cas9 screening data from the dependency map (DepMap) database were analyzed to explore proliferation-related essential genes and pathways in GBM. A five-gene prognostic signature-CLSPN, HSP90B1, MED10, SAMM50, and TOMM20-was constructed using univariate, LASSO, and multivariate Cox regression analyses, and its prognostic value was evaluated in independent cohorts. Weighted gene co-expression network analysis (WGCNA) and gene set enrichment analysis (GSEA) suggested that the E2F targets pathway may be involved in GBM proliferation, consistent with the CRISPR screening results. Among the identified genes, MED10 was preliminarily implicated in regulating GBM cell proliferation and migration, as supported by functional assays. These findings propose a proliferation-related gene signature with potential prognostic relevance in GBM and indicate the E2F targets pathway as a biological process potentially associated with tumor progression. MED10 warrants further investigation as a candidate gene in the context of GBM biology and therapy.},
}
@article {pmid40593724,
year = {2025},
author = {Hu, M and Zhang, B and Shan, Y and Cao, F and Wang, Y and Qi, W and Wang, X and Shen, Y and Guo, X and Zhang, M and Tian, T and Xie, W and Zhang, M and Liang, F and Pei, D and Zhou, X},
title = {Scalable modulation of CRISPR‒Cas enzyme activity using photocleavable phosphorothioate DNA.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {5939},
pmid = {40593724},
issn = {2041-1723},
support = {32150019//National Natural Science Foundation of China (National Science Foundation of China)/ ; 32300692//National Natural Science Foundation of China (National Science Foundation of China)/ ; 92068201//National Natural Science Foundation of China (National Science Foundation of China)/ ; 2024M760985//China Postdoctoral Science Foundation/ ; GZB20240235//China Postdoctoral Science Foundation/ ; 2024T170292//China Postdoctoral Science Foundation/ ; 2023M741235//China Postdoctoral Science Foundation/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; RNA, Guide, CRISPR-Cas Systems/genetics/metabolism ; *Phosphorothioate Oligonucleotides/metabolism/chemistry/genetics ; *DNA/metabolism/chemistry/genetics ; Light ; Humans ; },
abstract = {The regulation of CRISPR‒Cas activity is critical for developing advanced biotechnologies. Optical control of CRISPR‒Cas system activity can be achieved by modulation of Cas proteins or guide RNA (gRNA), but these approaches either require complex protein engineering modifications or customization of the optically modulated gRNAs according to the target. Here, we present a method, termed photocleavable phosphorothioate DNA (PC&amp;PS DNA)-mediated regulation of CRISPR‒Cas activity (DNACas), that is versatile and overcomes the limitations of conventional methods. In DNACas, CRISPR‒Cas activity is silenced by the affinity binding of PC&amp;PS DNA and restored through light-triggered chemical bond breakage of PC&amp;PS DNA. The universality of DNACas is demonstrated by adopting the PC&amp;PS DNA to regulate various CRISPR‒Cas enzymes, achieving robust light-switching performance. DNACas is further adopted to develop a light-controlled one-pot LAMP-BrCas12b detection method and a spatiotemporal gene editing strategy. We anticipate that DNACas could be employed to drive various biotechnological advances.},
}
@article {pmid40593661,
year = {2025},
author = {Hwang, HY and Lee, M and Yi, H and Seok, C and Lim, K and Na, YR and Kang, JS and Park, JH and Kim, D},
title = {Engineered Sdd7 cytosine base editors with enhanced specificity.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {5881},
pmid = {40593661},
issn = {2041-1723},
support = {2020R1A2C2101714//National Research Foundation of Korea (NRF)/ ; RS-2025-00521074//National Research Foundation of Korea (NRF)/ ; HI21C1314, HR22C1363, RS-2024-02507183,21A0202L1-12//Korea Health Industry Development Institute (KHIDI)/ ; },
mesh = {*Gene Editing/methods ; *Cytosine/metabolism ; Humans ; CRISPR-Cas Systems/genetics ; HEK293 Cells ; *Cytosine Deaminase/genetics/metabolism ; RNA, Guide, CRISPR-Cas Systems/genetics ; },
abstract = {Cytosine base editors (CBEs) revolutionize genome editing by enabling precise C-to-T conversions without double-strand breaks. Sdd7, a recently developed cytosine deaminase, exhibits high activity across a broad protospacer range but induces unintended off-target effects, including bystander mutations within and upstream of the protospacer and both gRNA-dependent and independent deamination. Here, we report that BE4max and Sdd7 induce bystander editing upstream of the protospacer. To overcome this, we engineer two Sdd7 variants, Sdd7e1 and Sdd7e2, enhancing specificity while preserving on-target efficiency. These variants display reduced bystander editing, narrowed editing windows, and significantly lower off-target activity. Delivery as ribonucleoproteins via engineered virus-like particles (eVLPs) further improves specificity, nearly eliminating bystander edits and increasing precise single-point mutations. Our findings establish Sdd7e1 and Sdd7e2, especially when delivered via eVLP, as high-fidelity CBEs poised for safe, precise therapeutic genome editing.},
}
@article {pmid40593653,
year = {2025},
author = {Pastuszka, A and Mazzuoli, MV and Crestani, C and Deborde, L and Sismeiro, O and Lemaire, C and Rong, V and Gominet, M and Jacquemet, E and Legendre, R and Lanotte, P and Firon, A},
title = {The virulence regulator CovR boosts CRISPR-Cas9 immunity in Group B Streptococcus.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {5678},
pmid = {40593653},
issn = {2041-1723},
support = {ANR-22-CE15-0024//Agence Nationale de la Recherche (French National Research Agency)/ ; LabEx IBEID, ANR-10-LABX-62-IBEID//Agence Nationale de la Recherche (French National Research Agency)/ ; },
mesh = {*Streptococcus agalactiae/genetics/immunology/pathogenicity ; *CRISPR-Cas Systems/genetics/immunology ; *Bacterial Proteins/genetics/metabolism/immunology ; Virulence/genetics ; Gene Expression Regulation, Bacterial ; Streptococcal Infections/immunology/microbiology ; Promoter Regions, Genetic ; *CRISPR-Associated Protein 9/genetics/metabolism ; Operon ; Host-Pathogen Interactions/immunology/genetics ; Mice ; Animals ; Humans ; },
abstract = {CRISPR-Cas9 immune systems protect bacteria from foreign DNA. However, immune efficiency is constrained by Cas9 off-target cleavages and toxicity. How bacteria regulate Cas9 to maximize protection while preventing autoimmunity is not understood. Here, we show that the master regulator of virulence, CovR, regulates CRISPR-Cas9 immunity against mobile genetic elements in Streptococcus agalactiae, a pathobiont responsible for invasive neonatal infections. We show that CovR binds to and represses a distal promoter of the cas operon, integrating immunity within the virulence regulatory network. The CovR-regulated promoter provides a controlled increase in off-target cleavages to counteract mutations in the target DNA, restores the potency of old immune memory, and stimulates the acquisition of new memory in response to recent infections. Regulation of Cas9 by CovR is conserved at the species level, with lineage specificities suggesting different adaptive trajectories. Altogether, we describe the coordinated regulation of immunity and virulence that enhances the bacterial immune repertoire during host-pathogen interaction.},
}
@article {pmid40593649,
year = {2025},
author = {Thorpe, C and Luo, W and Ji, Q and Eggenberger, AL and Chicowski, AS and Xu, W and Sandhu, R and Lee, K and Whitham, SA and Qi, Y and Wang, K and Jiang, S},
title = {Enhancing biolistic plant transformation and genome editing with a flow guiding barrel.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {5624},
pmid = {40593649},
issn = {2041-1723},
support = {2019-67013-29016//United States Department of Agriculture | Agricultural Research Service (USDA Agricultural Research Service)/ ; IOW04714//United States Department of Agriculture | Agricultural Research Service (USDA Agricultural Research Service)/ ; IOW04308//United States Department of Agriculture | Agricultural Research Service (USDA Agricultural Research Service)/ ; IOS-2132693//NSF | BIO | Division of Biological Infrastructure (DBI)/ ; },
mesh = {*Gene Editing/methods ; *Biolistics/methods/instrumentation ; Zea mays/genetics ; CRISPR-Cas Systems/genetics ; Plants, Genetically Modified/genetics ; Triticum/genetics ; *Transformation, Genetic ; Onions/genetics ; Genome, Plant ; Seedlings/genetics ; Genetic Engineering/methods ; },
abstract = {The biolistic delivery system is an essential tool in plant genetic engineering, capable of delivering DNAs, RNAs, and proteins independent of tissue type, genotype, or species. However, its efficiency and consistency remain longstanding challenges despite decades of widespread use. Here, through advanced simulations, we identify gas and particle flow barriers as the root cause of these limitations. We show that a flow guiding barrel (FGB) achieves a 22-fold enhancement in transient transfection efficiency, a 4.5-fold increase in CRISPR-Cas9 ribonucleoprotein editing efficiency in onion epidermis, and a 17-fold improvement in viral infection efficiency in maize seedlings. Furthermore, stable transformation frequency in maize using B104 immature embryos increases over 10-fold, while in planta CRISPR-Cas12a-mediated genome editing efficiency in wheat meristems doubles in both T0 and T1 generations. This study provides insights into the fundamental mechanisms underlying biolistic inefficiency and demonstrates a practical solution that enables broader and more reliable applications in plant genetic engineering.},
}
@article {pmid40593618,
year = {2025},
author = {Lin, M and Qiu, Z and Hao, M and Qi, W and Zhang, T and Shen, Y and Xiao, H and Liang, C and Xie, L and Jiang, Y and Cheng, M and Tian, T and Zhou, X},
title = {Cas12a Cis-cleavage mediated lateral flow assay enables multiplex and ultra-specific nucleic acid detection.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {5597},
pmid = {40593618},
issn = {2041-1723},
support = {2023M741238//China Postdoctoral Science Foundation/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; Humans ; *Endodeoxyribonucleases/metabolism/genetics ; *Nucleic Acid Amplification Techniques/methods ; *CRISPR-Associated Proteins/metabolism/genetics ; Sensitivity and Specificity ; *Bacterial Proteins/metabolism/genetics ; *Nucleic Acids/genetics/analysis ; Nucleic Acid Hybridization ; },
abstract = {CRISPR technology holds significant promise for advancing nucleic acid assays. However, current CRISPR diagnostic techniques, reliant on indiscriminate trans-cleavage mechanisms, face challenges in developing multiplex detection formats. Moreover, chaotic trans-cleavage activity often results from mismatched targets, leading to specificity issues. To address these limitations, here we exploit a double-key recognition mechanism based on CRISPR-Cas12a cis-cleavage and invasive hybridization identification of released sticky-end DNA products. By integrating multiplexed nucleic acid amplification, the double-key Cas12a detection mechanism, and a lateral flow detection platform, we develop a method termed Cas12a cis-cleavage mediated lateral flow assay (cc-LFA). We demonstrate that the cc-LFA exhibited superior specificity compared to three mainstream trans-cleavage-based CRISPR diagnostic techniques, achieving single-base resolution detection free from high-concentration wild-type DNA background interference. cc-LFA is also applied for highly specific detection of multiple respiratory pathogen samples and precise multiplexed detection of nine high-risk human papillomavirus (HPV) subtypes, achieving over 90% sensitivity and 100% specificity, respectively. Additionally, we present a portable device to automate nucleic acid amplification and strip detection procedures, showcasing the potential of cc-LFA for future applications in decentralized laboratory scenarios.},
}
@article {pmid40593576,
year = {2025},
author = {Kiernan, KA and Taylor, DW},
title = {Visualization of a multi-turnover Cas9 after product release.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {5681},
pmid = {40593576},
issn = {2041-1723},
support = {R35GM138348//U.S. Department of Health &amp; Human Services | NIH | National Institute of General Medical Sciences (NIGMS)/ ; },
mesh = {RNA, Guide, CRISPR-Cas Systems/metabolism/genetics ; *CRISPR-Cas Systems ; *CRISPR-Associated Protein 9/metabolism/genetics/chemistry/ultrastructure ; Gene Editing/methods ; Streptococcus pyogenes/enzymology/genetics ; Cryoelectron Microscopy ; Kinetics ; DNA/metabolism/chemistry/genetics ; Bacterial Proteins/metabolism/genetics ; },
abstract = {While the most widely used CRISPR-Cas enzyme is the Cas9 endonuclease from Streptococcus pyogenes (Cas9), it exhibits single-turnover enzyme kinetics which leads to long residence times on product DNA. This blocks access to DNA repair machinery and acts as a major bottleneck during CRISPR-Cas9 gene editing. Cas9 can eventually be removed from the product by extrinsic factors, such as translocating polymerases, but the mechanisms contributing to Cas9 dissociation following cleavage remain poorly understood. Here, we employ truncated guide RNAs as a strategy to weaken PAM-distal nucleic acid interactions and promote faster enzyme turnover. Using kinetics-guided cryo-EM, we examine the conformational landscape of a multi-turnover Cas9, including the first detailed snapshots of Cas9 dissociating from product DNA. We discovered that while the PAM-distal product dissociates from Cas9 following cleavage, tight binding of the PAM-proximal product directly inhibits re-binding of new targets. Our work provides direct evidence as to why Cas9 acts as a single-turnover enzyme and will guide future Cas9 engineering efforts.},
}
@article {pmid40593519,
year = {2025},
author = {Abe, I and Ohno, H and Mochizuki, M and Hayashi, K and Saito, H},
title = {Split RNA switch orchestrates pre- and post-translational control to enable cell type-specific gene expression.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {5362},
pmid = {40593519},
issn = {2041-1723},
support = {JP20H05626//MEXT | Japan Society for the Promotion of Science (JSPS)/ ; JP25H00970//MEXT | Japan Society for the Promotion of Science (JSPS)/ ; JP20H05701//MEXT | Japan Society for the Promotion of Science (JSPS)/ ; JP20K12644//MEXT | Japan Society for the Promotion of Science (JSPS)/ ; JP25K03464//MEXT | Japan Society for the Promotion of Science (JSPS)/ ; JP22bm0104001//Japan Agency for Medical Research and Development (AMED)/ ; JP23bm1223002h0002//Japan Agency for Medical Research and Development (AMED)/ ; JP23bm1123040//Japan Agency for Medical Research and Development (AMED)/ ; },
mesh = {Humans ; MicroRNAs/genetics/metabolism ; CRISPR-Cas Systems/genetics ; *Gene Expression Regulation ; Gene Editing/methods ; HEK293 Cells ; *Protein Processing, Post-Translational ; RNA, Messenger/genetics/metabolism ; },
abstract = {RNA switch is a synthetic RNA-based technology that controls gene expression in response to cellular RNAs and proteins, thus enabling cell type-specific gene regulation and holding promise for gene therapy, regenerative medicine, and cell therapy. However, individual RNA switches often lack the specificity required for practical applications due to low ON/OFF ratios and difficulty in finding distinct and single biomolecule targets. To address these issues, we present "split RNA switches" that integrate outputs from multiple RNA switches by exploiting protein splicing. We show that split RNA switches significantly improve the ON/OFF ratio of microRNA-responsive ON switch system by canceling leaky OFF level in human cells. Using this approach, we achieve efficient cell purification using drug-resistance genes based on endogenous microRNA profiles and CRISPR-mediated genome editing with minimal off-target-cell effects. Additionally, we demonstrate RNA-based synthetic circuits using split RNA switches to enable the detection of multiple microRNAs and proteins with logical operations. Split RNA switches highlight the potential of post-translational processing as a versatile and comprehensive strategy for advancing mRNA-based therapeutic technologies.},
}
@article {pmid40570069,
year = {2025},
author = {Speth, ZJ and Rehard, DG and Norton, PJ and Franz, AWE},
title = {Performance of two low-threshold population replacement gene drives in cage populations of the yellow fever mosquito, Aedes aegypti.},
journal = {PLoS genetics},
volume = {21},
number = {6},
pages = {e1011757},
doi = {10.1371/journal.pgen.1011757},
pmid = {40570069},
issn = {1553-7404},
mesh = {Animals ; *Aedes/genetics/virology ; CRISPR-Cas Systems/genetics ; *Gene Drive Technology/methods ; *Mosquito Vectors/genetics/virology ; Male ; Female ; *Yellow Fever/virology/transmission/genetics ; Promoter Regions, Genetic ; Mosquito Control/methods ; Animals, Genetically Modified ; },
abstract = {Aedes aegypti is the predominant vector for arboviruses including dengue, Zika, and chikungunya viruses, which infect over 100 million people annually. Mosquito population replacement in which arbovirus-susceptible mosquitoes in the field are replaced by laboratory-engineered refractory mosquitoes represents a novel genetic control measure to interrupt arboviral disease cycles. For this approach, the engineered mosquitoes need to harbor two genetic components: an antiviral effector construct which is linked to a gene drive (GD). We tested the performance of two single-locus CRISPR/Cas9 based GD for Ae. aegypti population replacement in small cage populations for up to 16 generations. Starting from a low release threshold of 1:9 GD bearing males to wild-type males, we observed two GD constructs in which Cas9 was expressed from two different germline promoters, nanos and zpg, to increase in frequency in all cage populations. By G16, an average of 72% and 82% of individuals from the zpg-GD and nanos-GD populations, respectively, harbored at least one GD copy with corresponding increases in allele frequencies. This indicated that the two single-locus, CRISPR/Cas9-based homing GD exhibited continuous super-Mendelian inheritance in populations of Ae. aegypti. Gene drive blocking indel (GDBI, a.k.a. "resistant alleles") frequency was measured for each discrete generation in pooled samples from the six populations harboring GD. We found that populations with Cas9 expression under control of the nanos-promoter accumulated GDBI at more than twice the rate of those populations harboring the zpg-promoter driven GD. Based on preexisting data sets for homing and GDBI frequencies in addition to the cage trial observations, the relative contributions of sex-specific homing rates, maternal Cas9 deposition and potential fitness effects were modeled in MGDrivE for both GD, further explaining their divergent performance. Our study demonstrates the feasibility of low-threshold, single-locus CRISPR/Cas9 based GD for Ae. aegypti population replacement.},
}
@article {pmid40488470,
year = {2025},
author = {Deng, Z and Weng, X and Tang, H and Zou, T and Zhou, X and Liu, H and Wen, P and Luo, G and Gan, T and He, J},
title = {Rapid and sensitive detection of Mycobacterium tuberculosis using the RPA/Cas12f1_ge4.1 system with fluorescence and lateral flow readouts.},
journal = {Microbiology spectrum},
volume = {13},
number = {7},
pages = {e0265224},
pmid = {40488470},
issn = {2165-0497},
mesh = {*Mycobacterium tuberculosis/isolation &amp; purification/genetics ; Humans ; *Tuberculosis/diagnosis/microbiology ; *Nucleic Acid Amplification Techniques/methods ; Sensitivity and Specificity ; Fluorescence ; CRISPR-Cas Systems ; *Molecular Diagnostic Techniques/methods ; Recombinases/genetics/metabolism ; DNA, Bacterial/genetics ; Limit of Detection ; },
abstract = {UNLABELLED: Tuberculosis remains a major global health threat, with existing detection methods often limited by efficiency and resource demands. Our previous PAM-dependent dsDNA Target-activated Cas12f1 Trans Reporter (PDTCTR) fluorescence sensing platform, while effective for PAM-dependent pathogen detection, was constrained by its reliance on specialized fluorescence equipment and lack of visual output, limiting its use in resource-limited settings. To overcome these limitations, we introduce an innovative RPA/CRISPR-Cas12f1_ge4.1 dual-mode system for rapid Mycobacterium tuberculosis detection. This system combines engineered Cas12f_ge4.1 with recombinase polymerase amplification (RPA), offering both fluorescent and lateral flow detection. It achieves high sensitivity with detection limits of 10 copies/µL (fluorescence) and 100 copies/µL (lateral flow), alongside 100% specificity. In clinical validation, compared with a commercial qPCR kit, the fluorescent and lateral flow approaches demonstrate sensitivities of 94.52% (69/73, 95% confidence interval [CI]: 85.84%-98.23%) and 90.41% (66/73, 95% CI: 80.67%-95.73%), respectively, while maintaining 100% (40/40, 95% CI: 89.09%-100%) specificity and high concordance (kappa values: 0.924 and 0.878). Detection is completed within 1 h, providing a rapid, sensitive, and specific solution for M. tuberculosis identification. This dual-mode capability represents a significant advancement in current tuberculosis diagnostics, enabling both sensitive laboratory confirmation and rapid point-of-care screening. Our versatile and efficient method promises to transform tuberculosis diagnostics, particularly in resource-constrained environments.<br><br>IMPORTANCE: Tuberculosis (TB) remains a significant global health challenge, demanding rapid and accurate detection for effective management. The innovative RPA/CRISPR-Cas12f1_ge4.1 dual-mode system represents a major advancement in TB diagnostics, offering highly sensitive and specific detection of Mycobacterium tuberculosis DNA. This adaptable system, incorporating both fluorescent and lateral flow detection modes, is designed for use in both advanced laboratories and resource-limited settings. Its high performance, rigorously validated through clinical trials, holds the potential to revolutionize TB diagnosis, particularly in high-burden, low-resource areas. By facilitating earlier treatment and enhancing control of TB transmission, this system could significantly contribute to global efforts in combating this persistent public health threat.},
}
@article {pmid40422178,
year = {2025},
author = {Siddiqui, FA and Chim-Ong, A and Wang, C and Miao, J and Cui, L},
title = {The PfK13 G533S mutation confers artemisinin partial resistance in multiple genetic backgrounds of Plasmodium falciparum.},
journal = {Antimicrobial agents and chemotherapy},
volume = {69},
number = {7},
pages = {e0016225},
doi = {10.1128/aac.00162-25},
pmid = {40422178},
issn = {1098-6596},
support = {U19AI181593/NH/NIH HHS/United States ; },
mesh = {*Plasmodium falciparum/genetics/drug effects ; *Artemisinins/pharmacology ; *Antimalarials/pharmacology ; *Drug Resistance/genetics ; *Protozoan Proteins/genetics ; Mutation/genetics ; Malaria, Falciparum/drug therapy/parasitology ; Humans ; CRISPR-Cas Systems ; },
abstract = {Mutations in the Plasmodium falciparum Kelch 13 (PfK13) protein are the key determinant of artemisinin partial resistance. While more than 200 PfK13 mutations have been identified in global parasite populations, only 13 have been validated to confer in vivo or in vitro artemisinin partial resistance. In the western Greater Mekong Subregion, the prevalence of the PfK13 G533S mutation has significantly increased in recent years. Field isolates carrying the PfK13 G533S mutation showed slower parasite clearance and higher day-3 positivity rates after artemisinin treatment, while culture-adapted isolates displayed significantly elevated ring-stage survival rates. Here, the PfK13 G533S mutation was introduced using CRISPR/Cas9 into four parasite strains: Dd2, 3D7, GB4, and F09N25 (a recent culture-adapted field isolate from the China-Myanmar border area). Across all four genetic backgrounds, the PfK13 G533S mutation conferred ring-stage survival rates of 12%-23% with a minimal fitness cost, explaining its rising prevalence in the region. In contrast, the PfK13 G533A mutation, sporadically detected in world P. falciparum populations, did not increase ring-stage survival rates when engineered into the 3D7 and Dd2 strains. These findings validate the PfK13 G533S mutation as a critical marker for artemisinin resistance surveillance and underscore the importance of monitoring its spread.},
}
@article {pmid40592986,
year = {2025},
author = {Elhamouly, NA and Atta, N and Liu, S and Tu, Y and Peng, D},
title = {A novel assay incorporating CRISPR with RPA in a single pot for visual and accurate detection of Aphelenchoides besseyi in soybean.},
journal = {Scientific reports},
volume = {15},
number = {1},
pages = {21217},
pmid = {40592986},
issn = {2045-2322},
mesh = {*Glycine max/parasitology ; *Nucleic Acid Amplification Techniques/methods ; Animals ; *Plant Diseases/parasitology ; CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; *Tylenchida/genetics/isolation &amp; purification ; },
abstract = {Aphelenchoides besseyi is a highly prevalent plant parasitic nematode which has a substantial impact and poses an economic risk to soybean cultivation, with a reported 2017 outbreak resulting in significant yield losses of up to 60%. Therefore, more effective control of this nematode depends on early and accurate nucleic acid detection. One of the promising detection approaches is to combine the CRISPR technology with the isothermal RPA. However, incorporating the RPA amplicon with the CRISPR ingredients in a single pot remains a significant challenge due to their incompatibility. In the current research, we propose a visual nucleic acid detection technique that takes less than thirty minutes and is highly sensitive for detecting A. besseyi. First, we conduct the RPA amplification, then we perform the CRISPR reaction using either a portable thermal cup or our body heat temperature. We tested this new assay on forty-four soybean samples exhibiting GSFR syndrome symptoms, and it effectively detected samples containing the A. besseyi. We designed three different ways for data collection and visualization to suit the requirements of various environments. Our findings confirm that the suggested new low-instrumentation portable single-pot RPA-CRISPR assay is durable, specific, and has strong nucleic acid sensitivity in the open field.},
}
@article {pmid40591069,
year = {2025},
author = {Anari, E and Anari, R and Pazoki-Toroudi, H},
title = {A decade of scientific advancements and collaborations on CRISPR-Cas9 application in cancer research: a bibliometric review.},
journal = {Discover oncology},
volume = {16},
number = {1},
pages = {1232},
pmid = {40591069},
issn = {2730-6011},
support = {25550//Iran University of Medical Sciences/ ; },
abstract = {The CRISPR-Cas system, a novel gene-editing technique, was a focus of research on cancer diagnosis and treatment during the last decade. A systematic review of the research trends and collaborations of the literature published in PubMed database between 2014 and 2024 resulted in 3241 papers. Bibliometric analysis and data visualization were performed using Excel, RStudio, and VOSviewer software. An increasing trend in the number of publications and citations has been observed since 2014, with a moderate fall after 2023. China and the USA have contributed the most publications and international collaborations, and high-income nations involved the highest contributions in the literature. The most productive institutes were Harvard Medical School and Dana-Farber Cancer Institute. Wang and Li contributed the maximum number of publications in this field. The prominent sources were Cancer Research, and Cancers. The application of CRISPR technique in breast and colorectal cancers has been frequently studied during the last decade. Future trials should explore the specificity and safety of CRISPR in various cancers.},
}
@article {pmid40590986,
year = {2025},
author = {K, MMP and Pagariya, MC and Jadhav, PR and Gawade, NS and Sarode, DK and Karkute, SG and Kardile, HB and Deshmukh, R and Penna, S and Kawar, PG},
title = {Advancing ornamental plant breeding through genomic technologies: opportunities, challenges, and future directions.},
journal = {Functional &amp; integrative genomics},
volume = {25},
number = {1},
pages = {140},
pmid = {40590986},
issn = {1438-7948},
mesh = {*Plant Breeding/methods ; *Genome, Plant ; Gene Editing ; *Genomics/methods ; CRISPR-Cas Systems ; *Plants/genetics ; },
abstract = {The ornamental plants constitute an important sector of horticulture industry, which are worth billions of dollars worldwide. There is a growing demand for new and improved cultivars and hence, breeders employ new tools and methods to address the problem of plant improvement. Recent advancements in Ornamental plant genomics have seen a great revolution due to new technologies of whole genome sequencing which have created previously unheard-of breeding program prospects. Research into gene regulation, genomic variations, genome evolution, and other biological processes are now aided by the use of complete genome sequencing data. The assembly of high-quality genomes for various ornamental species has facilitated the identification of genes controlling desirable traits such as flower color, shape, fragrance, biotic and abiotic stress resistance. The CRISPR/Cas9 based genome editing technology has offered immense scope for ornamental plant improvement through the enhancement of floral characteristics. Herein, we discuss how these genomic resources can be leveraged to improve breeding efficiency, accelerate the development of novel cultivars to augment the sustainability of the ornamental plant industry. This review aims to provide a viewpoint for the application of whole genome sequencing in ornamental plant breeding, highlighting the opportunities, challenges, and future prospects.},
}
@article {pmid40589866,
year = {2025},
author = {Chen, X and Zhang, S and Lin, S and Wang, S and Huang, M and Chen, S and Chen, S},
title = {A combination of recombinase polymerase amplification with CRISPR technology rapidly detects goose parvovirus with high accuracy and sensitivity.},
journal = {Frontiers in cellular and infection microbiology},
volume = {15},
number = {},
pages = {1566603},
pmid = {40589866},
issn = {2235-2988},
mesh = {Animals ; Sensitivity and Specificity ; *Parvoviridae Infections/diagnosis/veterinary/virology ; *CRISPR-Cas Systems ; *Nucleic Acid Amplification Techniques/methods ; *Recombinases/metabolism/genetics ; *Poultry Diseases/diagnosis/virology ; Geese/virology ; Ducks/virology ; *Parvovirinae/genetics/isolation &amp; purification ; *Parvovirus/genetics/isolation &amp; purification ; *Molecular Diagnostic Techniques/methods ; },
abstract = {BACKGROUND: Goose parvovirus (GPV) poses a significant threat to the waterfowl industry, necessitating reliable detection methods. However, conventional techniques are often time-consuming, equipment-dependent, or lack sufficient sensitivity for detecting early-stage infection. In contrast, emerging CRISPR/Cas12a-based systems offer a promising alternative for rapid, sensitive, and on-site diagnostics.<br><br>METHODS: We developed and optimized a recombinase polymerase amplification (RPA)-CRISPR/Cas12a assay targeting the conserved VP3 gene of GPV. The analytical and diagnostic performance of this assay was rigorously validated using plasmid standards and clinical specimens from both experimentally infected and field-collected ducklings.<br><br>RESULTS: Our developed assay combines RPA with CRISPR/Cas12a technology for rapid GPV nucleic acids detection. This method achieves a detection limit of 10 copies/&#x3bc;L of the VP3 gene within one hour, demonstrating high sensitivity and rapid turnaround. The assay exhibited exceptional specificity, with no cross-reactivity against other waterfowl viruses, and showed robust reproducibility, with intra- and inter-assay coefficients of variation consistently below 5.0%. Clinical validation using 42 field samples confirmed a diagnostic sensitivity of 100% and 95.5% specificity, showing superior performance to real-time quantitative PCR (qPCR) in both metrics. Furthermore, the assay supports flexible visual readouts using portable blue light transilluminators, facilitating on-site interpretation.<br><br>CONCLUSIONS: This study established a highly field-deployable RPA-CRISPR/Cas12a assay for rapid, visual detection of GPV with outstanding sensitivity and specificity. Its capability for instrument-free on-site diagnosis via blue light transillumination makes this approach particularly promising for resource-limited settings.},
}
@article {pmid40588792,
year = {2025},
author = {Ma, Q and Zhang, Y and Chen, L and Bao, Y and Guo, W and Feng, K and Huang, T and Cai, YD},
title = {Machine Learning-Driven Discovery of Essential Binding Preference in Anti-CRISPR Proteins.},
journal = {Proteomics. Clinical applications},
volume = {},
number = {},
pages = {e70013},
doi = {10.1002/prca.70013},
pmid = {40588792},
issn = {1862-8354},
support = {XDB38050200//Strategic Priority Research Program of Chinese Academy of Sciences/ ; XDA26040304//Strategic Priority Research Program of Chinese Academy of Sciences/ ; 2022YFF1203202//National Key R&amp;D Program of China/ ; GZNL2024A01003//Major Project of Guangzhou National Laboratory/ ; SRPG22-007//Self-Supporting Program of Guangzhou Laboratory/ ; 202002//Key Laboratory of Tissue Microenvironment and Tumor of Chinese Academy of Sciences/ ; ZR2022MC072//Shandong Provincial Natural Science Foundation/ ; 2024KCXTD081//Key Scientific Research Project of General Universities in Guangdong Province/ ; },
abstract = {PURPOSE: Anti-CRISPR (Acr) proteins can evade CRISPR-Cas immunity, yet their molecular determinants remain poorly understood. This study aimed to uncover key features driving Acr activity, thereby advancing both fundamental knowledge and the rational design of robust CRISPR-based tools.<br><br>EXPERIMENTAL DESIGN: We compiled a binary-encoded matrix of 761 InterPro-annotated domains and binding-site features for known Acr proteins. Seven feature ranking algorithms were applied to prioritize determinant features, and an incremental feature selection strategy, coupled with four distinct classifiers, was used to identify optimal subsets. Consensus key features were defined by intersecting the top subsets across all methods.<br><br>RESULTS: Key identified features include the DUF2829 domain, the Lambda repressor-like domain and Sulfolobus islandicus virus proteins, the Cro/C1-type helix-turn-helix domain, phage protein, and replication initiator A. These findings illuminate novel structural modules and regulatory motifs that underpin Acr inhibition.<br><br>CONCLUSIONS: This study provides critical theoretical support for deciphering Acr mechanisms and offers actionable insights for engineering next-generation CRISPR-Cas applications in clinical and biotechnological settings.<br><br>SUMMARY: The CRISPR system is a part of the antiviral immune defense initially discovered in bacteria and archaea. At present, the CRISPR system has become the cornerstone of genome editing technologies such as CRISPR-Cas9, widely used in clinical, agricultural, and biological research. Anti-CRISPR proteins are a group of proteins that inhibit the normal activity of CRISPR-Cas system in certain bacteria or archaea and avoid having the phages' genomes destroyed by the prokaryotic cells. The anti-CRISPR protein family has various components, but with similar functions to help exogenous DNA escape from the immune system. This study tried to uncover molecular mechanisms for anti-CRISPR proteins.},
}
@article {pmid40588529,
year = {2025},
author = {Park, BS and Lee, M and Kim, J and Kim, T},
title = {Perturbomics: CRISPR-Cas screening-based functional genomics approach for drug target discovery.},
journal = {Experimental &amp; molecular medicine},
volume = {},
number = {},
pages = {},
pmid = {40588529},
issn = {2092-6413},
support = {CAP23011-000//National Research Council of Science and Technology (National Research Council of Science &amp; Technology)/ ; 2E33131//Korea Institute of Science and Technology (KIST)/ ; RS-2024-00452719//National Research Foundation of Korea (NRF)/ ; },
abstract = {Despite more than two decades since the completion of the first draft of the Human Genome Project, a substantial proportion of human genes remain poorly characterized in terms of their functions. Functional genomics aims to elucidate the roles and interactions of genes and genetic elements, providing insights into their involvement in various biological processes. In this context, the perturbomics approach-a systematic analysis of phenotypic changes resulting from gene function modulation-offers valuable insights into the function of unannotated genes. With the advent of CRISPR-Cas-based genome and epigenome editing, CRISPR screens have become the method of choice for perturbomics studies, enabling the identification of target genes whose modulation may hold therapeutic potential for diseases such as cancer, cardiovascular disorders and neurodegeneration. These findings contribute to the development of targeted drug therapies and the design of gene and cell therapies for regenerative medicine. Here we highlight recent technical advances in CRISPR-based perturbomics, focusing on more physiologically relevant, single-cell-level analyses and their successful applications in discovering novel therapeutic strategies.},
}
@article {pmid40588516,
year = {2025},
author = {Malong, L and Roskosch, J and Hager, C and Fortin, JP and Schmucki, R and Callow, MG and Weile, C and Romeo, V and Patsch, C and Martin, S and Costa, M and Modrusan, Z and Villaseñor, R and Koller, E and Haley, B and Spang, A and Roudnicky, F},
title = {A CRISPR/Cas9 screen reveals proteins at the endosome-Golgi interface that modulate cellular anti-sense oligonucleotide activity.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {5378},
pmid = {40588516},
issn = {2041-1723},
mesh = {*Endosomes/metabolism ; *CRISPR-Cas Systems/genetics ; Humans ; *Golgi Apparatus/metabolism ; Animals ; Lysosomes/metabolism ; *Oligonucleotides, Antisense/metabolism/genetics ; Mice ; GTPase-Activating Proteins/metabolism/genetics ; HeLa Cells ; HEK293 Cells ; Adaptor Protein Complex 1/metabolism/genetics ; },
abstract = {Anti-sense oligonucleotides (ASOs) are modified synthetic single-stranded molecules with enhanced stability, activity, and bioavailability. They associate with RNA through sequence complementarity and can reduce or alter mRNA expression upon binding of splice site positions. To target RNA in the nucleus or cytoplasm, ASOs must cross membranes, a poorly understood process. We performed an unbiased CRISPR/Cas9 knockout screen with a genetic splice reporter to identify genes that can increase or decrease ASO activity, resulting in the most comprehensive catalog of ASO-activity modifier genes. Here we reveal distinct targets, including AP1M1 and TBC1D23, linking ASO activity to transport of cargo between the Golgi and endosomes. AP1M1 absence strongly increases ASO activity by delaying endosome-to-lysosome transport in vitro and in vivo. Prolonged ASO residence time in the endosomal system may increase the likelihood of ASO escape. This insight into AP1M1 role in ASO trafficking suggests a way for enhancing the therapeutic efficacy of ASOs by manipulating the endolysosomal pathways.},
}
@article {pmid40588042,
year = {2025},
author = {Long, R and Tang, D and Yang, T and Li, M and Lu, Y and Liu, W and Jiang, L},
title = {Advanced research and exploration of CRISPR technology in the field of directed evolution.},
journal = {Biotechnology advances},
volume = {},
number = {},
pages = {108633},
doi = {10.1016/j.biotechadv.2025.108633},
pmid = {40588042},
issn = {1873-1899},
abstract = {Directed molecular evolution is the key technology for obtaining enzymes, proteins, metabolic pathways, and other components of living organisms that have specific functions or desirable properties, which are indispensable in a variety of industrial and medical applications. Despite the success of traditional methods, they are often limited by low efficiency and the high cost of obtaining desired mutants. The advent of CRISPR technology has significantly advanced the field by enabling precise and efficient gene targeting, offering new prospects for directed evolution. This review provides a comprehensive overview of CRISPR tools and their applications in directed evolution, highlighting the principles, technological advancements, and specific applications of CRISPR-based mutation and screening platforms. We discuss the key findings from the use of CRISPR in enzyme and genome evolution, showcasing its ability to generate genetic diversity and select for improved phenotypes. The study underscores the unique value of CRISPR in directed evolution, particularly in its flexibility to target and edit various species' genomes, and its potential to accelerate the discovery of novel biomolecules with enhanced properties.},
}
@article {pmid40587425,
year = {2025},
author = {Li, X and Peng, Y and Xue, W and Liu, X and Luo, S and Wei, P and Wang, J},
title = {CRISPR-based Shuttle Cloning: A High-throughput Cloning Method.},
journal = {Journal of visualized experiments : JoVE},
volume = {},
number = {220},
pages = {},
doi = {10.3791/68503},
pmid = {40587425},
issn = {1940-087X},
mesh = {*Cloning, Molecular/methods ; Plasmids/genetics ; *Genetic Vectors/genetics ; *CRISPR-Cas Systems ; DNA/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {The development of genome-wide plasmid libraries using existing genomic repositories serves as a pivotal prerequisite for systematic functional characterization of genes across diverse biological processes. Current high-throughput methodologies for inter-vector DNA fragment transfer, however, necessitate PCR amplification of target sequences prior to cloning, rendering the generation of genome-scale plasmid collections technically demanding and time-intensive. By leveraging a CRISPRshuttle cassette, we developed a new high-throughput cloning method, CRISPR-based shuttle cloning (CRISPRshuttle cloning), which facilitates the transfer of many DNA fragments from donor plasmids sharing identical backbone sequences to a CRISPRshuttle-compatible vector without PCR amplification of the DNA fragments. Here, we present a protocol for CRISPRshuttle. This protocol involves two sequential test tube reactions prior to bacterial transformation. First, target DNA fragments are excised from donor plasmids by Cas9-mediated cleavage of their shared vector backbone sequence. Second, the excised DNA fragments are inserted into linearized CRISPRshuttle-compatible vectors through Gibson assembly. Our results demonstrate that the efficiency of CRISPRshuttle exceeds 94% and that two researchers can generate about 300 plasmids in 7 days using CRISPRshuttle. CRISPRshuttle facilitates efficient, adaptable, and cost-effective DNA fragment transfer between vectors, significantly streamlining genome-wide plasmid library generation.},
}
@article {pmid40587016,
year = {2025},
author = {Choudhary, A and Kumar, A and Munshi, A},
title = {Genetic variants in oncogenic miRNA and 3' untranslated region of tumor suppressor genes: emerging insight into cancer genetics.},
journal = {Medical oncology (Northwood, London, England)},
volume = {42},
number = {8},
pages = {303},
pmid = {40587016},
issn = {1559-131X},
support = {211610155703//University Grants Commission/ ; BT/PR45460/MED/12/952/2022//Department of Biotechnology, Ministry of Science and Technology, India/ ; },
mesh = {Humans ; *MicroRNAs/genetics ; *Genes, Tumor Suppressor ; *3' Untranslated Regions/genetics ; *Neoplasms/genetics ; *Breast Neoplasms/genetics ; *Genetic Variation ; Female ; Gene Expression Regulation, Neoplastic ; Oncogenes ; },
abstract = {The miRNAs are key regulators of post-transcriptional gene expression. These are associated with the different molecular mechanisms which are engaged in the pathogenesis of various cancers, including breast cancer. The tumor suppressor and oncogenic miRNAs have a significant impact on cell proliferation, metastasis, angiogenesis, and apoptotic pathways. Variation in oncogenic miRNA encoding and the 3' UTR of the tumor suppressor genes associated with the development and prognosis of the cancer is being explored. These genetic variants alter oncogenic miRNAs' stability, target recognition, and binding ability, thereby resulting in the dysregulation of their target tumor suppressor gene, leading to uncontrolled cell division, a significant hallmark of cancer. The current review has been compiled to explore the genetic variation reported in the oncogenic miRNA encoding and 3' UTR of their target tumor suppressor genes associated with the development of breast cancer and its progression, focusing on the associated molecular mechanisms. Further, an effort has been made to discuss the possible therapeutic strategies, especially anti-miR, RNA interference, CRISPR/Cas, and ASOs, that have the potential to restore the function of the dysregulated tumor suppressor as well as oncogenic miRNA encoding genes.},
}
@article {pmid40586829,
year = {2025},
author = {Hussain, MS and Maqbool, M and Arab, MM and Rana, AJ and Ashique, S and Khan, Y and Jakhmola, V and Gupta, G},
title = {Transforming Hemophilia Management: Lessons from Gene Therapy Clinical Trials.},
journal = {Molecular biotechnology},
volume = {},
number = {},
pages = {},
pmid = {40586829},
issn = {1559-0305},
abstract = {Gene therapy signifies a transformative revolution in hemophilia care, providing the possibility for sustained endogenous synthesis of coagulation factors and limiting the need for external factor supplementation. Preliminary experiments in hemophilia B via adeno-associated viral (AAV) vectors encountered constraints owing to immunological reactions and temporary translation. Progress in vector technology, particularly via self-complementary AAV innovation and codon-optimized mini-factor IX (FIX) concepts, has markedly improved transduction performance and prolonged FIX activity. Initial investigations have shown encouraging outcomes, with certain individuals sustaining consistent FIX expressions for more than 8 years; hence, decreasing yearly bleeding incidents and requiring preventive therapy. The development of gene therapy for hemophilia A has encountered substantial obstacles owing to the enormous size of the factor VIII (FVIII) gene. The recent experiments using AAV serotypes 5 (AAV5) vectors that encode B-domain-deleted FVIII constructs have shown sustained levels along with substantial decreases in hemorrhage incidents. Research has shown prolonged FVIII expression, with some individuals attaining almost normal coagulation efficiency. Phase III studies have validated long-term effectiveness and safety, with transient transaminase elevations being the most common adverse event. Notwithstanding these advancements, difficulties persist, including immunological reactions to vector capsids, hepatotoxicity, and unpredictability in translation levels. Innovative approaches including lentiviral vectors, gene-editing technologies, and novel customized connection strategies demonstrate possibilities for enhancing the effectiveness of gene therapy. Continuous clinical research and improvement in delivery systems will be crucial in substantiating gene therapy as a definitive approach for hemophilia.},
}
@article {pmid40586312,
year = {2025},
author = {Soderholm, A and Vunjak, M and de Almeida, M and Popitsch, N and Podvalnaya, N and Araguas-Rodriguez, P and Scinicariello, S and Nischwitz, E and Butter, F and Ketting, RF and Ameres, SL and Müller-McNicoll, M and Zuber, J and Versteeg, GA},
title = {ERH regulates type II interferon immune signaling through post-transcriptional regulation of JAK2 mRNA.},
journal = {Nucleic acids research},
volume = {53},
number = {12},
pages = {},
doi = {10.1093/nar/gkaf545},
pmid = {40586312},
issn = {1362-4962},
support = {10.55776/P36572//Austrian Science Fund/ ; 10.55776/P30415//Austrian Science Fund/ ; 10.55776/P30231//Austrian Science Fund/ ; 10.55776/P36945//Austrian Science Fund/ ; 10.55776/F79//Austrian Science Fund/ ; 10.55776/W1261//Austrian Science Fund/ ; //Austrian Academy of Sciences/ ; FFG-852936//Austrian Research Promotion Agency/ ; FFG-852936//&#xd6;sterreichische Forschungsf&#xf6;rderungsgesellschaft/ ; },
mesh = {*Janus Kinase 2/genetics/metabolism ; Humans ; *Interferon-gamma/metabolism/immunology/genetics ; Signal Transduction/genetics ; *RNA, Messenger/genetics/metabolism ; *RNA Processing, Post-Transcriptional ; Introns ; Immunity, Innate/genetics ; CRISPR-Cas Systems ; Serine-Arginine Splicing Factors/genetics ; HEK293 Cells ; RNA Splicing ; Animals ; },
abstract = {Type II interferon (IFNγ) signaling is essential for innate immunity and critical for effective immunological checkpoint blockade in cancer immunotherapy. Genetic screen identification of post-transcriptional regulators of this pathway has been challenging since such factors are often essential for cell viability. Here, we utilize our inducible CRISPR/Cas9 approach to screen for key post-transcriptional regulators of IFNγ signaling, and in this way, we identify ERH and the ERH-associated splicing and RNA export factors MAGOH, SRSF1, and ALYREF. Loss of these factors impairs post-transcriptional mRNA maturation of JAK2, a crucial kinase for IFNγ signaling, resulting in abrogated JAK2 protein levels and diminished IFNγ signaling. Further analysis highlights a critical role for ERH in preventing intron retention in AU-rich regions in specific transcripts, such as JAK2. This regulation is markedly different from previously described retention of GC-rich introns. Overall, these findings reveal that post-transcriptional JAK2 processing is a critical rate-limiting step for the IFNγ-driven innate immune response.},
}
@article {pmid40585259,
year = {2025},
author = {Zheng, Z and Yu, M and Ai, L and Wang, B and Lian, S and Liu, J and Li, L and Tsai, S and Kleinstiver, B and Ip, L},
title = {GenomePAM directs PAM characterization and engineering of CRISPR-Cas nucleases using mammalian genome repeats.},
journal = {Research square},
volume = {},
number = {},
pages = {},
doi = {10.21203/rs.3.rs-4552906/v1},
pmid = {40585259},
issn = {2693-5015},
abstract = {Characterizing the protospacer adjacent motif (PAM) requirements of different Cas enzymes is a bottleneck in the discovery of Cas proteins and their engineered variants in mammalian cell contexts. To overcome this challenge and to enable more scalable characterization of PAM preferences, we develop a method named GenomePAM that allows for direct PAM characterization in mammalian cells. GenomePAM leverages genomic repetitive sequences as target sites and does not require protein purification or synthetic oligos. GenomePAM uses a 20-nt protospacer that occurs ~16,942 times in every human diploid cell and is flanked by nearly random sequences. We demonstrate that GenomePAM can accurately characterize the PAM requirement of type II and type V nucleases, including the minimal PAM requirement of the near-PAMless SpRY and extended PAM for CjCas9. Beyond PAM characterization, GenomePAM allows for simultaneous comparison of activities and fidelities among different Cas nucleases on thousands of match and mismatch sites across the genome using a single gRNA and provides insight into the genome-wide chromatin accessibility profiles in different cell types.},
}
@article {pmid40585234,
year = {2025},
author = {Jain, P and Orosco, C and Rananaware, S and Huang, B and Hanna, M and Ahmadimashhadi, MR and Lewis, J and Baugh, M and Bodin, A and Flannery, S and Langue, I and Fang, Z and Karalkar, V and Meister, K},
title = {DNA-guided CRISPR/Cas12 for RNA targeting.},
journal = {Research square},
volume = {},
number = {},
pages = {},
pmid = {40585234},
issn = {2693-5015},
abstract = {CRISPR-Cas nucleases are transforming genome editing, RNA editing, and diagnostics but have been limited to RNA-guided systems. We present ΨDNA, a DNA-based guide for Cas12 enzymes, engineered for specific and efficient RNA targeting. ΨDNA mimics a crRNA but with a reverse orientation, enabling stable Cas12-RNA assembly and activating trans-cleavage without RNA components. ΨDNAs are effective in sensing short and long RNAs and demonstrated 100% accuracy for detecting HCV RNA in clinical samples. We discovered that ΨDNAs can guide certain Cas12 enzymes for RNA targeting in cells, enhancing mRNA degradation via ribosome stalling and enabling multiplex knockdown of multiple RNA transcripts. This study establishes ΨDNA as a robust alternative to RNA guides, augmenting CRISPR-Cas12's potential for diagnostic applications and for targeted RNA modulation in cellular environments.},
}
@article {pmid40581000,
year = {2025},
author = {Fraikin, N and Samuel, B and Burstein, D and Lesterlin, C},
title = {Strategies for zygotic gene expression during plasmid establishment.},
journal = {Plasmid},
volume = {},
number = {},
pages = {102754},
doi = {10.1016/j.plasmid.2025.102754},
pmid = {40581000},
issn = {1095-9890},
abstract = {Conjugative plasmids are key drivers of horizontal gene transfer and the spread of antimicrobial resistance. Their successful establishment in new hosts requires overcoming diverse bacterial defence mechanisms, such as restriction-modification systems, CRISPR-Cas systems, and the SOS response. Plasmids achieve this through a leading region-encoded zygotic program of anti-defence genes expressed early in conjugation. This program employs diverse strategies, including single-stranded promoters, repressed double-stranded promoters, and protein translocation. This review explores the diversity of these zygotic programs, the mechanisms underlying their timely regulation, and the array of anti-defence functions they encode. Further investigation of leading region genes is crucial for discovering novel counter-defence strategies and understanding their tailored regulation across diverse plasmid and bacterial species, ultimately enabling us to better understand and potentially manipulate plasmid transfer.},
}
@article {pmid40580284,
year = {2025},
author = {Manjunatha, C and Aditya, K and Prasannakumar, MK and Pramesh, D and Buella, P and Patil, SS and Venkateshbabu, G and Harish, J and Karan, R and Ramegowda, HV},
title = {Isothermal Amplification Techniques: An Emerging Tool for On-Site Detection of Phytopathogens in Field Conditions.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2943},
number = {},
pages = {47-64},
pmid = {40580284},
issn = {1940-6029},
mesh = {*Nucleic Acid Amplification Techniques/methods ; *Plant Diseases/microbiology/virology ; *Molecular Diagnostic Techniques/methods ; Point-of-Care Systems ; *Plants/microbiology ; CRISPR-Cas Systems ; },
abstract = {This chapter reviews various isothermal amplification techniques, which are alternative to PCR for detecting plant pathogens. These methods, including NASBA, SDA, LAMP, HDA, and RPA, amplify nucleic acids at a constant temperature, making them potentially more suitable for point-of-care (POC) applications. The review compares these techniques regarding their mechanisms, advantages, and limitations, highlighting their potential for rapid, cost-effective, and on-site plant disease diagnosis, including the integration of these techniques with CRISPR-Cas systems and lab-on-a-chip technologies.},
}
@article {pmid40580153,
year = {2025},
author = {Sharan, P and Kumar, BK and Kumar, A and Rai, P},
title = {Isothermal amplification for rapid and sensitive detection of hepatitis B virus: what we know so far? and way forward.},
journal = {Expert review of molecular diagnostics},
volume = {},
number = {},
pages = {},
doi = {10.1080/14737159.2025.2527634},
pmid = {40580153},
issn = {1744-8352},
abstract = {INTRODUCTION: Despite vaccine availability, Hepatitis B Virus (HBV) remains a major global health threat, especially in areas with low vaccination coverage and poor healthcare. Around 250 million people are chronically infected. Achieving the World Health Organisation's 2030 eradication goal is difficult, particularly due to diagnostic challenges in low-resource settings. While HBsAg detection is standard, low antigen levels and mutations hinder its reliability. Though molecular methods for HBV DNA offer high specificity, their cost and complexity limit use in under-resourced areas. Isothermal amplification emerges as a promising alternative, offering a more affordable, effective, and simplified approach to HBV detection, potentially improving access to timely diagnosis and care.<br><br>AREAS COVERED: This review evaluates the efficacy of various isothermal techniques to give insights into their benefits and limits, guiding researchers and clinicians in selecting the most effective assays for HBV molecular diagnostics.<br><br>EXPERT OPINION: RPA and PSR are the most promising isothermal assays for HBV detection in field settings. RPA is faster (&#x223c;20 min), works at low temperatures (37-42&#x202f;&#xb0;C), and uses stable lyophilized reagents, while PSR is simple, can be clubbed with visual detection, making both ideal for a low-resource setup.},
}
@article {pmid40580034,
year = {2025},
author = {Lee, H and Rashid, F and Hwang, J and London, JA and Fishel, R and Berger, JM and Myong, S and Ha, T},
title = {A high-throughput single-molecule platform to study DNA supercoiling effect on protein-DNA interactions.},
journal = {Nucleic acids research},
volume = {53},
number = {12},
pages = {},
doi = {10.1093/nar/gkaf581},
pmid = {40580034},
issn = {1362-4962},
support = {R35 GM122569/NH/NIH HHS/United States ; R35 CA263778/NH/NIH HHS/United States ; R37 GM071747/NH/NIH HHS/United States ; R35 CA263778/NH/NIH HHS/United States ; R01 GM149729/NH/NIH HHS/United States ; R01 CA067007/NH/NIH HHS/United States ; /HHMI/Howard Hughes Medical Institute/United States ; },
mesh = {*DNA, Superhelical/metabolism/chemistry/genetics ; DNA Gyrase/metabolism ; Plasmids/genetics/chemistry/metabolism ; Protein Binding ; *MutS DNA Mismatch-Binding Protein/metabolism/genetics ; *Single Molecule Imaging/methods ; Escherichia coli Proteins/metabolism ; Adenosine Triphosphate/metabolism ; CRISPR-Associated Protein 9/metabolism ; CRISPR-Cas Systems ; DNA/metabolism/chemistry ; },
abstract = {DNA supercoiling significantly influences DNA metabolic pathways. To examine its impact on DNA-protein interactions at the single-molecule level, we developed a highly efficient and reliable protocol to modify plasmid DNA at specific sites, allowing us to label plasmids with fluorophores and biotin. We then induced physiological levels of negative or positive supercoiling in these plasmids using gyrase or reverse gyrase, respectively. By comparing supercoiled DNA with relaxed circular DNA, we assessed the effects of supercoiling on CRISPR-Cas9 and the mismatch repair protein MutS. We found that negative DNA supercoiling exacerbates off-target effects in DNA unwinding by Cas9. For MutS, we observed that both negative and positive DNA supercoiling enhance the binding interaction between MutS and a mismatched base pair but do not affect the rate of ATP-induced sliding clamp formation. These findings not only underscore the versatility of our protocol but also open new avenues for exploring the intricate dynamics of protein-DNA interactions under the influence of supercoiling.},
}
@article {pmid40532220,
year = {2025},
author = {Lu, Q and Ye, C and Mao, W and Zeng, F and Liu, Z and Chen, R and Cheng, X and Gao, Y and Wang, M and Liu, M and Tang, S and Song, Y},
title = {Targeting Senescent Alveolar Type 2 Cells with a Gene-Editable FePt Dual-Atom Catalyst for Mitigating Idiopathic Pulmonary Fibrosis.},
journal = {ACS nano},
volume = {19},
number = {25},
pages = {23162-23176},
doi = {10.1021/acsnano.5c04686},
pmid = {40532220},
issn = {1936-086X},
mesh = {*Idiopathic Pulmonary Fibrosis/drug therapy/pathology/metabolism/genetics ; *Cellular Senescence/drug effects ; Animals ; Mice ; Humans ; Hyaluronic Acid/chemistry ; Gene Editing ; *Alveolar Epithelial Cells/drug effects/metabolism/pathology ; *Platinum/chemistry/pharmacology ; CRISPR-Cas Systems ; Catalysis ; *Iron/chemistry ; Mice, Inbred C57BL ; },
abstract = {Idiopathic pulmonary fibrosis (IPF) remains an age-related, fatal, incurable, epithelial-driven fibrotic lung disease despite the availability of approved antifibrotic drugs. The medical need for effective antipulmonary fibrotic therapies is thus very high. A promising therapeutic intervention for IPF is to target key cellular senescence processes in alveolar type 2 (AT2) cells. Herein, we introduce an inhalable gene-editable nanoplatform, comprising a CRISPR-Cas9 gene-editing system linked to a core FePt diatomic catalyst, encapsulated within a biocompatible hyaluronic acid (HA) surface layer (FePtR@HA). The FePt diatomic site facilitates H2O2 bridge adsorption, enabling efficient O-O bond cleavage and rapid catalytic conversion. The strong Fe-Pt interaction modulates the metal's d-band center, optimizing the adsorption of oxygen-containing intermediates. This precise regulation efficiently clears ROS, delivering robust antioxidant and antisenescence effects to AT2 cells. Simultaneously, the CRISPR-Cas9 gene editing system knocks down the pro-aging gene KAT7, reducing senescence-associated secretory phenotype (SASP) factors and further reversing AT2 cell senescence. Additionally, we demonstrated the antifibrotic efficacy of FePtR@HA in a lung-on-a-chip model, where it reprogrammed the fibrotic microenvironment, prevented myofibroblast recruitment to AT2 cells. Moreover, FePtR@HA showed satisfactory results in IPF mouse models, alleviating fibrosis and presenting a highly promising approach to combat the progression of IPF.},
}
@article {pmid40517335,
year = {2025},
author = {Wang, Q and Yang, S and Chen, X and Song, S and Wu, Y and Li, Y and Meng, X and Dai, L and Yang, Z and Wu, H and Xia, J and Xu, J},
title = {Mutually Activated Dual-Exponential Amplification DNA Machine Enhances Robust CRISPR/Cas12a Feedback Propagation for Ultrasensitive miRNA Detection.},
journal = {Analytical chemistry},
volume = {97},
number = {25},
pages = {13504-13513},
doi = {10.1021/acs.analchem.5c01953},
pmid = {40517335},
issn = {1520-6882},
mesh = {*MicroRNAs/analysis/genetics/blood ; Humans ; *CRISPR-Cas Systems ; *Nucleic Acid Amplification Techniques/methods ; *DNA/genetics/chemistry ; *Breast Neoplasms/diagnosis/genetics ; Limit of Detection ; Female ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {Breast cancer (BC) remains a critical global health challenge, necessitating ultrasensitive methods for detecting biomarkers such as miR-155, a key regulator in BC progression. Here, we present a mutually activated dual-exponential amplification DNA machine (MADEA-DNA machine) for ultrasensitive miR-155 detection. This system integrates exponential rolling circle amplification (E-RCA) and autocatalytic incremental strand displacement amplification (AI-SDA), driven by a bidirectional activation mechanism. Target miR-155 initiates E-RCA via a functional primer probe (FPP) or AI-SDA through a functional hairpin probe (FHP), with amplification products cross-activating the counterpart system to establish a self-reinforcing loop. The resultant amplicons further activate CRISPR/Cas12a, enabling the trans-cleavage of fluorescent reporters for signal amplification. The MADEA-DNA machine achieves a detection limit of 1.26 fM, with a dynamic range spanning 5 fM-10 nM, and demonstrates exceptional specificity against mismatched and nontarget miRNAs. Validation in human serum revealed significantly elevated miR-155 levels in BC patients versus healthy donors, corroborated by qRT-PCR. This system combines machine-like operational efficiency, dual-amplification synergy, and CRISPR-enhanced sensitivity, offering a robust platform for liquid biopsy applications in early BC diagnostics.},
}
@article {pmid40511702,
year = {2025},
author = {Sun, X and Li, X and Jiang, H and Shan, Y and Zhou, S and Wang, Z},
title = {Accurate Diagnosis of Colorectal Cancer Using a Combination of Lectin-Induced Recombinase Polymerase Amplification and CRISPR/Cas12a Assay on a Point-of-Care Testing Platform with Deep Learning Assistant.},
journal = {Analytical chemistry},
volume = {97},
number = {25},
pages = {13086-13094},
doi = {10.1021/acs.analchem.5c00468},
pmid = {40511702},
issn = {1520-6882},
mesh = {*Colorectal Neoplasms/diagnosis ; Humans ; *CRISPR-Cas Systems ; Animals ; *Point-of-Care Testing ; Mice ; *Deep Learning ; *Nucleic Acid Amplification Techniques/methods ; *Lectins/metabolism/chemistry ; *Recombinases/metabolism ; Exosomes/metabolism ; Cell Line, Tumor ; Limit of Detection ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {Specific glycosylation patterns on exosome surfaces represent novel diagnostic biomarkers for cancer liquid biopsy. Lectins can induce exosome aggregation through multiple bindings with exosomal glycoproteins. In this work, we developed a one-pot lectin-induced recombinase polymerase amplification (RPA) and CRISPR/Cas12a-mediated cleavage assay (LI-RPA-CRISPR/Cas12a) for diagnosing colorectal cancer (CRC) through the interactions of abundant α-fucose residues on CRC cell-derived exosome surfaces with Ulex Europaeus Agglutinin I (UEA-I). The combination of a homemade portable isothermal amplification device, the as-proposed LI-RPA-CRISPR/Cas12a exhibits a wide detection range from 2 × 10[6] to 1 × 10[2] extracellular vehicles (EVs) μL[-1] with a visual limit of detection (LOD) as low as 1.0 × 10[2] EVs μL[-1], and has been successfully utilized to dynamically monitor the progression of tumors in mice-bearing SW480 CRC subtype at an early stage. After integration with a long short-term memory (LSTM) deep learning model, the LI-RPA-CRISPR/Cas12a achieves accurate diagnosis of primary colorectal cancer with a drop of blood through a smartphone-based data analysis application, reaching an accuracy of 95% in 100 clinical samples. This rapid, sensitive, and user-friendly approach provides a promising platform for point-of-care testing (POCT) diagnosis of CRC, enabling early detection and monitoring of disease progression through a minimally invasive liquid biopsy.},
}
@article {pmid40491322,
year = {2025},
author = {Cao, J and Guo, Z and Xu, X and Li, P and Fang, Y and Deng, S},
title = {Advances in CRISPR-Cas9 in lineage tracing of model animals.},
journal = {Animal models and experimental medicine},
volume = {8},
number = {6},
pages = {1004-1022},
pmid = {40491322},
issn = {2576-2095},
support = {22SH19//Institute of Laboratory Animal Sciences, Chinese Academy of Medical Sciences and Comparative Medicine Center, Peking Union Medical College, Collaborative Innovation Program of the Chinese Academy of Sciences/ ; 2023-PT180-01//Non-profit Central Research Institute Fund of Chinese Academy of Medical Sciences/ ; },
mesh = {Animals ; *CRISPR-Cas Systems ; *Gene Editing/methods ; Humans ; *Cell Lineage/genetics ; *Models, Animal ; Mice ; Swine ; Zebrafish ; *Cell Tracking/methods ; },
abstract = {Cell lineage tracing is a key technology for describing the developmental history of individual progenitor cells and assembling them to form a lineage development tree. However, traditional methods have limitations of poor stability and insufficient resolution. As an efficient and flexible gene editing tool, CRISPR-Cas9 system has been widely used in biological research. Furthermore, CRISPR-Cas9 gene editing-based tracing methods can introduce fluorescent proteins, reporter genes, or DNA barcodes for high-throughput sequencing, enabling precise lineage analysis, significantly improving precision and resolution, and expanding its application range. In this review, we summarize applications of CRISPR-Cas9 system in cell lineage tracing, with special emphasis on its successful applications in traditional model animals (e.g., zebrafish and mice), large animal models (pigs), and human cells or organoids. We also discussed its potential prospects and challenges in xenotransplantation and regenerative medicine.},
}
@article {pmid40481182,
year = {2025},
author = {Yu, L and Liu, Y and Lin, X},
title = {Transitioning from native to synthetic receptors: broadening T-cell engineering and beyond.},
journal = {Cellular &amp; molecular immunology},
volume = {22},
number = {7},
pages = {712-729},
pmid = {40481182},
issn = {2042-0226},
support = {82341212//National Natural Science Foundation of China (National Science Foundation of China)/ ; 82293664//National Natural Science Foundation of China (National Science Foundation of China)/ ; },
mesh = {Humans ; *T-Lymphocytes/immunology ; Animals ; *Receptors, Antigen, T-Cell/genetics/immunology ; Gene Editing ; *Immunotherapy, Adoptive/methods ; *Cell Engineering/methods ; *Receptors, Chimeric Antigen/genetics/immunology ; CRISPR-Cas Systems ; *Receptors, Artificial/genetics/immunology ; },
abstract = {T-cell immunotherapy has progressed rapidly, evolving from native T-cell receptor biology to the development of innovative synthetic receptors that extend therapeutic applications beyond cancer. This review explores engineering strategies, ranging from natural TCRs to synthetic receptors, that increase T-cell activation and therapeutic potential. We begin by highlighting the foundational role of native receptors in the T-cell response, emphasizing how these structural and functional insights inform the design of next-generation synthetic receptors. Comparisons between CAR and TCR-like synthetic receptors underscore their respective advantages in specificity, efficacy, and safety, as well as potential areas for further improvement. In addition, gene editing technologies such as CRISPR-Cas9 enable precise modifications to the T-cell genome, enhancing receptor performance and minimizing immunogenic risks. In addition to tumors, these engineered T cells can be directed against viral infections, autoimmune disorders, and other diseases. We also explore advanced strategies that engage multiple immune cell types to achieve synergistic, durable responses. By demonstrating how native and synthetic receptors collectively drive innovation, this review aims to inspire new research directions and ultimately expand the scope of T-cell engineering for universal therapeutic applications.},
}
@article {pmid40434897,
year = {2025},
author = {Chen, L and Chen, X and Kashina, A},
title = {Amino acid-level differences in alpha-tubulin sequences are uniquely required for meiosis.},
journal = {Molecular biology of the cell},
volume = {36},
number = {7},
pages = {ar89},
doi = {10.1091/mbc.E24-11-0529},
pmid = {40434897},
issn = {1939-4586},
mesh = {Schizosaccharomyces/metabolism/genetics ; *Meiosis/physiology/genetics ; *Tubulin/metabolism/genetics ; Schizosaccharomyces pombe Proteins/metabolism/genetics ; Amino Acid Sequence ; CRISPR-Cas Systems ; Protein Isoforms/metabolism/genetics ; Amino Acids/metabolism/genetics ; Spores, Fungal/metabolism ; Spindle Apparatus/metabolism ; },
abstract = {Members of the tubulin gene family members are essential components of the cytoskeleton; however, functional diversity of tubulin isoforms is poorly understood. Here, we addressed this question using Schizosaccharomyces pombe as a model system. These yeast encode two α-tubulins, nda2 and atb2, that are very similar at the amino acid level but differ in their roles in organism's survival: nda2 deletion is lethal, while lack of atb2 does not interfere with cell viability. Using CRISPR-Cas9 gene editing, we generated a yeast strain expressing atb2 amino acid sequence utilizing nda2 codon usage in the native nda2 locus. Such nda2-coded atb2 (NCA) yeast, unlike nda2 knockout, were viable and displayed no visible abnormalities in vegetative life cycle. Instead, they displayed strong impairments in sporulation and meiosis, linked to altered balance of several spindle proteins. Our data indicate that nda2 protein is uniquely required for normal meiosis, and identify novel protein- and nucleotide-level determinants driving functional distinction between closely related tubulin isoforms.},
}
@article {pmid40378737,
year = {2025},
author = {Zheng, H and Guo, J and Wang, H and Wang, Y and Zhou, W and Liu, K and Ma, Q and Qi, Y},
title = {A zeolitic imidazolate framework-90 enhanced ultrasensitive ATP sensing platform with HCR and CRISPR-Cas12a dual signal amplification for live bacteria detection.},
journal = {Journal of hazardous materials},
volume = {494},
number = {},
pages = {138612},
doi = {10.1016/j.jhazmat.2025.138612},
pmid = {40378737},
issn = {1873-3336},
mesh = {*Adenosine Triphosphate/analysis ; *Zeolites/chemistry ; *Imidazoles/chemistry ; CRISPR-Cas Systems ; *Staphylococcus aureus/isolation &amp; purification ; *Biosensing Techniques/methods ; Aptamers, Nucleotide/chemistry ; Limit of Detection ; Nucleic Acid Hybridization ; *Metal-Organic Frameworks/chemistry ; },
abstract = {Bacteria are prevalent environmental pollutants. Live bacteria can proliferate and spread under appropriate conditions, presenting higher risks compared to non-viable counterparts. However, detecting live bacteria remains a challenge. Adenosine triphosphate (ATP), an energy currency in organisms, offers a reliable biomarker for the live bacteria sensing. Herein, we developed a zeolitic imidazolate framework-90 (ZIF-90) enhanced ATP sensing platform to detect live bacteria. The nanosystem which based on ZIF-90, encapsulating the DNA decorated magnetic beads (MB@S1) through self-assembly. When the S. aureus aptamers on ZIF-90 bonded to bacteria, the skeleton structure of ZIF-90 was disrupted by ATP leakage from live bacteria, leading to the release of MB@S1. Then, the MB@S1 initiated the hybridization chain reaction (HCR) and they were transduced by CRISPR-Cas12a to amplify the signal twice. The proposed ZIF-90 blocking sensing strategy (ZIF-90 strategy) exhibited remarkable sensitivity with the limit of detection (LOD) down to 0.223 pM for ATP detection, about 500 folds lower than the traditional ATP aptamer blocking sensing strategy (aptamer strategy). Furthermore, we used a smartphone for on-site analysis, realizing the quantification of live S. aureus with the LOD of 2.0 CFU/mL. Therefore, the approach possessed great application potential for public health, environment monitoring, bioanalysis and food safety.},
}
@article {pmid40299756,
year = {2025},
author = {An, Y and Wang, SQ and Jia, XY and Jiao, X and Qu, MQ and Dong, Y and Wang, ZY and Ma, ZY and Yang, S and Han, X and Huang, LC and Chen, NN and Jiang, C and Lu, MZ and Dai, JF and Zhang, J},
title = {Bioengineered poplar fibres via PagGLR2.8 editing: A synergistic design for high-performance biocomposites.},
journal = {Plant biotechnology journal},
volume = {23},
number = {7},
pages = {2824-2838},
pmid = {40299756},
issn = {1467-7652},
support = {32371902//National Natural Science Foundation of China/ ; 51903222//National Natural Science Foundation of China/ ; 2021YFD2200205//National Key Research and Development Program of China/ ; 2021C02070-1//the Key Scientific and Technological Grant of Zhejiang for Breeding New Agricultural Varieties/ ; 2018FR013//the research foundation of Zhejiang A&amp;F University/ ; //Zhejiang students' technology and innovation program/ ; },
mesh = {*Populus/genetics/metabolism ; Plants, Genetically Modified/genetics ; Polyesters ; Gene Editing ; *Plant Proteins/genetics/metabolism ; CRISPR-Cas Systems ; Bioengineering ; },
abstract = {The urgent need to replace petroleum-derived materials with sustainable alternatives drives innovation at the nexus of plant biotechnology and materials science. Here, we engineered Populus alba × P. glandulosa '84 K' through CRISPR-Cas9-mediated knockout of PagGLR2.8, a glutamate receptor gene regulating vascular development, to investigate its role in fibre biosynthesis and composite performance. Knockout of PagGLR2.8 improved the quality of poplar fibre by altering the structure and development mode of poplar vascular tissue. Our study established the relationship between fibre quantity and structure and the performance of polylactic acid (PLA) composites. The mechanical and fire-resistance properties of these transgenic plant fibres/PLA composites significantly outperformed those of pure PLA, demonstrating the potential of phloem fibres to reinforce toughened composites. Notably, we also evaluated flammability and dripping behaviours, with findings indicating that our optimised fibre/PLA composites exhibit superior strengths, modulus, fire resistance, and anti-dripping, surpassing those of PLA. This research unveils a groundbreaking approach to regulating composite properties through genetic manipulation and highlights the promising potential of plant-derived materials in enriching forest resources and advancing the sustainable utilisation of poplar fibres and polymers.},
}
@article {pmid39397560,
year = {2025},
author = {Gou, S and Liu, Y and Li, Q and Yang, J and Qiu, L and Zhao, Y},
title = {CRISPR/Cas12 System-Based Assay for Rapid, Sensitive Detection of Rotavirus in Food Samples.},
journal = {Foodborne pathogens and disease},
volume = {22},
number = {7},
pages = {459-466},
doi = {10.1089/fpd.2024.0078},
pmid = {39397560},
issn = {1556-7125},
mesh = {*Rotavirus/isolation &amp; purification/genetics ; *CRISPR-Cas Systems ; *Nucleic Acid Amplification Techniques/methods ; Sensitivity and Specificity ; *Food Microbiology/methods ; *Molecular Diagnostic Techniques/methods ; Humans ; *Food Contamination/analysis ; RNA, Viral/genetics ; },
abstract = {Foodborne viruses have become an important threat to food safety and human health. Among the foodborne viruses, group A rotavirus is the most important pathogen of diarrhea in autumn and winter. The field detection of rotavirus is crucial for the early control of infection and patient management. Quantitative real-time reverse transcription-polymerase chain reaction is the most widely used in virus detection. However, the technique relies on high-cost instruments and trained personnel, which limit its use in field detection. In this study, we developed accurate, realizable, and simple detection methods by combining optimized CRISPR (clustered regularly interspaced short palindromic repeats) Cas12 and reverse transcription loop-mediated isothermal amplification (RT-LAMP) (reverse transcription loop-mediated isothermal amplification) to reduce the requirements for temperature control and costly real-time fluorescence polymerase chain reaction instruments. We investigated two nucleic acid detection systems combining RT-LAMP with CRISPR Cas12a and RT-LAMP with CRISPR Cas12b and compared them with reverse transcription-quantitative polymerase chain reaction. The resulting detection system only needs a reaction temperature and in single tube to react for 60 min with the detection sensitivity of 38 copies/μL. Overall, this study developed an innovative method for the rapid detection of rotavirus in food samples, which will help to effectively identify food contaminated by pathogens and prevent human infections and economic losses caused by disease outbreaks.},
}
@article {pmid40579649,
year = {2025},
author = {Liu, ZG and Wang, N and Wang, WJ and Huang, L and Chi, SS and Nie, YX and Yuan, MS and Sun, YR and Mu, YL and Li, K},
title = {Transcriptome analysis reveals no obvious unintended effects in the spleen of CRISPR/Cas9-mediated CD163 and pAPN double-knockout pigs.},
journal = {Functional &amp; integrative genomics},
volume = {25},
number = {1},
pages = {139},
pmid = {40579649},
issn = {1438-7948},
support = {2022ZD04020//Biological Breeding-National Science and Technology Major Project/ ; CAAS-CSAB-202401//Innovation Program of Chinese Academy of Agricultural Sciences/ ; 202102//Outstanding Talents Training Fund in Shenzhen/ ; },
mesh = {Animals ; *CRISPR-Cas Systems ; CD163 Antigen ; *Antigens, Differentiation, Myelomonocytic/genetics/metabolism ; *Receptors, Cell Surface/genetics/metabolism ; *Spleen/metabolism ; Swine/genetics ; *Antigens, CD/genetics/metabolism ; *Transcriptome ; Gene Editing ; Gene Knockout Techniques ; Gene Expression Profiling ; Membrane Proteins ; },
abstract = {Gene editing provides powerful tools for farm animal breeding. Our group previously obtained CD163/pAPN double-knockout (DKO) pigs via CRISPR/Cas9 and somatic cell nuclear transfer. These pigs are not only resistant to three infectious viruses but also maintain normal production performance. However, unintended effects of CRISPR/Cas9 tools may pose potential risks to animal well-being or safety. This study aimed to characterize the differences in splenic gene expression between wild-type (WT) and DKO pigs, providing a basis for safety evaluation of gene-edited animals. A comprehensive transcriptional panorama reflected considerable congruence in the aggregate gene expression profiles of the DKO and WT pigs. Comparisons between 35-day-old and 11-month-old DKO pigs and their WT equivalents revealed 225 and 242 differentially expressed genes (DEGs), respectively, a count significantly lower than that of the DEGs in the disparate developmental stage comparison groups. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses revealed that the majority of DEGs between DKO and WT pigs correlated with the biological functions of CD163 and pAPN, without any alterations in the expression of tumor suppressor genes in DKO pigs. This revealed a less pronounced effect of dual gene editing on the gene expression profile of porcine spleens than the effect of animal maturation, with no evident unanticipated consequences observed in DKO pigs.},
}
@article {pmid40579133,
year = {2025},
author = {Mo, Y and Shu, Y and Mo, Y and Liu, J and Xu, O and Deng, H and Wang, Q},
title = {[CRISPR-Cas9-mediated CDC20 gene knockout inhibits cervical cancer cell proliferation, invasion and metastasis].},
journal = {Nan fang yi ke da xue xue bao = Journal of Southern Medical University},
volume = {45},
number = {6},
pages = {1200-1211},
doi = {10.12122/j.issn.1673-4254.2025.06.09},
pmid = {40579133},
issn = {1673-4254},
mesh = {Humans ; *Uterine Cervical Neoplasms/pathology/genetics/metabolism ; Female ; *Cdc20 Proteins/genetics ; *Cell Proliferation ; Animals ; Cell Movement ; Neoplasm Invasiveness ; Apoptosis ; Mice, Nude ; beta Catenin/metabolism ; *CRISPR-Cas Systems ; Mice ; Cell Line, Tumor ; Gene Knockout Techniques ; Neoplasm Metastasis ; },
abstract = {OBJECTIVES: To study the effect of CDC20 knockdown on proliferation, migration and invasion of cervical cancer cells and its underlying mechanism.<br><br>METHODS: CDC20 expression in cervical cancer tissues was analyzed using the TCGA database, and the protein expressions of CDC20 and &#x3b2;-Catenin in clinical specimens of cervical cancer and adjacent tissues were detected using immunohistochemistry. A dual target sgRNA2&amp;7 sequence for CDC20 gene was designed for CDC20 gene knockdown in cervical cancer C33A cells using CRISPR/Cas9 technology, and CDC20 mRNA and protein expression levels in the transfected cells were detected using qRT-PCR and Western blotting. The changes in proliferation, cell cycle, apoptosis, migration and invasiveness of the transfected cells were evaluated using colony-forming assay, fluorescence activated cell sorting (FACS) and Transwell assay. In the animal experiment, na&#xef;ve C33A cells and the cells with CDC20 knockdown were injected subcutaneously into the left and right axillae of nude mice (n=5) to observe tumor growth. The expressions of CDC20 and &#x3b2;-Catenin proteins in transfected cells and the xenograft were analyzed using Western blotting, and their interaction was confirmed by co-immunoprecipitation (CoIP) and immunofluorescence co-localization assays.<br><br>RESULTS: Cervical cancer tissues expressed significantly higher CDC20 and &#x3b2;&#x2011;Catenin levels than the adjacent tissues. C33A cells with CDC20 knockdown showed reduced proliferation, increased apoptosis, and lowered migration and invasion abilities. CDC20 knockdown significantly suppressed the growth of C33A cell xenograft in nude mice, and the tumor-bearing mice did not exhibit obvious body mass changes. CDC20 and &#x3b2;-Catenin levels were both significantly lowered in C33A cells with CDC20 knockdown. Co-immunoprecipitation and co-localization assays confirmed the interaction between CDC20 and &#x3b2;&#x2011;Catenin.<br><br>CONCLUSIONS: CDC20 is highly expressed in cervical cancer tissues, and CDC20 knockdown can suppress proliferation, invasion, and metastasis while enhancing apoptosis of C33A cells, which is closely related with the regulation of the Wnt/&#x3b2;-Catenin signaling pathway.},
}
@article {pmid40578341,
year = {2025},
author = {Mahler, M and Cui, L and Smith, LM and Wandera, KG and Dietrich, O and Mayo-Muñoz, D and Balamkundu, S and Lee, ME and Ye, H and Liu, CF and Wu, J and Mathew, J and Dubrulle, J and Malone, LM and Jackson, SA and Fairbanks, AJ and Dedon, PC and Brouns, SJJ and Fineran, PC},
title = {Phage arabinosyl-hydroxy-cytosine DNA modifications result in distinct evasion and sensitivity responses to phage defense systems.},
journal = {Cell host &amp; microbe},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.chom.2025.06.005},
pmid = {40578341},
issn = {1934-6069},
abstract = {Bacteria encode diverse anti-phage systems, such as CRISPR-Cas and restriction modification (RM), which limit infection by targeting phage DNA. We identified a DNA modification in phages, i.e., 5-arabinosyl-hydroxy-cytosine (5ara-hC), which adds arabinose to cytosines via a hydroxy linkage and protects phage from DNA targeting. The hydroxy linkage was common among arabinoslyated phages, with some arabinosylated phages encoding arabinose-5ara-hC transferases (Aat) that add a second or third arabinose to DNA. DNA arabinosylation enables evasion from DNA-targeting type I CRISPR-Cas and type II RM systems. However, arabinosylated phages remain sensitive to RNA-targeting CRISPR-Cas (type III and VI) and promiscuous type IV restriction endonucleases. 5ara-hC enables evasion of glycosylase defenses that target phages with glucosylated hydroxymethyl cytosines, and 5ara-ara-hC protects against some defenses capable of targeting 5ara-hC-modified phages. Collectively, this work identifies DNA modifications that enable phages to evade multiple defenses yet remain vulnerable to some systems that target RNA or modified nucleobases.},
}
@article {pmid40575705,
year = {2025},
author = {Chen, S and Liu, Z and Lo, CH and Wang, Q and Ning, K and Zhang, Q and Zhao, J and Shen, Y and Sun, Y},
title = {Gene therapy for ocular hypertension using hfCas13d-mediated mRNA targeting.},
journal = {PNAS nexus},
volume = {4},
number = {6},
pages = {pgaf168},
pmid = {40575705},
issn = {2752-6542},
abstract = {Glaucoma is a major global cause of irreversible vision loss. It is marked by elevated intraocular pressure (IOP) and the loss of retinal ganglion cells (RGC). While there are medical and surgical therapies for glaucoma aiming to reduce aqueous humor production or enhance its drainage, these treatments are often inadequate for effectively managing the disease. In this study, we developed a targeted therapy for glaucoma by knocking down two genes associated with aqueous humor production (aquaporin 1 [AQP1] and carbonic anhydrase type 2 [CA2]) using Cas13 RNA editing systems. We demonstrate that hfCas13d-mediated knockdown of AQP1 and CA2 significantly lowers IOP in wild-type mice and in a corticosteroid-induced glaucoma mouse model. We show that the lowered IOP results from decreasing aqueous production without affecting the outflow facility; this treatment also significantly promotes RGC survival as compared with untreated control groups. Therefore, CRISPR-Cas-based gene editing may be an effective treatment to lower IOP for glaucomatous optic neuropathy.},
}
@article {pmid40575544,
year = {2025},
author = {Choi, JH and Yoon, J and Chen, M and Shin, M and Goldston, LL and Lee, KB and Choi, JW},
title = {CRISPR/Cas-Based Nanobiosensor Using Plasmonic Nanomaterials to Detect Disease Biomarkers.},
journal = {Biochip journal},
volume = {19},
number = {2},
pages = {167-181},
pmid = {40575544},
issn = {1976-0280},
abstract = {The development of clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein (Cas) technology (CRISPR/Cas) as a gene-editing tool has the potential to revolutionize nucleic acid analysis. Recently, CRISPR/Cas systems have demonstrated considerable promise in the development of biosensors for the detection of essential disease biomarkers because they exhibit nonspecific collateral cleavage properties upon target sequence recognition. However, the CRISPR/Cas-based biosensors developed thus far have limitations, such as complicated steps, low sensitivity, low selectivity, and low signal-to-noise ratios. These limitations can be overcome by incorporating the unique characteristics of plasmonic nanomaterials into CRISPR/Cas systems to enhance the signal and improve the sensitivity of these biosensors. From this perspective, current interdisciplinary studies on CRISPR/Cas-based nanobiosensors comprising plasmonic nanomaterials can contribute to the development of highly sensitive CRISPR/Cas-based nanobiosensors. These nanobiosensors can detect attractive disease biomarkers, such as viral nucleic acids, small molecules, and proteins. This review article provides a thorough overview of nanobiosensors that incorporate CRISPR/Cas systems combined with plasmonic nanomaterials to enhance biosensing performance. We believe this review will inspire novel approaches and further innovation in the fields of molecular diagnostics and biomedicine aimed at using CRISPR/Cas systems and plasmonic nanomaterials for more personalized and effective medical treatments.},
}
@article {pmid40573878,
year = {2025},
author = {Ullah, F and Ali, S and Siraj, M and Akhtar, MS and Zaman, W},
title = {Plant Microbiomes Alleviate Abiotic Stress-Associated Damage in Crops and Enhance Climate-Resilient Agriculture.},
journal = {Plants (Basel, Switzerland)},
volume = {14},
number = {12},
pages = {},
doi = {10.3390/plants14121890},
pmid = {40573878},
issn = {2223-7747},
abstract = {Plant microbiomes, composed of a diverse array of microorganisms such as bacteria, fungi, archaea, and microalgae, are critical to plant health and resilience, playing key roles in nutrient cycling, stress mitigation, and disease resistance. Climate change is expected to intensify various abiotic stressors, such as drought, salinity, temperature extremes, nutrient deficiencies, and heavy metal toxicity. Plant-associated microbiomes have emerged as a promising natural solution to help mitigate these stresses and enhance agricultural resilience. However, translating laboratory findings into real-world agricultural benefits remains a significant challenge due to the complexity of plant-microbe interactions under field conditions. We explore the roles of plant microbiomes in combating abiotic stress and discuss advances in microbiome engineering strategies, including synthetic biology, microbial consortia design, metagenomics, and CRISPR-Cas, with a focus on enhancing their practical application in agriculture. Integrating microbiome-based solutions into climate-smart agricultural practices may contribute to long-term sustainability. Finally, we underscore the importance of interdisciplinary collaboration in overcoming existing challenges. Microbiome-based solutions hold promise for improving global food security and promoting sustainable agricultural practices in the face of climate change.},
}
@article {pmid40573432,
year = {2025},
author = {Hu, G and Wei, Z and Guo, J and Zhao, K and Qiao, Q and Zhu, X and Wu, T and Rong, H and Ning, S and Hao, Z and Chi, Y and Cui, L and Ge, Y},
title = {A Single-Tube Two-Step MIRA-CRISPR/Cas12b Assay for the Rapid Detection of Mpox Virus.},
journal = {Viruses},
volume = {17},
number = {6},
pages = {},
doi = {10.3390/v17060841},
pmid = {40573432},
issn = {1999-4915},
support = {2023YFC2605100, 2023YFC2605104//the National Key R&amp;D Program of China/ ; BK20231374, BK20221413//the natural science foundation of Jiangsu Province/ ; },
mesh = {*CRISPR-Cas Systems ; *Nucleic Acid Amplification Techniques/methods ; *Molecular Diagnostic Techniques/methods ; Sensitivity and Specificity ; Animals ; Humans ; Limit of Detection ; *Capripoxvirus/isolation &amp; purification/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats ; CRISPR-Associated Proteins/genetics ; },
abstract = {Mpox is a zoonotic disease caused by the Mpox virus (MPXV). The rapid and accurate diagnosis of MPXV is essential for the timely and effective prevention, control, and treatment of the disease. In this study, we combined Multienzyme Isothermal Rapid Amplification (MIRA) (at 42 °C) and Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated protein 12b(CRISPR/Cas12b) (at 60 °C) to develop a single-tube two-step assay for rapid MPXV detection, leveraging the distinct physical states of tricosane at these temperatures. MIRA amplification primers and CRISPR/cas12b SgRNA were designed based on the MPXV F3L gene. After screening the primers and sgRNAs, the reaction conditions were optimized, and the performances of the assay were evaluated. The detection limit (LOD) of this single-tube two-step MIRA-CRISPR/Cas12b assay for MPXV is four copies of DNA molecules. No cross-reactivity with other pathogens (herpes simplex virus (HSV), Epstein-Barr virus (EBV), Coxsackievirus A16 (CVA16), Enterovirus A71 (EV-A71), and measles virus (MeV)) was found. The assay also showed good consistency with quantitative real-time PCR (qPCR) (Kappa = 0.9547, p < 0.05, n = 100) in the detection of clinical samples, with a sensitivity of 98.5% and a specificity of 97.0%. The single-tube two-step MIRA-CRISPR/Cas12b assay permits the rapid (within 45 min), sensitive, and specific detection of MPXV. The lack of need for opening the reaction tube eliminates the risk of product contamination.},
}
@article {pmid40573367,
year = {2025},
author = {Workman, AM and Heaton, MP and Vander Ley, BL},
title = {CD46 Gene Editing Confers Ex Vivo BVDV Resistance in Fibroblasts from Cloned Angus Calves.},
journal = {Viruses},
volume = {17},
number = {6},
pages = {},
doi = {10.3390/v17060775},
pmid = {40573367},
issn = {1999-4915},
support = {3040-32000-036-00D//USDA-ARS appropriated funds/ ; },
mesh = {Animals ; Cattle ; *Gene Editing ; *Fibroblasts/virology/immunology ; *Bovine Virus Diarrhea-Mucosal Disease/genetics/virology/immunology/prevention &amp; control ; *Membrane Cofactor Protein/genetics ; Cloning, Organism ; *Diarrhea Viruses, Bovine Viral ; CRISPR-Cas Systems ; *Disease Resistance/genetics ; Female ; },
abstract = {A previous study demonstrated that a 19-nucleotide edit, encoding a six amino acid substitution in the bovine CD46 gene, dramatically reduced bovine viral diarrhea virus (BVDV) susceptibility in a cloned Gir (Bos indicus) heifer. The present study aimed to replicate this result in American Angus (Bos taurus) using genetically matched controls and larger sample sizes. CRISPR/Cas9-mediated homology-directed repair introduced the identical CD46 edit, encoding the A82LPTFS amino acid sequence, into exon 2 of CD46 in primary Angus fibroblasts. Thirty-three cloned embryos (22 CD46-edited and 11 unedited) were transferred to recipient cows. However, all pregnancies resulted in pre- and perinatal losses due to cloning-related abnormalities, preventing in vivo BVDV challenge. Consequently, ex vivo BVDV susceptibility assays were performed on primary fibroblast cell lines rescued from deceased cloned Angus calves. Infection studies revealed significantly reduced susceptibility in the edited lines, comparable to the resistance previously observed from the edited Gir heifer. These studies extend the applicability of this finding from Gir to the most common US beef breed, Angus, suggesting the potential for broad application of CD46 editing in BVDV control. Continued advancements in cloning technology will enhance the potential of gene-editing for producing disease-resistant livestock.},
}
@article {pmid40572275,
year = {2025},
author = {Kalu, MC and Acharya, A and Jorth, P and Wong-Beringer, A},
title = {ESBL-Producing Escherichia coli and Klebsiella pneumoniae Exhibit Divergent Paths During In-Human Evolution Towards Carbapenem Resistance.},
journal = {Microorganisms},
volume = {13},
number = {6},
pages = {},
pmid = {40572275},
issn = {2076-2607},
support = {not applicable//Merck &amp; Co., Inc., Rahway, NJ, USA (United States)/ ; },
abstract = {Treatment of infections caused by ESBL-producing Escherichia coli (EC) and Klebsiella pneumoniae (KP) with carbapenem antibiotics can lead to the development of carbapenem resistance over time through the acquisition of porin mutations and plasmids bearing blaKPC. However, the impact of genetic background and the presence of CRISPR-Cas systems on the evolutionary path towards carbapenem resistance in EC and KP has yet to be investigated. The in-human evolution following repeated carbapenem treatment among ESBL-producing Escherichia coli (EC) and Klebsiella pneumoniae (KP) clinical pairs (n = 45 pairs) was examined to determine the relationship between strain genetic background (MLST, CRISPR-Cas) and the evolved genetic mutations related to resistance, virulence, and metabolism by whole genome sequencing. ST131 and ST258 were predominant among seven distinct STs in EC (70%, 19/27) and 11 STs in KP (33%, 6/18), respectively. Complete CRISPR-Cas systems were present in 22% EC (6/27) and 27.8% (5/18) KP pairs, but none in strains belonging to ST131 or ST258; partial loss of CRISPR-Cas was associated with increased carbapenem resistance. Porin, virulence, and metabolism-related genetic mutations were present on the chromosome in both the EC and KP evolved strains, but their presence was differentially associated with the CRISPR-Cas system. Future research on the role of antibiotic exposure in the species-specific resistance evolution of the Enterobacterales could guide antimicrobial stewardship efforts.},
}
@article {pmid40572209,
year = {2025},
author = {Antequera-Zambrano, L and Parra-Sánchez, &#xc1; and González-Paz, L and Fernandez, E and Martinez-Navarrete, G},
title = {Distribution of Genetic Determinants Associated with CRISPR-Cas Systems and Resistance to Antibiotics in the Genomes of Archaea and Bacteria.},
journal = {Microorganisms},
volume = {13},
number = {6},
pages = {},
pmid = {40572209},
issn = {2076-2607},
abstract = {The CRISPR-Cas system represents an adaptive immune mechanism found across diverse Archaea and Bacteria, allowing them to defend against invading genetic elements such as viruses and plasmids. Despite its broad distribution, the prevalence and complexity of CRISPR-Cas systems differ significantly between these domains. This study aimed to characterize and compare the genomic distribution, structural features, and functional implications of CRISPR-Cas systems and associated antibiotic resistance genes in 30 archaeal and 30 bacterial genomes. Through bioinformatic analyses of CRISPR arrays, cas gene architectures, direct repeats (DRs), and thermodynamic properties, we observed that Archaea exhibit a higher number and greater complexity of CRISPR loci, with more diverse cas gene subtypes exclusively of Class 1. Bacteria, in contrast, showed fewer CRISPR loci, comprising a mix of Class 1 and Class 2 systems, with Class 1 representing the majority (~75%) of the detected systems. Notably, Bacteria lacking CRISPR-Cas systems displayed a higher prevalence of antibiotic resistance genes, suggesting a possible inverse correlation between the presence of these immune systems and the acquisition of such genes. Phylogenetic and thermodynamic analyses further highlighted domain-specific adaptations and conservation patterns. These findings support the hypothesis that CRISPR-Cas systems play a dual role: first, as a defense mechanism preventing the integration of foreign genetic material-reflected in the higher complexity and diversity of CRISPR loci in Archaea-and second, as a regulator of horizontal gene transfer, evidenced by the lower frequency of antibiotic resistance genes in organisms with active CRISPR-Cas systems. Together, these results underscore the evolutionary and functional diversification of CRISPR-Cas systems in response to environmental and selective pressures.},
}
@article {pmid40493452,
year = {2025},
author = {Xue, H and Mishra, MK and Liu, Y and Liu, P and Grzybowski, M and Pandey, R and Usa, K and Vanden Avond, MA and Bala, N and Alli, AA and Cowley, AW and Qiu, Q and Greene, AS and Rao, S and O'Meara, CC and Geurts, AM and Liang, M},
title = {Physiological role and mechanisms of action for a long noncoding haplotype region.},
journal = {Cell reports},
volume = {44},
number = {6},
pages = {115805},
doi = {10.1016/j.celrep.2025.115805},
pmid = {40493452},
issn = {2211-1247},
mesh = {Humans ; *Haplotypes/genetics ; Polymorphism, Single Nucleotide/genetics ; Animals ; Induced Pluripotent Stem Cells/metabolism ; Blood Pressure/genetics ; Gene Editing/methods ; Mice ; CRISPR-Cas Systems ; },
abstract = {Direct targeting of noncoding genomic regions harboring common sequence variants associated with human traits through in vivo animal model studies and precise genome editing in human cells is essential for closing the critical gap between genetic discoveries and physiological understanding. However, such investigation has been impractical for many of these variants as they are in haplotypes containing multiple single-nucleotide polymorphisms (SNPs) spanning thousands of base pairs and have small effect sizes. We developed an integrated approach to address this challenge, combining an efficient two-step technique to precisely edit large haplotypes in human induced pluripotent stem cells and orthologous region deletion in phenotypically permissive animal models. As proof of principle, we applied this approach to examine a blood pressure-associated locus with a noncoding haplotype containing 11 SNPs spanning 17.4 kbp. We found a robust blood pressure effect of nearly 10 mmHg and identified the physiological and molecular mechanisms involved.},
}
@article {pmid40434888,
year = {2025},
author = {Guo, J and Zhou, Z and Li, R and Xing, Z and Zhang, L and Zhao, S and Wei, W and Wang, J and Deng, T},
title = {A genome-wide base-editing screen uncovers a pivotal role of paxillin δ ubiquitination in influenza virus infection.},
journal = {Cell reports},
volume = {44},
number = {6},
pages = {115748},
doi = {10.1016/j.celrep.2025.115748},
pmid = {40434888},
issn = {2211-1247},
mesh = {Animals ; *Ubiquitination ; *Paxillin/metabolism/genetics ; Humans ; Mice ; Virus Replication ; *Influenza A virus/physiology ; *Gene Editing ; *Orthomyxoviridae Infections/virology/genetics/metabolism ; Madin Darby Canine Kidney Cells ; CRISPR-Cas Systems ; Virus Internalization ; Dogs ; Protein Isoforms/metabolism ; },
abstract = {Dissecting host factors critical for viral infection and understanding their mechanisms of action is critical for identifying drug targets. Here, we leverage a genome-wide CRISPR base-editing screen to identify functional lysine residues in host factors required for influenza A virus (IAV) replication. Multiple host genes, including GSTM4, FLNC, HMGB1, ZNF236, GRIP1, and PXN, along with regulatory lysine codons, are identified. Among these, paxillin (encoded by PXN) is identified as an important host entry factor. Depletion of paxillin significantly reduces IAV infection in both cell cultures and mice. Further analysis suggests that the δ isoform of paxillin, rather than the canonical β isoform, plays the key role. Additionally, our data indicate that lysine 68 of paxillin δ undergoes K6-linked ubiquitination and regulates influenza virus replication via modulating endosome-dependent viral entry. These observations contribute to understanding how influenza viruses interact with host factors and may inform therapeutic development.},
}
@article {pmid40384639,
year = {2025},
author = {Ye, J and Shen, Y and Lin, Z and Xu, L and Wang, L and Lin, X and Huang, B and Ma, Z and Yu, Z and Lin, D and Chen, W and Feng, S},
title = {A CRISPR/Cas12a-Assisted SERS Nanosensor for Highly Sensitive Detection of HPV DNA.},
journal = {ACS sensors},
volume = {10},
number = {6},
pages = {4286-4296},
doi = {10.1021/acssensors.5c00547},
pmid = {40384639},
issn = {2379-3694},
mesh = {*Spectrum Analysis, Raman/methods ; *CRISPR-Cas Systems ; *DNA, Viral/analysis/genetics ; *Human papillomavirus 16/genetics/isolation &amp; purification ; *Biosensing Techniques/methods ; Humans ; *Human papillomavirus 18/genetics/isolation &amp; purification ; Biotin/chemistry ; Limit of Detection ; },
abstract = {The lack of timely and effective screening and diagnosis is a major contributing factor to the high mortality rate of cervical cancer in low-income countries and resource-limited regions. Therefore, the development of a rapid, sensitive, and easily deployable diagnostic tool for HPV DNA is of critical importance. In this study, we present a novel high-sensitivity and high-specificity detection method for HPV16 and HPV18 by integrating the CRISPR/Cas12a system with surface-enhanced Raman scattering (SERS) technology. This method leverages the trans-cleavage activity of the CRISPR/Cas12a system, which cleaves biotin-modified spherical nucleic acids (Biotin-SNA) in the presence of target DNA, releasing free Biotin-DNA. The released Biotin-DNA preferentially binds to streptavidin-modified magnetic beads (SAV-MB), reducing the capture of Biotin-SNA by SAV-MB and thereby significantly enhancing detection sensitivity. This method offers the potential for point-of-care diagnostics as it operates efficiently at 37 °C without the need for thermal cycling. Using standard DNA samples, we demonstrated that this biosensor achieved detection limits as low as 209 copies/μL and 444 copies/μL within 95 min. When combined with recombinase polymerase amplification (RPA), the sensor demonstrated enhanced sensitivity, enabling detection of target DNA at concentrations as low as 1 copy/μL within approximately 50 min. Furthermore, validation with clinical samples confirmed the feasibility and practical applicability of this method. This novel SERS-based sensor offers a new and effective tool in the prevention and detection of cervical cancer.},
}
@article {pmid40300601,
year = {2025},
author = {Kang, X and Li, X and Zhou, J and Zhang, Y and Qiu, L and Tian, C and Deng, Z and Liang, X and Zhang, Z and Du, S and Hu, S and Wang, N and Yue, Z and Xu, Y and Gao, Y and Dai, J and Wang, Z and Yu, C and Chen, J and Wu, Y and Chen, L and Yao, Y and Yao, S and Yang, X and Yan, L and Wen, Q and Depies, OM and Chan, K and Liang, X and Li, G and Zi, Z and Liu, X and Gan, H},
title = {Extrachromosomal DNA replication and maintenance couple with DNA damage pathway in tumors.},
journal = {Cell},
volume = {188},
number = {13},
pages = {3405-3421.e27},
doi = {10.1016/j.cell.2025.04.012},
pmid = {40300601},
issn = {1097-4172},
mesh = {Humans ; *DNA Replication ; Ataxia Telangiectasia Mutated Proteins/metabolism ; DNA End-Joining Repair ; DNA Breaks, Double-Stranded ; *Neoplasms/genetics/metabolism/pathology ; *DNA Damage ; Cell Line, Tumor ; DNA Ligase ATP/metabolism ; DNA-Directed DNA Polymerase/metabolism ; DNA, Circular/metabolism/genetics ; DNA Polymerase theta ; CRISPR-Cas Systems ; },
abstract = {Extrachromosomal DNA (ecDNA) drives the evolution of cancer cells. However, the functional significance of ecDNA and the molecular components involved in its replication and maintenance remain largely unknown. Here, using CRISPR-C technology, we generated ecDNA-carrying (ecDNA+) cell models. By leveraging these models alongside other well-established systems, we demonstrated that ecDNA can replicate and be maintained in ecDNA+ cells. The replication of ecDNA activates the ataxia telangiectasia mutated (ATM)-mediated DNA damage response (DDR) pathway. Topoisomerases, such as TOP1 and TOP2B, play a role in ecDNA replication-induced DNA double-strand breaks (DSBs). A subset of these elevated DSBs persists into the mitotic phase and is primarily repaired by the alternative non-homologous end joining (alt-NHEJ) pathway, which involves POLθ and LIG3. Correspondingly, ecDNA maintenance requires DDR, and inhibiting DDR impairs the circularization of ecDNA. In summary, we demonstrate reciprocal interactions between ecDNA maintenance and DDR, providing new insights into the detection and treatment of ecDNA+ tumors.},
}
@article {pmid40571317,
year = {2025},
author = {Karinen, S and Forero-Rodríguez, J and Järvinen, A and Eklund, KK and Barreto, G and Salem, A},
title = {CRISPR/Cas9-mediated Knockout of LYVE1 In Human Tongue Cancer Cells Reveals Transcriptomic Changes in Metastasis-associated Pathways.},
journal = {Cancer genomics &amp; proteomics},
volume = {22},
number = {4},
pages = {525-537},
doi = {10.21873/cgp.20519},
pmid = {40571317},
issn = {1790-6245},
mesh = {Humans ; *Tongue Neoplasms/genetics/pathology/metabolism ; *CRISPR-Cas Systems ; *Transcriptome ; *Vesicular Transport Proteins/genetics/metabolism ; Gene Expression Regulation, Neoplastic ; Epithelial-Mesenchymal Transition ; Cell Line, Tumor ; Gene Knockout Techniques ; Neoplasm Metastasis ; *Squamous Cell Carcinoma of Head and Neck/genetics ; },
abstract = {BACKGROUND/AIM: Tongue squamous cell carcinoma (TSCC), a highly aggressive subtype of head and neck cancers, is characterized by frequent lymphatic metastasis and poor prognosis. Recently, we showed that lymphatic vessel endothelial hyaluronan receptor 1 (LYVE-1) is involved in TSCC progression, yet the underlying molecular mechanisms remain unclear.<br><br>MATERIALS AND METHODS: CRISPR/Cas9 gene editing was employed to generate LYVE1 knockout (KO) TSCC cell lines. Single-cell clones were isolated, screened, and validated through sequencing and Inference of CRISPR Edits (ICE) analysis and qRT-PCR. RNA sequencing was performed on LYVE1 KO and wild-type (WT) cells to identify differentially expressed genes (DEGs). Bioinformatic analyses, including Gene Ontology (GO) enrichment and protein-protein interaction (PPI) network mapping, were conducted to explore affected pathways. Finally, network topology was examined using NetworkAnalyzer and cytoHubba plugins.<br><br>RESULTS: Transcriptomic analysis revealed significant down-regulation of pro-metastatic pathways, including epithelial-mesenchymal transition (EMT), extracellular matrix remodeling, and immune modulation. DEG analysis identified 263 genes, with key down-regulated targets such as WNT5A, TGFB2, and MMP2, and up-regulation of tumor-suppressive genes including PTGS2. GO and PPI analyses highlighted LYVE1's pivotal role in regulating cell adhesion, migration, and immune response.<br><br>CONCLUSION: LYVE1 KO reduces TSCC invasive potential by disrupting EMT and tumor-stroma interactions, aligning with previous experimental findings. These results suggest LYVE1 as a critical driver of metastasis, highlighting its potential as a therapeutic target.},
}
@article {pmid40571168,
year = {2025},
author = {Jeong, JH and Kim, SH and Kim, JY},
title = {Empowering Agrobacterium: Ternary vector systems as a new arsenal for plant transformation and genome editing.},
journal = {Biotechnology advances},
volume = {},
number = {},
pages = {108631},
doi = {10.1016/j.biotechadv.2025.108631},
pmid = {40571168},
issn = {1873-1899},
abstract = {The continuous evolution of plant transformation technologies is pivotal for accelerating genetic advancements in agriculture. Among these, ternary vector systems have emerged as a transformative innovation, significantly enhancing Agrobacterium-mediated plant transformation by overcoming critical biological barriers. Unlike traditional binary vectors, ternary vector systems incorporate accessory virulence genes and immune suppressors that overcome the intrinsic transformation barriers of recalcitrant crops. This has enabled 1.5- to 21.5-fold increases in stable transformation efficiency in species previously resistant to Agrobacterium-mediated transformation, such as maize, sorghum, and soybean, thereby expanding the effective host range of plant genetic engineering. Furthermore, the fusion of ternary vectors with advanced genome editing technologies like CRISPR/Cas is revolutionizing precision breeding, facilitating unprecedented control over genetic modifications. Future innovations are likely to focus on expanding the capabilities of ternary vectors, including transient delivery of morphogenic factors to enhance regeneration and organelle-targeted transformation for broader genetic modifications. Additionally, refining Agrobacterium engineering, such as developing auxotrophic strains for improved biosafety and optimizing secretion systems for enhanced protein delivery, presents exciting opportunities for further advancements. This review highlights the recent breakthroughs in ternary vector engineering, including its potential to optimize regeneration pathways via morphogenic factors and extend genetic transformation to previously unamenable plant species. By bridging the gap between transformation efficiency and targeted genome modifications, these advancements are reshaping the landscape of plant biotechnology, driving more resilient and high-performing crops in an era of global agricultural challenges.},
}
@article {pmid40569097,
year = {2025},
author = {Jenkins, K and Layton, D and Gough, T and O'Neil, T and Malaver Otega, L and Mishra, K and Bruce, K and Morris, K and Wise, T and Challagulla, A and Doran, T and Bean, A},
title = {Production of immune receptor knockout chickens via direct in vivo transfection of primordial germ cells.},
journal = {Animal biotechnology},
volume = {36},
number = {1},
pages = {2523027},
doi = {10.1080/10495398.2025.2523027},
pmid = {40569097},
issn = {1532-2378},
mesh = {Animals ; *Chickens/genetics ; *Receptor, Interferon alpha-beta/genetics ; *Gene Knockout Techniques/veterinary/methods ; *Transfection/veterinary/methods ; *Animals, Genetically Modified/genetics ; *Germ Cells ; *Receptors, Interleukin-1 Type I/genetics ; CRISPR-Cas Systems ; Chick Embryo ; Male ; Female ; },
abstract = {The advancement of genetic engineering in chickens has enabled significant advancement in developmental biology, bioreactors, and disease resilience. The development of CRISPR/Cas9 genome engineering technology has further expanded the potential applications of genetic engineering in poultry. In this study we aimed to evaluate the efficacy of a direct in vivo transfection method, previously demonstrated to produce transgenic chickens, in generating gene knockout (KO) chickens. Specifically, we targeted the Interferon-α/β Receptor 1 (IFNAR1) and Interleukin 1 receptor, type I (IL1R1), both critical pathways in the inflammatory and antiviral responses. We designed guide RNAs targeting the genes and validated their efficiency in vivo via microinjection into the developing embryos. PCR analysis confirmed the presence of gene deletions in chimeric roosters, which were subsequently bred to produce G1 germline heterozygote KO offspring. Homozygous KO chickens were generated and subjected to phenotypic and functional analyses. Our results demonstrated successful generation of functional knockouts of both IFNAR1 and IL1R1 using a direct in vivo transfection. Overall, this study demonstrates that direct in vivo transfection provides a robust and predictable method for generating KO chickens, facilitating further research into avian immune responses and the development of antiviral strategies.},
}
@article {pmid40568938,
year = {2025},
author = {Liu, X and Tan, H and Wang, J and Cao, Y and Li, P and Fan, X and Wang, Q and Zhang, H and Zhang, J and Yang, T and Zhao, G and Zhang, X and Duan, X and Zi, L and Liu, L and Ma, L and Chen, Z and Liang, L and Liu, R},
title = {SELECT: high-precision genome editing strategy via integration of CRISPR-Cas and DNA damage response for cross-species applications.},
journal = {Nucleic acids research},
volume = {53},
number = {12},
pages = {},
doi = {10.1093/nar/gkaf595},
pmid = {40568938},
issn = {1362-4962},
support = {2023YFC3402300//National Key R&amp;D Program of China/ ; 22208044//National Natural Science Foundation of China/ ; 22278058//National Natural Science Foundation of China/ ; XLYC2203075//Xingliao Talent Plan/ ; 2023JJ12SN030//Science and Technology Innovation Foundation of Dalian/ ; 2024-MSBA-09//Natural Science Foundation of Liaoning Province/ ; DUT24YG131//Fundamental Research Funds for the Central Universities/ ; DUT23YG219//Fundamental Research Funds for the Central Universities/ ; },
mesh = {*CRISPR-Cas Systems ; *Gene Editing/methods ; Saccharomyces cerevisiae/genetics ; Escherichia coli/genetics ; *DNA Damage ; Promoter Regions, Genetic ; DNA Breaks, Double-Stranded ; },
abstract = {CRISPR-based methods enable genome modifications for diverse applications but often face challenges, such as inconsistent efficiencies, reduced performance in iterative modifications, and difficulties generating high-quality datasets for high-throughput genome engineering. Here, we present SELECT (SOS Enhanced programmabLE CRISPR-Cas ediTing), a novel strategy integrating the CRISPR-Cas system with the DNA damage response. By employing designed and optimized double-strand break induced promoters that are activated upon genome editing, SELECT enables a counter-selection process to eliminate unedited cells, ensuring high-fidelity editing. This approach achieves up to 100% efficiency for point mutations, iterative knockouts, and insertions. In high-throughput library editing, SELECT achieved up to 94.2% efficiency and preserved higher library diversity compared with conventional methods. Application of SELECT in flaviolin biosynthesis resulted in a 3.97-fold increase in production. Furthermore, integration with machine learning tools allowed rapid mapping of genotype-phenotype relationships. SELECT provides a versatile platform for precision genome engineering in Escherichia coli and Saccharomyces cerevisiae.},
}
@article {pmid40567005,
year = {2025},
author = {Zeng, Z and Wang, H and Luo, Y and Chen, W and Xu, M and Wei, H and Chen, Z and Xiang, T and Wang, L and Han, N and Huang, X and Bian, H},
title = {CRISPR/Cas9-mediated editing of barley lipoxygenase genes promotes grain fatty acid accumulation and storability.},
journal = {GM crops &amp; food},
volume = {16},
number = {1},
pages = {482-497},
doi = {10.1080/21645698.2025.2523069},
pmid = {40567005},
issn = {2164-5701},
mesh = {*Hordeum/genetics/metabolism/enzymology ; *CRISPR-Cas Systems ; Gene Editing ; *Lipoxygenase/genetics/metabolism ; *Fatty Acids/metabolism ; *Edible Grain/genetics/metabolism ; Plants, Genetically Modified/genetics/metabolism ; *Plant Proteins/genetics/metabolism ; Seeds/genetics/metabolism ; Gene Expression Regulation, Plant ; },
abstract = {Plant lipoxygenases (LOXs) catalyze the oxidation of polyunsaturated fatty acids, which can adversely affect grain storability. Although the genetic engineering of LOXs holds great potential for improving grain storage quality, this approach remains largely unexplored in barley. In this study, we identified five LOX genes in the barley genome: HvLOXA, HvLOXB, and HvLOXC1-3. HvLOXC1 exhibited the highest expression in early developing grains, roots, and shoots; HvLOXA was predominantly expressed in embryos, whereas HvLOXB and HvLOXC3 were weakly expressed across tissues. Transgene-free homozygous barley mutants of loxB, loxC1, and loxAloxC1 were generated using CRISPR/Cas9-mediated genome editing. Compared to the wild-type, all mutants displayed normal plant height, tiller number, and grain size, although the loxC1 and loxAloxC1 mutants exhibited significantly lower thousand grain weights. Notably, the total LOX activity in mature grains decreased by 36-42% in loxC1 mutants and by 94% in loxAloxC1 mutants, with no significant change observed in loxB mutants. Additionally, the loxAloxC1 double mutants had a significantly lower malondialdehyde content and accumulated 10-21% more fatty acids than the wild-type. Artificial aging treatment experiments revealed that loxAloxC1 mutants had enhanced grain storability, demonstrated by significantly higher germination rates, reduced lipid peroxidation, and improved seedling growth. Our findings highlight that the targeted knockout of LOX genes, particularly the double mutation of HvLOXA and HvLOXC1, represents a promising genetic strategy for improving grain storability and nutritional value in barley.},
}
@article {pmid40566742,
year = {2025},
author = {Ni, H and Kelley, K and Xie, N and Zou, H and Friedel, RH},
title = {Generation of Plexin-B1 Conditional Knockout Mouse With CRISPR/Cas9 Technology.},
journal = {Genesis (New York, N.Y. : 2000)},
volume = {63},
number = {3},
pages = {e70019},
doi = {10.1002/dvg.70019},
pmid = {40566742},
issn = {1526-968X},
support = {NS092735/NS/NINDS NIH HHS/United States ; NS134159/NS/NINDS NIH HHS/United States ; AG077828/AG/NIA NIH HHS/United States ; CA196521/CA/NCI NIH HHS/United States ; DOH01-C39068GG//New York State Department of Health/ ; DOH01-C38330GG//New York State Department of Health/ ; },
mesh = {Animals ; Mice ; *CRISPR-Cas Systems ; Mice, Knockout ; *Nerve Tissue Proteins/genetics ; Alleles ; Gene Targeting ; *Receptors, Cell Surface/genetics ; Gene Editing/methods ; Female ; },
abstract = {Plexins are axon guidance transmembrane receptors that control cytoskeleton and membrane dynamics in development and adult physiology. As plexins are expressed in multiple cell types in various tissues, floxed alleles that enable conditional deletion are needed to facilitate cell type-specific functional analysis. We report here the generation of a conditional floxed allele of Plexin-B1 (gene symbol Plxnb1) in mouse using CRISPR/Cas9 technology to insert two loxP sites flanking critical exons. Targeting reagents (Cas9 protein, sgRNAs, ssODNs) were delivered into single-cell embryos by electroporation. After screening a total of 128 mouse pups by PCR and Sanger sequencing, two mice were identified carrying both loxP sites in the targeted Plxnb1 locus (success rate ~ 1.6%). The usage of Alt-R modified ssODNs increased targeting frequencies at one loxP site, but not the other. We also tested homology directed repair (HDR) enhancer V2 reagent, but addition of the enhancer reduced the viability of mouse embryos. The Plxnb1[flox] allele was successfully transmitted through the germline in Mendelian ratios, and effective excision of the floxed region was confirmed by breeding with Cre recombinase strains.},
}
@article {pmid40566685,
year = {2025},
author = {Liu, J and Zhang, R and Chai, N and Su, L and Zheng, Z and Liu, T and Guo, Z and Ma, Y and Xie, Y and Xie, X and Lin, Q and Chen, L and Liu, YG and Zhu, Q},
title = {Programmable genome engineering and gene modifications for plant biodesign.},
journal = {Plant communications},
volume = {},
number = {},
pages = {101427},
doi = {10.1016/j.xplc.2025.101427},
pmid = {40566685},
issn = {2590-3462},
abstract = {Plant science has entered a transformative era with genome editing by enabling precise DNA alterations to address global challenges such as climate adaptability and food safety. These alterations are primarily driven by the integration of three modular components that can be activated or suppressed: DNA-targeting modules, effector modules, and control modules. The field has evolved from protein-centric systems (zinc finger nucleases and transcription activator-like effector nucleases) to RNA-focused platforms (CRISPR-Cas and other nucleases), which facilitate diverse control over genetic and epigenetic contexts. The modular design of DNA-targeting modules paired with effector domains, with or without inducible systems, provides scientists with superior precision in regulating transcription and altering chromatin states. The present review article examines these three modules and highlights various optimization methods. Additionally, it outlines innovative tools such as optogenetic systems and receptor-integrated systems that enable spatiotemporal control of genome editor expression. These modular instruments overcome traditional boundaries and allow scientists to create plants with favorable characteristics, decipher complex gene networks, and adopt sustainable farming practices.},
}
@article {pmid40565548,
year = {2025},
author = {Xavier, KVM and Silva, AMA and Luz, ACO and da Silva, FSC and de Melo, BST and Pitta, JLLP and Leal-Balbino, TC},
title = {Diversity and Role of Prophages in Pseudomonas aeruginosa: Resistance Genes and Bacterial Interactions.},
journal = {Genes},
volume = {16},
number = {6},
pages = {},
doi = {10.3390/genes16060656},
pmid = {40565548},
issn = {2073-4425},
mesh = {*Pseudomonas aeruginosa/genetics/virology/drug effects/pathogenicity ; *Prophages/genetics ; CRISPR-Cas Systems/genetics ; *Drug Resistance, Bacterial/genetics ; Humans ; Pseudomonas Infections/microbiology ; Genome, Bacterial ; Brazil ; },
abstract = {Pseudomonas aeruginosa is a major pathogen associated with hospital-acquired infections, and the spread of carbapenem-resistant isolates highlights the urgency of developing non-conventional therapies, such as phage therapy. For this alternative to be effective, understanding phage-host interactions is crucial for the selection of candidate phages and offers new insights into these dynamics. Background/Objectives: This study aimed to characterize prophage diversity in clinical P. aeruginosa genomes, assess the relationship between phages and the CRISPR/Cas system, and investigate the potential role of prophages in disseminating resistance genes. Methods: A total of 141 genomes from Brazilian hospitals were analyzed. Prophage detection was performed using VIBRANT, and in silico analyses were conducted to evaluate taxonomic diversity, the presence of resistance genes, phage life cycle, genomic distribution, and the presence of the CRISPR/Cas system. Results: A total of 841 viral sequences were identified by the VIBRANT tool, of which 498 were confirmed by CheckV, with a predominance of the class Caudoviricetes and high overall phage diversity. No statistically significant difference was observed in the number of prophages between isolates with and without CRISPR/Cas systems. Prophages carrying resistance genes such as rsmA, OXA-56, SPM-1, and others were detected in isolates harboring the type I-C CRISPR/Cas system. Additionally, prophages showed no preference for specific insertion sites along the bacterial genome. Conclusions: These findings provide evidence of a well-established phage-host relationship. The dual role of prophages-as vectors of antimicrobial resistance and as potential therapeutic agents-reflects their dynamic impact on bacterial communities and reinforces their importance in developing new strategies to combat antimicrobial resistance.},
}
@article {pmid40565267,
year = {2025},
author = {Chakraborty, M and Nielsen, L and Nash, D and Bruder, MR and Nissimov, JI and Charles, TC and Aucoin, MG},
title = {Baculovirus Variant Detection from Transient CRISPR-Cas9-Mediated Disruption of gp64 at Different Gene Locations.},
journal = {International journal of molecular sciences},
volume = {26},
number = {12},
pages = {},
doi = {10.3390/ijms26125805},
pmid = {40565267},
issn = {1422-0067},
support = {RGPIN 355513-2017/RGPIN-2023-03666//Natural Sciences and Engineering Research Council/ ; },
mesh = {*CRISPR-Cas Systems ; Animals ; *Baculoviridae/genetics ; *Viral Envelope Proteins/genetics ; Genetic Vectors/genetics ; Genome, Viral ; RNA, Guide, CRISPR-Cas Systems/genetics ; Gene Editing ; Sf9 Cells ; Mutation ; },
abstract = {The Baculovirus Expression Vector System (BEVS) is an important protein and complex biologics production platform. The baculovirus GP64 protein is the major envelope glycoprotein that aids in virus entry and is required for cell-to-cell transmission in cell culture. Several studies have developed strategies around gp64 gene disruption in an attempt to minimize baculovirus co-production. Here, we investigate the result of transiently targeting the baculovirus gp64 gene with CRISPR-Cas9 during infection. Because not all genomes are effectively disrupted, we describe a variant calling methodology that allows the detection of the targeted mutations in gp64 even though these mutations are not the dominant sequences. Using a transfection-infection assay (T-I assay), the AcMNPV gp64 gene was targeted at six different locations to evaluate the effects of single and multiple targeting sites, and we demonstrated a reduction in the levels of baculovirus vectors while maintaining or enhancing foreign protein production when protein was driven by a p6.9 promoter. Viral genomes were subsequently isolated from the supernatant and cell pellet fractions, and our sequencing pipeline successfully detected indel mutations within gp64 for most of the single-guide RNA (sgRNA) targets. We also observed that 68.8% of variants found in the virus stock were conserved upon virus propagation in cell culture, thus indicating that they are not detrimental to viral fitness. This work provides a comprehensive assessment of CRISPR-Cas9 genome editing of baculovirus vectors, with potential applications in enhancing the efficiency of the BEVS.},
}
@article {pmid40565111,
year = {2025},
author = {Bouchard, C and Rousseau, J and Lamothe, G and Dubost, M and Bastrenta, L and Ramezani, S and Tremblay, JP},
title = {In Vitro Correction of Point Mutations in the DYSF Gene Using Prime Editing.},
journal = {International journal of molecular sciences},
volume = {26},
number = {12},
pages = {},
doi = {10.3390/ijms26125647},
pmid = {40565111},
issn = {1422-0067},
support = {TREM-01//Jain Foundation/ ; },
mesh = {Humans ; *Point Mutation ; *Gene Editing/methods ; HEK293 Cells ; *Dysferlin/genetics ; Genetic Therapy/methods ; Myoblasts/metabolism ; Fibroblasts/metabolism ; CRISPR-Cas Systems ; },
abstract = {Dysferlinopathy is caused by over 500 mutations in the gene encoding dysferlin, including close to 300 point mutations. One option to cure the disease is to use a gene therapy to correct these mutations at the root. Prime editing is a technique which can replace the mutated nucleotide with the wild-type nucleotide. In this article, prime editing is used to correct several point mutations in the DYSF gene responsible for dysferlinopathy. In vitro editing of HEK293T cells reaches up to 31%. Notably, editing was more efficient in myoblasts than in patient-derived fibroblasts. The prime editing technique was also used to create a new myoblast clone containing a patient mutation from a healthy myoblast cell line.},
}
@article {pmid40521923,
year = {2025},
author = {Cao, Y and Huang, C and Li, K and Zhang, X and Tong, M and Tu, J and Yang, K and Yuan, S and Zhang, H},
title = {CRISPR/Cas12a-assisted electrochemiluminescent detection of ochratoxin A based on COF@Ru coupled with a DNA tetrahedral scaffold.},
journal = {Analytical methods : advancing methods and applications},
volume = {17},
number = {25},
pages = {5298-5307},
doi = {10.1039/d5ay00427f},
pmid = {40521923},
issn = {1759-9679},
mesh = {*Ochratoxins/analysis ; *CRISPR-Cas Systems ; *Electrochemical Techniques/methods ; *Biosensing Techniques/methods ; *Luminescent Measurements/methods ; *Metal-Organic Frameworks/chemistry ; Limit of Detection ; Gold/chemistry ; *DNA/chemistry ; Metal Nanoparticles/chemistry ; Aptamers, Nucleotide/chemistry ; DNA Probes/chemistry ; },
abstract = {To sensitively detect ochratoxin A (OTA), a CRISPR/Cas12a-assisted electrochemiluminescence (ECL) aptasensor based on COF@Ru coupled with a DNA tetrahedral scaffold (DTS) was successfully fabricated. The covalent organic framework (COF) acted as a confined micro-reactor for the tris(2,2'-bipyridyl)ruthenium(II)/tri-n-propylamine (Ru(bpy)3[2+]/TPrA) system and hence, evidently enhanced ECL signals. Au nanoparticles (AuNPs) coupled with DTS caused more ferrocene (Fc)-modified quenching DNA probe molecules to be attached to the surface of the sensing electrode, which could reduce the detection background. Activated CRISPR/Cas12a could effectively amplify the ECL signal since it could cut off a considerable amount of quenching DNA probe molecules and make them move away from the electrode. Owing to these strategies, the fabricated ECL aptasensor could detect OTA as low as 3.5 fg mL[-1] in a linear detection range of 10[-5]-100 ng mL[-1]. Furthermore, the CRISPR/Cas12a and COF@Ru coupled with DTS-based ECL biosensor possessed high stability and specificity. More importantly, the ECL biosensor could effectively detect OTA in food samples, indicating that it can play a role in monitoring OTA levels in the field of food safety.},
}
@article {pmid40518166,
year = {2025},
author = {Ge, L and Xiong, H},
title = {[Advances in CRISPR-Cas9 genome editing for the treatment of muscular dystrophies].},
journal = {Zhonghua er ke za zhi = Chinese journal of pediatrics},
volume = {63},
number = {7},
pages = {808-811},
doi = {10.3760/cma.j.cn112140-20250421-00356},
pmid = {40518166},
issn = {0578-1310},
mesh = {Humans ; *Genetic Therapy/methods ; *CRISPR-Cas Systems ; *Muscular Dystrophies/therapy/genetics ; Animals ; *Gene Editing/methods ; Muscular Dystrophy, Duchenne/therapy/genetics ; Disease Models, Animal ; },
}
@article {pmid40501421,
year = {2025},
author = {Zheng, Y and Liu, B and Zuo, Q and Deng, F and Lan, C},
title = {Detection of Streptococcus anginosus in fecal samples using PCR-CRISPR /Cas12a system.},
journal = {Bioanalysis},
volume = {17},
number = {11},
pages = {737-745},
doi = {10.1080/17576180.2025.2518042},
pmid = {40501421},
issn = {1757-6199},
mesh = {*Feces/microbiology ; Humans ; *CRISPR-Cas Systems/genetics ; *Polymerase Chain Reaction/methods ; *Streptococcus anginosus/genetics/isolation &amp; purification ; DNA, Bacterial/genetics ; RNA, Ribosomal, 16S/genetics ; },
abstract = {OBJECTIVE: To develop a highly sensitive and specific detection method based on PCR-CRISPR/Cas12a for the detection of Streptococcus anginosus (S. anginosus) in feces and to evaluate its detection rate in the general population as well as its potential as a gastrointestinal tumor marker.<br><br>MATERIALS AND METHODS: Specific primers and crRNA targeting the 16S rDNA of S. anginosus were designed to construct a PCR-CRISPR/Cas12a detection system. A total of 230 fecal samples were collected from the general population, and bacterial DNA was extracted. The target gene was detected using this system to verify its sensitivity, specificity, and stability.<br><br>RESULTS: The established detection system demonstrated strong specificity, with stable recognition of S. anginosus, and a minimum detection limit of 10[-7] ng/&#x3bc;L. The detection rate of S. anginosus in fecal samples from the general population was 51.7%.<br><br>CONCLUSION: The PCR-CRISPR/Cas12a system can efficiently detect S. anginosus in feces, providing a reliable technical tool for exploring its association with gastrointestinal tumors.},
}
@article {pmid40490360,
year = {2025},
author = {Kagawa, N and Umesono, Y and Suzuki, KT and Mochii, M},
title = {Step-by-Step Protocol for Making a Knock-In Xenopus laevis to Visualize Endogenous Gene Expression.},
journal = {Development, growth &amp; differentiation},
volume = {67},
number = {5},
pages = {293-302},
doi = {10.1111/dgd.70011},
pmid = {40490360},
issn = {1440-169X},
support = {//University of Hyogo/ ; JP18K06266//Japan Society for the Promotion of Science/ ; JP21H03829//Japan Society for the Promotion of Science/ ; 22NIBB331//NIBB Collaborative Research Program/ ; 23NIBB340//NIBB Collaborative Research Program/ ; JPMJCR 2025//JST, CREST/ ; },
mesh = {Animals ; *Xenopus laevis/genetics ; *Gene Knock-In Techniques/methods ; CRISPR-Cas Systems/genetics ; Green Fluorescent Proteins/genetics/metabolism ; },
abstract = {We established a novel knock-in technique, New and Easy Xenopus Targeted integration (NEXTi), to recapitulate endogenous gene expression by reporter expression. NEXTi is a CRISPR-Cas9-based method to integrate a donor DNA containing a reporter gene (egfp) into the target 5' untranslated region (UTR) of the Xenopus laevis genome. It enables us to track eGFP expression under the regulation of endogenous promoter/enhancer activities. We obtained about 2% to 13% of knock-in vector-injected embryos showing eGFP signal in a tissue-specific manner, targeting krt.12.2.L, myod1.S, sox2.L, and bcan.S loci, as previously reported. In addition, F1 embryos which show stable eGFP signals were obtained by outcrossing the matured injected frogs with wild-type animals. Integrations of donor DNAs into target 5' UTRs were confirmed by PCR amplification and sequencing. Here, we describe the step-by-step protocol for preparation of donor DNA and single guide RNA, microinjection, and genotyping of F1 animals for the NEXTi procedure.},
}
@article {pmid39883121,
year = {2025},
author = {Paryani, M and Gupta, N and Jain, SK and Butani, S},
title = {Lowering LDL cholesterol by PCSK9 inhibition: a new era of gene silencing, RNA, and alternative therapies.},
journal = {Naunyn-Schmiedeberg's archives of pharmacology},
volume = {398},
number = {6},
pages = {6597-6615},
pmid = {39883121},
issn = {1432-1912},
mesh = {Humans ; *Antibodies, Monoclonal/pharmacology/therapeutic use ; Cholesterol, LDL/blood ; CRISPR-Cas Systems ; Gene Silencing ; Hypercholesterolemia/drug therapy/economics ; *Oligonucleotides, Antisense/pharmacology/therapeutic use ; *Plant Extracts/pharmacology/therapeutic use ; Proprotein Convertase 9/genetics/metabolism ; *Proprotein Convertases/antagonists &amp; inhibitors ; Protein Subunit Vaccines/pharmacology/therapeutic use ; *RNA, Small Interfering/pharmacology/therapeutic use ; *Anticholesteremic Agents/pharmacology/therapeutic use ; Fibronectin Type III Domain ; Recombinant Proteins ; },
abstract = {Proprotein convertase subtilisin/kexin type 9 (PCSK9) discovery has added a new paradigm to our understanding of cholesterol homeostasis and lipid metabolism. Since its discovery, PCSK9 inhibitors have become a widely investigated therapeutic class for lipid management in cardiovascular diseases and hypercholesterolemia. Scientists have explored different approaches for PCSK9 inhibition, such as monoclonal antibodies (mAbs), gene silencing and gene editing techniques, vaccines, mimetic peptides, and small molecules. European Medicines Agency (EMA) and United States Food and Drug Administration (US FDA) have approved only three PCSK9 inhibitors, including two monoclonal antibodies and one small interfering ribonucleic acid (siRNA). Despite the efficacy of approved large molecules, high costs and the need for regular injection have limited their adherence to the patient. This review aims to provide an understanding of PCSK9's function in Low-Density Lipoprotein Cholesterol (LDL-C) management, its current treatment, recent advancements, and potential future development of small molecules in the class of PCSK9 inhibitors.},
}
@article {pmid30965672,
year = {2019},
author = {Song, HY and Chien, CS and Yarmishyn, AA and Chou, SJ and Yang, YP and Wang, ML and Wang, CY and Leu, HB and Yu, WC and Chang, YL and Chiou, SH},
title = {Generation of GLA-Knockout Human Embryonic Stem Cell Lines to Model Autophagic Dysfunction and Exosome Secretion in Fabry Disease-Associated Hypertrophic Cardiomyopathy.},
journal = {Cells},
volume = {8},
number = {4},
pages = {},
pmid = {30965672},
issn = {2073-4409},
mesh = {Apoptosis ; *Autophagy ; CRISPR-Cas Systems ; Cardiomyopathy, Hypertrophic/etiology/*genetics/*pathology ; Cell Line ; Exosomes ; Fabry Disease/*complications ; Gene Knockout Techniques ; Human Embryonic Stem Cells/metabolism ; Humans ; *Models, Biological ; Myocytes, Cardiac/metabolism ; Reactive Oxygen Species/metabolism ; Trihexosylceramides/metabolism ; alpha-Galactosidase/*genetics ; },
abstract = {Fabry disease (FD) is a rare inherited disorder characterized by a wide range of systemic symptoms; it is particularly associated with cardiovascular and renal problems. Enzyme replacement therapy and pharmacological chaperone migalastat are the only approved and effective treatment strategies for FD patients. It is well documented that alpha-galactosidase A (GLA) enzyme activity deficiency causes globotriaosylceramide (Gb3) accumulation, which plays a crucial role in the etiology of FD. However, the detailed mechanisms remain unclear, and the lack of a reliable and powerful disease model is an obstacle. In this study, we created such a model by using CRISPR/Cas9-mediated editing of GLA gene to knockout its expression in human embryonic stem cells (hESCs). The cardiomyocytes differentiated from these hESCs (GLA-null CMs) were characterized by the accumulation of Gb3 and significant increases of cell surface area, the landmarks of FD-associated cardiomyopathy. Furthermore, we used mass spectrometry to compare the proteomes of GLA-null CMs and parental wild type CMs and found that the Rab GTPases involved in exocytotic vesicle release were significantly downregulated. This caused impairment of autophagic flux and protein turnover, resulting in an increase of reactive oxygen species and apoptosis. To summarize, we established a FD model which can be used as a promising tool to study human hypertrophic cardiomyopathy in a physiologically and pathologically relevant manner and to develop new therapies by targeting Rab GTPases signaling-related exosomal vesicles transportation.},
}
@article {pmid28565744,
year = {2017},
author = {Cheng, LH and Liu, Y and Niu, T},
title = {[Chromosomal large fragment deletion induced by CRISPR/Cas9 gene editing system].},
journal = {Zhonghua xue ye xue za zhi = Zhonghua xueyexue zazhi},
volume = {38},
number = {5},
pages = {427-431},
pmid = {28565744},
issn = {0253-2727},
mesh = {Animals ; *CRISPR-Cas Systems ; Chromosome Deletion ; DNA ; Gene Deletion ; *Gene Editing ; HEK293 Cells ; Humans ; Mice ; Multigene Family ; Mutagenesis, Insertional ; NIH 3T3 Cells ; Plasmids ; },
abstract = {Objective: Using CRISPR-Cas9 gene editing technology to achieve a number of genes co-deletion on the same chromosome. Methods: CRISPR-Cas9 lentiviral plasmid that could induce deletion of Aloxe3-Alox12b-Alox8 cluster genes located on mouse 11B3 chromosome was constructed via molecular clone. HEK293T cells were transfected to package lentivirus of CRISPR or Cas9 cDNA, then mouse NIH3T3 cells were infected by lentivirus and genomic DNA of these cells was extracted. The deleted fragment was amplified by PCR, TA clone, Sanger sequencing and other techniques were used to confirm the deletion of Aloxe3-Alox12b-Alox8 cluster genes. Results: The CRISPR-Cas9 lentiviral plasmid, which could induce deletion of Aloxe3-Alox12b-Alox8 cluster genes, was successfully constructed. Deletion of target chromosome fragment (Aloxe3-Alox12b-Alox8 cluster genes) was verified by PCR. The deletion of Aloxe3-Alox12b-Alox8 cluster genes was affirmed by TA clone, Sanger sequencing, and the breakpoint junctions of the CRISPR-Cas9 system mediate cutting events were accurately recombined, insertion mutation did not occur between two cleavage sites at all. Conclusion: Large fragment deletion of Aloxe3-Alox12b-Alox8 cluster genes located on mouse chromosome 11B3 was successfully induced by CRISPR-Cas9 gene editing system.},
}
@article {pmid28549865,
year = {2017},
author = {Powell, SK and Gregory, J and Akbarian, S and Brennand, KJ},
title = {Application of CRISPR/Cas9 to the study of brain development and neuropsychiatric disease.},
journal = {Molecular and cellular neurosciences},
volume = {82},
number = {},
pages = {157-166},
pmid = {28549865},
issn = {1095-9327},
support = {P50 MH096890/MH/NIMH NIH HHS/United States ; R01 MH101454/MH/NIMH NIH HHS/United States ; R01 MH106056/MH/NIMH NIH HHS/United States ; },
mesh = {Brain/*growth &amp; development ; Brain Diseases/therapy ; CRISPR-Cas Systems/*genetics ; *Gene Editing/methods ; Gene Expression/*genetics ; Humans ; Induced Pluripotent Stem Cells/*cytology ; },
abstract = {CRISPR/Cas9 technology has transformed our ability to manipulate the genome and epigenome, from efficient genomic editing to targeted localization of effectors to specific loci. Through the manipulation of DNA- and histone-modifying enzyme activities, activation or repression of gene expression, and targeting of transcriptional regulators, the role of gene-regulatory and epigenetic pathways in basic biology and disease processes can be directly queried. Here, we discuss emerging CRISPR-based methodologies, with specific consideration of neurobiological applications of human induced pluripotent stem cell (hiPSC)-based models.},
}
@article {pmid28549739,
year = {2017},
author = {Deng, H and Gao, R and Liao, X and Cai, Y},
title = {Characterization of a major facilitator superfamily transporter in Shiraia bambusicola.},
journal = {Research in microbiology},
volume = {168},
number = {7},
pages = {664-672},
doi = {10.1016/j.resmic.2017.05.002},
pmid = {28549739},
issn = {1769-7123},
mesh = {Ascomycota/chemistry/drug effects/*genetics/pathogenicity ; CRISPR-Cas Systems ; Membrane Transport Proteins/*genetics/*metabolism ; Mutation ; Perylene/*analogs &amp; derivatives/metabolism/pharmacology ; Phenol ; Quinones/*metabolism/pharmacology ; Reactive Oxygen Species/metabolism ; Sasa/microbiology ; },
abstract = {Reactive oxygen species (ROS) generated by photo-activated hypocrellin from Shiraia bambusicola are detrimental to cellular macromolecules. However, S. bambusicola can still maintain excellent morphology during continuous hypocrellin production, indicating an extraordinary autoresistance system that protects against the harmful ROS. In this study, a major facilitator superfamily transporter (MFS) was isolated from S. bambusicola and deleted using the clustered regularly interspaced short palindromic repeat sequences (CRISPR)/Cas9 system. The ΔMFS mutant abolished hypocrellin production and was slightly sensitive to 40-μM hypocrellin, while the ΔMFS compliment strain restored hypocrellin production and resistance. Hypocrellin treatment also enhanced the relative expression of MFS in wild-type S. bambusicola. Subsequent pathogenicity assays showed that MFS deletion reduced damage to bamboo leaves. By contrast, restoration of hypocrellin production in the MFS compliment strain generated similar necrotic lesions on bamboo leaves to those observed with the wild-type strain. These results revealed that the identified MFS is involved in efflux of hypocrellin from cells, which reduces the hypocrellin toxicity. Furthermore, hypocrellin contributed to the virulence of S. bambusicola on bamboo leaves. These findings could help to reduce plant loss by disrupting hypocrellin biosynthesis in S. bambusicola, or overexpressing the associated resistance gene in transgenic plants.},
}
@article {pmid40562814,
year = {2025},
author = {Qiao, JH and Zang, Y and Gao, Q and Liu, S and Zhang, XW and Hu, W and Wang, Y and Han, C and Li, D and Wang, XB},
title = {Transgene- and tissue culture-free heritable genome editing using RNA virus-based delivery in wheat.},
journal = {Nature plants},
volume = {},
number = {},
pages = {},
pmid = {40562814},
issn = {2055-0278},
support = {32425046//National Natural Science Foundation of China (National Science Foundation of China)/ ; },
abstract = {CRISPR-Cas genome editing technology is a cutting-edge strategy for crop breeding. However, the delivery of genome-editing reagents remains to be a technological bottleneck in monocot plants[1]. Here we engineered barley yellow striate mosaic virus (BYSMV) into a negative-strand RNA virus-based vector system[2] for delivery of both Cas9 and single guide RNA to achieve heritable gene editing in different wheat cultivars. We found that fusion of a mobile transfer RNA sequence[3] to the Cas9 messenger RNA and single guide RNAs could deliver them into the growth points of axillary meristems to achieve gene editing before tiller generation. The resulting nascent tillers contained simultaneous mutations in the three homoeoalleles. Moreover, the progeny seedlings are virus-free and harbour bi-allelic or homozygous mutations. Given BYSMV infects 26 monocot species[4], the BYSMV delivery system could have wide applicability for achieving highly efficient, non-transgenic and less genotype-dependent heritable genome editing, thereby facilitating genomic studies and crops breeding.},
}
@article {pmid40560955,
year = {2025},
author = {Leprince, A and Moineau, S},
title = {CRISPR-Cas in the Cheese Industry.},
journal = {Annual review of virology},
volume = {},
number = {},
pages = {},
doi = {10.1146/annurev-virology-092623-111016},
pmid = {40560955},
issn = {2327-0578},
abstract = {Bacteria have evolved a wide range of defense systems to combat phage infections. In the cheese industry, lactic acid bacteria (LAB) used for milk fermentation continuously face threats from phages. Therefore, selecting or developing industrial strains with enhanced phage resistance requires a focus on robust defense systems. Among these systems, the clustered regularly interspaced short palindromic repeats (CRISPR) and their CRISPR-associated proteins (Cas) are notably prevalent in LAB. The early characterization of this adaptive immune system was closely tied to the cheese industry, particularly with Streptococcus thermophilus in which CRISPR-Cas systems are ubiquitous and highly active. This review underscores the contributions of S. thermophilus and its virulent phages to our understanding of the function and mechanisms of CRISPR-Cas systems. Additionally, we review the diversity of CRISPR-Cas systems in LAB used in the cheese industry, the counter-defense strategies employed by dairy phages, and the applications of CRISPR-Cas systems within this sector.},
}
@article {pmid40560862,
year = {2025},
author = {Nakamura, K and Ito, S and Ohguchi, Y and Jimbo, T and Wada, Y and Nakajima, R and Kanou, M and Yamana, K and Ueda, H},
title = {Establishment of Cre/LoxP-mediated multifunctional reporter knock-in rats with the CRISPR system.},
journal = {PloS one},
volume = {20},
number = {6},
pages = {e0325444},
doi = {10.1371/journal.pone.0325444},
pmid = {40560862},
issn = {1932-6203},
mesh = {Animals ; Rats ; *Integrases/genetics/metabolism ; *CRISPR-Cas Systems/genetics ; *Gene Knock-In Techniques/methods ; *Genes, Reporter ; Rats, Transgenic ; Male ; },
abstract = {Rats and mice are essential experimental animals in preclinical research, serving as models for various human diseases and contributing significantly to drug development. Rats offer distinct advantages over mice due to their larger size, which allows for more complex surgical procedures, repeated blood sampling, or sophisticated behavioral analysis. However, unlike the case with mice, genetically modified rat lines for achieving complex experimental objectives-such as tissue-specific gene knockout or visualization of specific cell populations-are still limited. We here established LoxP-mediated multifunctional reporter KI rats, enabling us to evaluate fluorescence, bioluminescence, and cell-killing assays simultaneously with only one gene-modified rat line. CRISPR/Cas12a, also known as CRISPR/Cpf1, was successfully used to insert the Cre sequence into a target locus to generate Cre driver rats. These results will contribute to the application of gene-modified rats for a more comprehensive understanding of physiology, and for extrapolation of their capabilities in preclinical research.},
}
@article {pmid40560492,
year = {2025},
author = {Lv, D and Xu, Y and Wang, ZX and Zhang, QL and Yan, JP and Xu, JW},
title = {CRISPR/Cas9-mediated genome editing in Ganoderma lucidum: recent advances and biotechnological opportunities.},
journal = {World journal of microbiology &amp; biotechnology},
volume = {41},
number = {7},
pages = {223},
pmid = {40560492},
issn = {1573-0972},
support = {32360014//National Natural Science Foundation of China/ ; },
mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems ; *Reishi/genetics/metabolism ; *Biotechnology/methods ; RNA, Guide, CRISPR-Cas Systems/genetics ; Genome, Fungal ; },
abstract = {Ganoderma lucidum is a well-known traditional medicinal mushroom that has attracted considerable attention due to its potential as a promising cell factory for producing high-value bioactive compounds. However, conventional methods for the genetic manipulation of G. lucidum are often time-consuming and labor-intensive, hindering research into the biosynthesis and regulatory mechanisms of its valuable natural products. In recent years, the clustered regularly interspaced short palindromic repeats/CRISPR-associated nuclease 9 (CRISPR/Cas9) system has emerged as a powerful genome editing tool, extensively utilized in life sciences research due to its high efficiency and user-friendliness. This review provides a structured overview of advancements in CRISPR/Cas9-mediated genome editing technology in G. lucidum. We first discuss the development and optimization of the CRISPR system, focusing on the various expression strategies for Cas9 and guide RNA established in G. lucidum. Furthermore, we highlight the application of this system for targeted gene deletion, insertion, and replacement in genome editing, as well as its use in the functional analysis of genes in G. lucidum. In addition, we discuss the limitations and challenges associated with employing CRISPR/Cas9 tools in G. lucidum and provide an outlook on the future development of the CRISPR/Cas9 system and its applications in this organism.},
}
@article {pmid40560319,
year = {2025},
author = {Peer, LA and Mir, BA},
title = {Molecular mechanisms and genetic regulation of self-incompatibility in flowering plants: implications for crop improvement and evolutionary biology.},
journal = {Plant molecular biology},
volume = {115},
number = {4},
pages = {76},
pmid = {40560319},
issn = {1573-5028},
mesh = {*Self-Incompatibility in Flowering Plants/genetics ; *Crops, Agricultural/genetics ; Biological Evolution ; *Gene Expression Regulation, Plant ; Evolution, Molecular ; Pollination/genetics ; Self-Fertilization/genetics ; Plant Proteins/genetics/metabolism ; },
abstract = {Self-incompatibility is a fundamental biological mechanism in flowering plants that prevents self-fertilization, thereby promoting outcrossing and enhancing genetic diversity. This complex system has independently evolved across multiple angiosperm lineages and is crucial in maintaining plant reproductive success. Recent research has expanded our understanding of self-incompatibility's molecular basis and uncovered key genes and signaling pathways involved in self-incompatibility responses, such as S-RNase in Solanaceae and PrsS-PrpS in Papaveraceae, as well as the SRK-SCR interaction in Brassicaceae. However, despite significant advances, many aspects of self-incompatibility, such as the interplay between gene duplications, polyploidization, and the evolution of novel self-incompatibility mechanisms, remain underexplored. This review integrates findings from various plant families, including Solanaceae, Rosaceae, Papaveraceae, and Brassicaceae, and discusses the evolutionary dynamics of self-incompatibility systems, highlighting the role of gene duplication, recombination, and translocation events in shaping self-incompatibility diversity. Special emphasis is placed on understanding how modern molecular techniques, such as CRISPR/Cas9 and marker-assisted selection, can be employed to transition self-incompatibility to self-compatibility in economically significant crops. Additionally, the role of epigenetic changes and modifier genes in mediating transitions from self-incompatibility to self-compatibility is addressed, offering insights into how these mechanisms can be leveraged for crop breeding and hybrid seed production. Future research should focus on elucidating the molecular mechanisms underlying self-incompatibility responses, exploring the potential of targeted gene editing to overcome reproductive barriers, and understanding the evolutionary resilience of self-incompatibility systems to environmental changes.},
}
@article {pmid40559612,
year = {2025},
author = {Jurić, I and Jelić, M and Markanović, M and Kanižaj, L and Bošnjak, Z and Budimir, A and Kuliš, T and Tambić-Andrašević, A and Ivančić-Baće, I and Mareković, I},
title = {CRISPR-Cas Dynamics in Carbapenem-Resistant and Carbapenem-Susceptible Klebsiella pneumoniae Clinical Isolates from a Croatian Tertiary Hospital.},
journal = {Pathogens (Basel, Switzerland)},
volume = {14},
number = {6},
pages = {},
doi = {10.3390/pathogens14060604},
pmid = {40559612},
issn = {2076-0817},
support = {10106-24-1295 to I.M. and 20286539 to I.I.B.//School od Medicine, University of Zagreb and Faculty of Science, University of Zagreb/ ; },
mesh = {*Klebsiella pneumoniae/genetics/drug effects/isolation &amp; purification/classification ; Humans ; *CRISPR-Cas Systems/genetics ; *Carbapenems/pharmacology ; *Klebsiella Infections/microbiology ; beta-Lactamases/genetics ; Croatia ; Tertiary Care Centers ; Anti-Bacterial Agents/pharmacology ; Microbial Sensitivity Tests ; Bacterial Proteins/genetics ; Multilocus Sequence Typing ; Drug Resistance, Multiple, Bacterial/genetics ; *Carbapenem-Resistant Enterobacteriaceae/genetics/drug effects/isolation &amp; purification ; },
abstract = {(1) Background: CRISPR-Cas systems provide adaptive immunity against mobile genetic elements (MGEs) carrying antimicrobial resistance (AMR) genes. Carbapenem-resistant (CR) Klebsiella pneumoniae is a major public health concern, and the role of CRISPR-Cas in its resistance is understudied. This study explored CRISPR-Cas associations with multidrug resistance in clinical K. pneumoniae. (2) Methods: 400 K. pneumoniae isolates (200 CR and 200 carbapenem susceptible (CS)) were analyzed. Carbapenemase genes (blaOXA-48, blaNDM-1, blaKPC-2), cas1, rpoB, and CRISPR1-3 loci were identified by PCR, while only CRISPR loci were sequenced. Genetic relatedness was assessed via PFGE, MLST, and spacer analysis. Statistical analysis utilized chi-squared and Fisher's exact tests. (3) Results: CRISPR-Cas was present in 15.8% of isolates, mainly subtypes I-E and I-E* (93.3%), with CRISPR3 loci showing the greatest spacer diversity. Clonal complexes ST14/15/101 (CR) and ST35 (CS) were identified. blaOXA-48 was linked to CRISPR-Cas-negative strains, while blaNDM-1 and blaKPC-2 were more frequent in CRISPR-Cas-positive strains (p < 0.0001). Imipenem/relebactam resistance was higher in CRISPR-Cas-negative isolates. (4) Conclusions: K. pneumoniae CRISPR-Cas systems correlate with specific carbapenemase profiles, suggesting pressure against blaOXA-48 acquisition. The coexistence of I-E and I-E* subtypes highlight synergies in targeting MGEs. CRISPR loci could be tools for subtyping organisms following MLST.},
}
@article {pmid40558493,
year = {2025},
author = {Parikh, SJ and Terron, HM and Burgard, LA and Butler, DD and LaFerla, FM and Lane, S and Leissring, MA},
title = {5' DREDGE: Direct Repeat-Enabled Downregulation of Gene Expression via the 5' UTR of Target Genes.},
journal = {Cells},
volume = {14},
number = {12},
pages = {},
doi = {10.3390/cells14120866},
pmid = {40558493},
issn = {2073-4409},
support = {1R01AG066928-04A1/NH/NIH HHS/United States ; },
mesh = {*5' Untranslated Regions/genetics ; *Down-Regulation/genetics ; Humans ; CRISPR-Cas Systems/genetics ; RNA, Messenger/genetics/metabolism ; },
abstract = {Despite the availability of numerous methods for controlling gene expression, there remains a strong need for technologies that maximize two key properties: selectivity and reversibility. To this end, we developed a novel approach that exploits the highly sequence-specific nature of CRISPR-associated endoribonucleases (Cas RNases), which recognize and cleave short RNA sequences known as direct repeats (DRs). In this approach, referred to as DREDGE (direct repeat-enabled downregulation of gene expression), selective control of gene expression is enabled by introducing one or more DRs into the untranslated regions (UTRs) of target mRNAs, which can then be cleaved upon expression of the cognate Cas RNase. We previously demonstrated that the expression of target genes with DRs in their 3' UTRs are efficiently controlled by the DNase-dead version of Cas12a (dCas12a) with a high degree of selectivity and complete reversibility. Here, we assess the feasibility of using DREDGE to regulate the expression of genes with DRs inserted in their 5' UTRs. Among the five different Cas RNases tested, Csy4 was found to be the most efficient in this context, yielding robust downregulation with rapid onset in doxycycline-regulatable systems targeting either a stably expressed fluorescent protein or an endogenous gene, both in a fully reversible manner. Unexpectedly, dCas12a was also found to be modestly effective despite binding essentially irreversibly to the cut mRNA on its 5' end and thereby boosting mRNA levels. Our results expand the utility of DREDGE as an attractive method for regulating gene expression in a targeted, highly selective, and fully reversible manner.},
}
@article {pmid40558461,
year = {2025},
author = {Jeddoub, O and Touil, N and Nyabi, O and El Fahime, E and Ennibi, K and Gala, JL and Benjouad, A and Belayachi, L},
title = {The Use of CRISPR-Cas Systems for Viral Detection: A Bibliometric Analysis and Systematic Review.},
journal = {Biosensors},
volume = {15},
number = {6},
pages = {},
doi = {10.3390/bios15060379},
pmid = {40558461},
issn = {2079-6374},
mesh = {*CRISPR-Cas Systems ; Bibliometrics ; Humans ; *Viruses/isolation &amp; purification/genetics ; *Virus Diseases/diagnosis ; },
abstract = {Viral infections impose a significant burden on global public health and the economy. This study examines the current state of CRISPR-Cas system research, focusing on their applications in viral detection and their evolution over recent years. A bibliometric analysis and systematic review were conducted using articles published between 2019 and 2024, retrieved from Web of Science, Scopus, and PubMed databases. Out of 2713 identified articles, 194 were included in the analysis. The findings reveal substantial growth in scientific output related to CRISPR-Cas systems, with the United States leading in research and development in this field. The rapid increase in CRISPR-Cas research during this period underscores its immense potential to transform viral diagnostics. With advantages such as speed, precision, and suitability for deployment in resource-limited settings, CRISPR-Cas systems outperform many traditional diagnostic methods. The concerted efforts of scientists worldwide further highlight the promising future of this technology. CRISPR-Cas systems are emerging as a powerful alternative, offering the possibility of expedited and accessible point-of-care testing and paving the way for more equitable and effective diagnostics on a global scale.},
}
@article {pmid40558442,
year = {2025},
author = {Du, K and Zeng, Q and Jiang, M and Hu, Z and Zhou, M and Xia, K},
title = {CRISPR/Cas12a-Based Biosensing: Advances in Mechanisms and Applications for Nucleic Acid Detection.},
journal = {Biosensors},
volume = {15},
number = {6},
pages = {},
doi = {10.3390/bios15060360},
pmid = {40558442},
issn = {2079-6374},
support = {No. 82330035//National Natural Science Foundation of China/ ; No. 82130043//National Natural Science Foundation of China/ ; No. 82361138573//National Natural Science Foundation of China/ ; No. 2021SK1010//National Natural Science Foundation of Hunan Province/ ; No. 2024JJ5340//Natural Science Foundation of Hunan Province/ ; No. 2024RC3207//Science and Technology innovation Program of Hunan Province/ ; No. 23B0437//Scientific Research Fund of Hunan Provincial Education Department/ ; },
mesh = {*Biosensing Techniques/methods ; *CRISPR-Cas Systems ; Humans ; *Nucleic Acids/analysis ; DNA/analysis ; },
abstract = {Nucleic acid detection technology is crucial for molecular diagnosis. The advent of CRISPR/Cas12a-based nucleic acid detection has considerably broadened its scope, from the identification of infectious disease-causing microorganisms to the detection of disease-associated biomarkers. This innovative system capitalizes on the non-specific single-strand cleavage activity of Cas12a upon target DNA recognition. By employing a fluorescent probe in the form of a single-stranded DNA/RNA, this technology enables the observation of fluorescence changes resulting from nonspecific cleavage, thereby facilitating detection. CRISPR/Cas12a-based detection systems can be regarded as a new type of biosensor, offering a practical and efficient approach for nucleic acid analysis in various diagnostic settings. CRISPR/Cas12a-based biosensors outperform conventional nucleic acid detection methods in terms of portability, simplicity, speed, and efficiency. In this review, we elucidate the detection principle of CRISPR/Cas12a-based biosensors and their application in disease diagnostics and discuss recent innovations and technological challenges, aiming to provide insights for the research and further development of CRISPR/Cas12a-based biosensors in personalized medicine. Our findings show that although CRISPR/Cas12a-based biosensors have considerable potential for various applications and theoretical research, certain challenges remain. These include simplifying the reaction process, enhancing precision, broadening the scope of disease detection, and facilitating the translation of research findings into clinical practice. We anticipate that ongoing advancements in CRISPR/Cas12a-based biosensors will address these challenges.},
}
@article {pmid40558428,
year = {2025},
author = {De Silva, PIT and Hiniduma, K and Canete, R and Bhalerao, KS and Shawky, SM and Gunathilaka, H and Rouge, JL and Mosa, IM and Steffens, DC and Manning, K and Diniz, BS and Rusling, JF},
title = {Multiplexed CRISPR Assay for Amplification-Free Detection of miRNAs.},
journal = {Biosensors},
volume = {15},
number = {6},
pages = {},
doi = {10.3390/bios15060346},
pmid = {40558428},
issn = {2079-6374},
support = {2231490//U.S. National Science Foundation/ ; },
mesh = {*MicroRNAs/blood/analysis ; Humans ; *Biosensing Techniques ; *CRISPR-Cas Systems ; Limit of Detection ; },
abstract = {CRISPR-Cas proteins from bacteria are powerful tools for gene editing and molecular diagnostics. Expanding capacity of CRISPR to low cost, multiplexed assays of biomarkers is a key to future disease diagnostics, since multiple biomarker detection is essential for reliable diagnostics. Herein we describe a multiplexed assay in a 3D-printed 96-well plate with CRISPR-Cas13a immobilized in each well to target three circulating blood biomarker microRNAs (miRNAs 34c-5p, 200c-3p, and 30e-5p) for Alzheimer's disease (ALZ). Immobilized Cas13a is equipped with different crRNAs complementary to each miRNA target. MiRNA binding to crRNA complements activates the collateral RNase activity of Cas13a, cleaving a quenched fluorescent reporter (RNaseAlert) with fluorophore and quencher connected by an RNA oligonucleotide to enable fluorescence measurements. We achieved ultralow limits of detection (LOD) of 0.74 fg/mL for miRNA 34c-5p, 0.70 fg/mL for miRNA 30e-5p, and 7.4 fg/mL for miRNA 200c-3p, with dynamic ranges from LODs up to about 1800 pg/mL. The accuracy of the assay was validated by spike-recovery studies and good correlation of levels of patient plasma samples vs. a referee method. This new approach provides selective, sensitive multiplex miRNA biosensing, and simultaneously accommodates analysis of standards and controls.},
}
@article {pmid40558037,
year = {2025},
author = {Taran, JA and Mintaev, RR and Glazkova, DV and Belugin, BV and Bogoslovskaya, EV and Shipulin, GA},
title = {[Influence of Homology Arm Length and Structure on the Efficiency of Long Transgene Integration into a Cleavage Site Induced by SpCas9 or AsCpf1].},
journal = {Molekuliarnaia biologiia},
volume = {59},
number = {2},
pages = {255-265},
pmid = {40558037},
issn = {0026-8984},
mesh = {Humans ; *Receptors, CCR5/genetics/metabolism ; Dependovirus/genetics ; *CRISPR-Cas Systems ; *Transgenes ; Green Fluorescent Proteins/genetics/metabolism ; *CRISPR-Associated Protein 9/genetics/metabolism ; Genetic Vectors/genetics ; *Bacterial Proteins/genetics/metabolism ; },
abstract = {One of the promising new approaches to the treatment of HIV infection is CRISPR/Cas-mediated knockout of the CCR5 receptor gene followed by the integration of an anti-HIV gene into the break site. Numerous studies have focused on the knockout of the CCR5 gene; however, the efficiency of subsequent targeted integration of long fragments remains poorly studied. To evaluate the efficiency of this approach, we used HT1080 cells and investigated the integration of a cassette expressing the EGFP gene into the CCR5 locus using two different nucleases (SpCas9 and AsCpf1) and various donor DNA constructs delivered by recombinant adeno-associated viral vectors (rAAV). For each nuclease, we designed five variants of donor DNA differing in the length (ranging from 150 to 1000 bp) or structure of the homology arms. The efficiency of transgene integration with 150 bp homology arms was the lowest for both nucleases and differed significantly from constructs with longer homology arms. Furthermore, it was shown that the presence of nuclease cleavage sites in the donor DNA flanking the cassette with homology arms did not affect the efficiency of transgene integration during AAV delivery. We demonstrated that the AsCpf1 nuclease provided higher efficiency of EGFP transgene integration than SpCas9, despite the lower efficiency of CCR5 knockout. The maximum percentage of cells with the integrated transgene was achieved using the AsCpf1 nuclease and an expression cassette with 600 bp homology arms, reaching 59 ± 6%.},
}
@article {pmid40558035,
year = {2025},
author = {Mintaev, RR and Glazkova, DV and Taran, JA and Bogoslovskaya, EV and Shipulin, GA},
title = {[Improving the Efficiency and Safety of Human CCR5 Gene Editing by Selection of Optimal Guide RNAs for SpCAS9 and CAS12A].},
journal = {Molekuliarnaia biologiia},
volume = {59},
number = {2},
pages = {234-243},
pmid = {40558035},
issn = {0026-8984},
mesh = {*Receptors, CCR5/genetics/metabolism ; Humans ; *Gene Editing/methods ; *RNA, Guide, CRISPR-Cas Systems/genetics/metabolism ; *CRISPR-Cas Systems ; *CRISPR-Associated Protein 9/genetics/metabolism ; *HIV Infections/genetics/therapy ; *CRISPR-Associated Proteins/genetics/metabolism ; HEK293 Cells ; *Endodeoxyribonucleases/genetics/metabolism ; *Bacterial Proteins/genetics/metabolism ; },
abstract = {Advances in CRISPR/Cas-mediated genome editing have opened up treatment alternatives for many human diseases, including HIV infection. Knockout of the CCR5 gene as a potential way to treat HIV infection has long been studied. Here we analyzed guide RNAs for SpCas9 and AsCas12a nucleases targeting CCR5 gene which had been previously studied and selected the most effective among them. We also designed novel guide RNAs for the same nucleases using bioinformatics resources. We compared the efficiency of target site cleavage for all selected gRNAs using three nucleases: wt SpCas9, SpCas9-HF1-plus, and AsCas12a, as well as their off- target activities. We demonstrated that among the tested guide RNAs two for SpCas9- HF1-plus and three for AsCas12a exhibited high cleavage activity, cutting CCR5 gene in 60-72% of cells, and had off-target activities below the limit of detection. Thus, these guide RNAs may be candidates for future development of gene therapies against HIV infection.},
}
@article {pmid40557869,
year = {2025},
author = {Li, X and Zhu, Z and Wu, J and Li, C and Liu, Z and Wang, J and Li, P and Zhang, Z and Huang, Y and Hong, J and Wu, T},
title = {PAM-free hairpin target binding activates trans-cleavage activity of Cas12a.},
journal = {Nucleic acids research},
volume = {53},
number = {12},
pages = {},
doi = {10.1093/nar/gkaf596},
pmid = {40557869},
issn = {1362-4962},
support = {22474045//National Natural Science Foundation of China/ ; 82172372//National Natural Science Foundation of China/ ; 82303589//National Natural Science Foundation of China/ ; 82000512//National Natural Science Foundation of China/ ; 2024JYCXJJ010//Central Universities, HUST/ ; },
mesh = {*CRISPR-Associated Proteins/metabolism/chemistry/genetics ; *CRISPR-Cas Systems ; Molecular Docking Simulation ; *Endodeoxyribonucleases/metabolism/chemistry/genetics ; *Bacterial Proteins/metabolism/chemistry/genetics ; *DNA/chemistry/metabolism/genetics ; Calcium/analysis ; Nucleic Acid Conformation ; Protein Binding ; },
abstract = {CRISPR-Cas12a has been demonstrated to be activated for its trans-cleavage activity by single- and double-stranded DNA containing a protospacer adjacent motif (PAM), but other types of activators have remained undiscovered. In this work, we found that a hairpin-structured substrate can activate the trans-cleavage activity of Cas12a without a PAM, and the parameters of the hairpin loop obviously affect the activity. Cas12a exhibits sequence preference for proximal loops, preferring to recognize polyadenine hairpin loop activators. Molecular docking and dynamic calculations provide a theoretical basis for the activation of Cas12a by hairpin activators. Leveraging the efficient activation capability of the hairpin activator, we constructed an allosteric detection platform for non-nucleic acid targets, capable of sensitively and specifically detecting hypochlorous acid and calcium ions. This novel activator of Cas12a holds enormous potential for the development of multi-functional biological platforms.},
}
@article {pmid40482035,
year = {2025},
author = {Dai, P and Liu, T and Yang, D and Luo, Y and Liu, LD and Xie, X and Shang, Y and Liu, X and Meng, FL},
title = {Protocol for genetic dissection of class switch recombination using genome-editing tools.},
journal = {STAR protocols},
volume = {6},
number = {2},
pages = {103882},
pmid = {40482035},
issn = {2666-1667},
mesh = {*Gene Editing/methods ; CRISPR-Cas Systems/genetics ; *Immunoglobulin Class Switching/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; Humans ; Animals ; Electroporation/methods ; *Recombination, Genetic ; Mice ; },
abstract = {Antibody class switch recombination is achieved through programmed DNA damage, and the processing of programmed DNA lesions requires the coordinated action of many DNA metabolic factors. Here, we present a protocol for inducing class switch recombination using base editors or CRISPR-Cas9. We provide optimized guide RNA (gRNA) sequences and describe steps for cytokine activation, electroporation, surface immunoglobulin detection, and data analysis. This method allows researchers to investigate the involvement of specific factors in antibody diversification and elucidate their functional roles. For complete details on the use and execution of this protocol, please refer to Dai et al.[1].},
}
@article {pmid40436039,
year = {2025},
author = {Tang, S and Žedaveinytė, R and Burman, N and Pandey, S and Ramirez, JL and Kulber, LM and Wiegand, T and Wilkinson, RA and Ma, Y and Zhang, DJ and Lampe, GD and Berisa, M and Jovanovic, M and Wiedenheft, B and Sternberg, SH},
title = {Protein-primed homopolymer synthesis by an antiviral reverse transcriptase.},
journal = {Nature},
volume = {},
number = {},
pages = {},
pmid = {40436039},
issn = {1476-4687},
abstract = {Bacteria defend themselves from viral predation using diverse immune systems, many of which target foreign DNA for degradation[1]. Defence-associated reverse transcriptase (DRT) systems provide an intriguing counterpoint to this strategy by using DNA synthesis instead[2,3]. We and others recently showed that DRT2 systems use an RNA template to assemble a de novo gene that encodes the antiviral effector protein Neo[4,5]. It remains unclear whether similar mechanisms of defence are used by other related DRT families. Here, we show that DRT9 systems defend against phage using DNA homopolymer synthesis. Viral infection triggers polydeoxyadenylate (poly-dA) accumulation in the cell, driving abortive infection and population-level immunity. Cryo-electron microscopy structures reveal how a non-coding RNA serves as both a structural scaffold and reverse transcription template to direct hexameric complex assembly and poly-dA synthesis. Notably, biochemical and functional experiments identify tyrosine residues within the reverse transcriptase itself that probably prime DNA synthesis, leading to the formation of protein-DNA covalent adducts. Synthesis of poly-dA by DRT9 in vivo is regulated by the competing activities of phage-encoded triggers and host-encoded silencers. Collectively, our study identifies a nucleic-acid-driven defence system that expands the paradigm of bacterial immunity and broadens the known functions of reverse transcriptases.},
}
@article {pmid40418630,
year = {2025},
author = {Biar, CG and Bodkin, N and Carvill, GL and Calhoun, JD},
title = {Protocol to perform multiplexed assays of variant effect using curated loci prime editing.},
journal = {STAR protocols},
volume = {6},
number = {2},
pages = {103851},
pmid = {40418630},
issn = {2666-1667},
mesh = {*Gene Editing/methods ; Humans ; *Genetic Variation/genetics ; CRISPR-Cas Systems/genetics ; High-Throughput Nucleotide Sequencing/methods ; },
abstract = {Multiplexed assays of variant effect (MAVEs) perform simultaneous characterization of many variants. Here, we present a protocol to perform MAVEs using curated loci prime editing (cliPE), an accessible experimental pipeline that enables prime editing of a target gene. We describe steps for designing prime editing reagents, screening for genome editing efficiency, selecting a pool of cells edited to harbor different genetic variants, and sequencing. Lastly, we detail procedures for performing enrichment analysis to identify variants with normal or aberrant activity.},
}
@article {pmid40397576,
year = {2025},
author = {Zhong, X and Wu, H and Wang, G and Sun, Z},
title = {Protocol for CRISPR-mediated deletion of cis-regulatory element in murine Th17 cells for in vivo assessment of effector function.},
journal = {STAR protocols},
volume = {6},
number = {2},
pages = {103831},
pmid = {40397576},
issn = {2666-1667},
support = {P30 CA033572/CA/NCI NIH HHS/United States ; R01 AI109644/AI/NIAID NIH HHS/United States ; R21 AI163256/AI/NIAID NIH HHS/United States ; },
mesh = {Animals ; Mice ; *Th17 Cells/metabolism/immunology/cytology ; Encephalomyelitis, Autoimmune, Experimental/genetics/immunology ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; Cell Differentiation ; *Regulatory Sequences, Nucleic Acid/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Mice, Inbred C57BL ; },
abstract = {Studying the cis-regulatory elements (CREs) of genes in Th17 cells during autoimmune disease progression, such as experimental autoimmune encephalomyelitis (EAE), is often limited by the availability of gene-edited mice. Here, we present a protocol for CRISPR-mediated deletion of a CRE in murine Th17 cells for in vivo assessment of effector function in EAE. We describe steps for dual U6gRNA construction, preparation of retroviruses, viral delivery, and Th17 differentiation. We then detail procedures for in vivo functionality analysis. For complete details on the use and execution of this protocol, please refer to Zhong et al.[1][,][2].},
}
@article {pmid40310725,
year = {2025},
author = {Hao, M and Lu, P and Guan, JL},
title = {Protocol for in vivo CRISPR knockout screening of autophagy genes to identify breast cancer metastasis suppressors.},
journal = {STAR protocols},
volume = {6},
number = {2},
pages = {103798},
pmid = {40310725},
issn = {2666-1667},
mesh = {Animals ; *Autophagy/genetics ; *Breast Neoplasms/genetics/pathology ; Mice ; Female ; *CRISPR-Cas Systems/genetics ; *Gene Knockout Techniques/methods ; Humans ; Neoplasm Metastasis/genetics ; Lung Neoplasms/secondary/genetics ; },
abstract = {Metastasis represents the primary cause of mortality among patients with breast cancer. Here, we present a protocol for utilizing an in vivo custom CRISPR-Cas9 knockout library to systematically investigate autophagy regulatory genes implicated in breast cancer metastasis to the lung in mice. We describe steps for library synthesis, cloning, and virus packaging and transfection. We then detail procedures for genome DNA collection and transplantation, followed by analysis of screening data. This protocol enables efficient identification of potential suppressors of breast cancer metastasis in vivo. For complete details on the use and execution of this protocol, please refer to Hao et al.[1].},
}
@article {pmid40215169,
year = {2025},
author = {Ortega, P and Sanchez, A and Seldin, M and Buisson, R},
title = {Oligo-seq protocol for mapping DNA motifs targeted by base editors.},
journal = {STAR protocols},
volume = {6},
number = {2},
pages = {103758},
pmid = {40215169},
issn = {2666-1667},
mesh = {*Gene Editing/methods ; *Nucleotide Motifs/genetics ; CRISPR-Cas Systems/genetics ; *Sequence Analysis, DNA/methods ; *DNA/genetics/chemistry ; Humans ; High-Throughput Nucleotide Sequencing/methods ; },
abstract = {Determining which DNA sequences are preferentially targeted by base editors is critical for understanding how APOBECs, AID, and other CRISPR-Cas9 base editors edit DNA in cells or improve their editing efficiency. We have developed Oligo-seq, an in vitro sequencing-based method to identify the preferred sequence motifs targeted by these enzymes. This assay monitors DNA deaminase activity on DNA oligonucleotides containing random nucleotides and/or DNA structures and determines by sequencing which sequences are preferentially deaminated. For complete details on the use and execution of this protocol, please refer to Sanchez et al.[1].},
}
@article {pmid40198219,
year = {2025},
author = {Obolenski, S and Olvera-León, R and Sun, D and Adams, DJ and Waters, AJ},
title = {Protocol for the functional evaluation of genetic variants using saturation genome editing.},
journal = {STAR protocols},
volume = {6},
number = {2},
pages = {103710},
pmid = {40198219},
issn = {2666-1667},
mesh = {*Gene Editing/methods ; CRISPR-Cas Systems/genetics ; Humans ; *Genetic Variation/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; High-Throughput Nucleotide Sequencing/methods ; Gene Library ; },
abstract = {Saturation genome editing (SGE) employs CRISPR-Cas9 and homology-directed repair (HDR) to introduce exhaustive nucleotide modifications at specific genomic sites in multiplex, enabling the functional analysis of genetic variants while preserving their native genomic context. Here, we present a protocol for SGE-based variant evaluation in HAP1-A5 cells. We describe the steps for designing variant libraries, single-guide RNAs (sgRNAs), and oligonucleotide primers for PCR. We also detail the sample preparation before the SGE screen, the cellular screening process, and subsequent next-generation sequencing (NGS) library preparation. For complete details on the use and execution of this protocol, please refer to Radford et al.,[1] Waters et al.,[2] and Olvera-León et al.[3].},
}
@article {pmid40173037,
year = {2025},
author = {Della Volpe, L and Vacca, R and Di Micco, R},
title = {Protocol for optimizing culture conditions for ex vivo activation during CRISPR-Cas9 gene editing in human hematopoietic stem and progenitor cells.},
journal = {STAR protocols},
volume = {6},
number = {2},
pages = {103722},
pmid = {40173037},
issn = {2666-1667},
mesh = {Humans ; *Gene Editing/methods ; *Hematopoietic Stem Cells/cytology/metabolism ; *CRISPR-Cas Systems/genetics ; *Cell Culture Techniques/methods ; },
abstract = {Long-range correction strategies require ex vivo activation of hematopoietic stem and progenitor cells (HSPCs) to engage the homology-directed repair (HDR) mechanism, but prolonged culture causes harmful cellular responses, reducing the long-term functionality of gene-edited (GE) HSPCs. Here, we present a protocol for optimizing culture conditions for ex vivo activation during CRISPR-Cas9 gene editing in human HSPCs. We describe steps for HSPC thawing, ex vivo treatments, gene editing, and downstream in vitro and in vivo analyses to assess the functionality of GE-HSPCs. For complete details on the use and execution of this protocol, please refer to della Volpe et al.[1].},
}
@article {pmid40120111,
year = {2025},
author = {Liang, Z and Wu, X and Ye, Z and She, L and Ma, X and Huo, YX},
title = {Protocol for identification of sgRNA mutants using high-throughput screening technique and multiplex genome editing.},
journal = {STAR protocols},
volume = {6},
number = {2},
pages = {103690},
pmid = {40120111},
issn = {2666-1667},
mesh = {*Gene Editing/methods ; *RNA, Guide, CRISPR-Cas Systems/genetics ; *CRISPR-Cas Systems/genetics ; *High-Throughput Screening Assays/methods ; *Mutation ; Humans ; Gene Library ; },
abstract = {In the CRISPR-Cas9 system, tandem expression of multiple identical single-guide RNAs (sgRNAs) often triggers homologous sequences loss, which affects multiplex genome editing efficiencies. Here, we present a protocol for high-throughput screening of functional sgRNAs with nonrepetitive mutants. We describe steps for constructing the screening platform, designing and constructing sgRNA libraries, and screening sgRNA mutants. These mutants can interact with the Cas9 protein, enabling multiplex genome editing. For complete details on the use and execution of this protocol, please refer to Liang et al.[1].},
}
@article {pmid40088451,
year = {2025},
author = {Kohabir, KAV and Linthorst, J and Wolthuis, RMF and Sistermans, EA},
title = {Protocol for high-precision CRISPR-Cas12a-based SNV detection on synthetic DNA, cell line cfDNA models, and liquid biopsies.},
journal = {STAR protocols},
volume = {6},
number = {2},
pages = {103696},
pmid = {40088451},
issn = {2666-1667},
mesh = {*CRISPR-Cas Systems/genetics ; Humans ; Liquid Biopsy/methods ; *Cell-Free Nucleic Acids/genetics ; *Polymorphism, Single Nucleotide/genetics ; *DNA/genetics ; Cell Line ; Gene Editing/methods ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {CRISPR-based diagnostics (CRISPRdx) offer promising tools for rapid and cost-effective genetic testing, but achieving single-nucleotide fidelity remains a challenge. Here, we present a protocol for high-precision detection of single-nucleotide variants (SNVs) using a Cas12a-based approach. We describe how to apply our publicly available ARTEMIS algorithm to identify targetable SNVs, design optimized CRISPR RNAs (crRNAs), and perform fluorescence-based CRISPRdx assays on synthetic DNA, cell line-derived cell-free DNA (cfDNA), and liquid biopsy samples. For complete details on the use and execution of this protocol, please refer to Kohabir et al.[1].},
}
@article {pmid40555885,
year = {2025},
author = {Asah-Asante, R and Tang, L and Gong, X and Fan, S and Yan, C and Asante, JO and Zeng, Q},
title = {Exploring pigment-producing Streptomyces as an alternative source to synthetic pigments: diversity, biosynthesis, and biotechnological applications. A review.},
journal = {World journal of microbiology &amp; biotechnology},
volume = {41},
number = {7},
pages = {211},
pmid = {40555885},
issn = {1573-0972},
mesh = {*Streptomyces/metabolism/genetics/classification ; *Pigments, Biological/biosynthesis/chemistry ; Biosynthetic Pathways/genetics ; *Biotechnology/methods ; Multigene Family ; CRISPR-Cas Systems ; Melanins/biosynthesis ; Gene Editing ; Anthraquinones/metabolism ; Prodigiosin/analogs &amp; derivatives/biosynthesis ; Synthetic Biology ; Fermentation ; Benzoisochromanequinones ; },
abstract = {The increasing health and environmental concerns associated with synthetic pigments have intensified the global search for natural, eco-friendly alternatives. Among microbial sources, Streptomyces, a genus within the class Actinomycetia, has emerged as a prolific source of bioactive pigments with wide-ranging industrial applications. The review provides a comprehensive synthesis of pigment-producing Streptomyces, focusing on their ecological diversity, biosynthetic pathways, and taxonomic relevance. This review has discussed key pigment classes, including melanin, prodiginine, quinone, and actinorhodin, with their bioactive properties, such as antioxidants, antimicrobials, and anticancer. The review further emphasizes recent advancements in synthetic biology, including clustered regularly interspaced short palindromic repeats/CRISPR-associated (CRISPR/Cas)-based gene editing, which has facilitated the activation of silent biosynthetic gene clusters (BGCs) that can enhance pigment yield. Additionally, this review discusses the optimization and fermentation protocols, industrial scalability, and the application of pigments in food, pharmaceutical, cosmetic, textile, and environmental sectors. Despite these advancements, critical research gaps persist, particularly in toxicological evaluation, pigment stability under industrial conditions, high-throughput screening of unexplored Streptomyces strains, and the integration of AI-based predictive tools for pathway optimization. Addressing these gaps is essential to unlocking the full potential of Streptomyces-derived pigments to replace harmful synthetic colorants.},
}
@article {pmid40554662,
year = {2025},
author = {Li, C and Liu, X and Li, Y and Peng, Q and Miao, J and Liu, X},
title = {The Tetracycline-Inducible/CRISPR-Cas9 System is an Efficient Tool for Studying Gene Function in Phytophthora sojae.},
journal = {Molecular plant pathology},
volume = {26},
number = {6},
pages = {e70114},
doi = {10.1111/mpp.70114},
pmid = {40554662},
issn = {1364-3703},
support = {32372602//National Natural Science Foundation of China/ ; 32272599//National Natural Science Foundation of China/ ; 2024ZC-KJXX-062//the Project of Shaanxi Young Stars in Science and Technology/ ; },
mesh = {*Phytophthora/genetics/drug effects ; *CRISPR-Cas Systems/genetics/drug effects ; *Tetracycline/pharmacology ; Promoter Regions, Genetic/genetics ; },
abstract = {The present study presents a novel approach combining a tetracycline-inducible system (Tet-On) and CRISPR-Cas9 techniques to investigate the function of two essential genes in Phytophthora sojae. We constructed a donor vector in which the reverse tetracycline transactivator (rtTA) is driven by an oomycete promoter. Additionally, it contains a fused TetR binding site and the minimum oomycete promoter, as well as 1000-bp homologous arms of the promoter upstream and downstream sequences. The promoter of the target gene was replaced with a tetracycline-responsive promoter (Ptet) using a CRISPR-Cas9 system. In the native transformants, the target gene was induced by the administration of tetracycline and repressed in its absence. Using the Tet-On/CRISPR-Cas9 system, we obtained inducible transformants of PsAF5 and PsCesA3. The phenotype of PsAF5 inducible transformants without doxycycline was consistent with that of ΔPsAF5 transformants, specifically characterised by an increase in oospore production and heightened sensitivity to H2O2. PsCesA3 inducible transformants could not grow in the absence of doxycycline, which means PsCesA3 is an essential protein for P. sojae. In conclusion, the Tet-On/CRISPR-Cas9 system represents an effective approach for investigating crucial genes in P. sojae.},
}
@article {pmid40554553,
year = {2025},
author = {Wei, SC and Cantor, AJ and Walleshauser, J and Mepani, R and Melton, K and Bans, A and Khekare, P and Gupta, S and Wang, J and Soares, C and Kiwan, R and Lee, J and McCawley, S and Jani, V and Leong, WI and Shahi, PK and Chan, J and Boivin, P and Otoupal, P and Pattnaik, BR and Gamm, DM and Saha, K and Gowen, BG and Haak-Frendscho, M and Janatpour, MJ and Silverman, AP},
title = {Evaluation of subretinally delivered Cas9 ribonucleoproteins in murine and porcine animal models highlights key considerations for therapeutic translation of genetic medicines.},
journal = {PloS one},
volume = {20},
number = {6},
pages = {e0317387},
doi = {10.1371/journal.pone.0317387},
pmid = {40554553},
issn = {1932-6203},
mesh = {Animals ; Mice ; *Gene Editing/methods ; *Ribonucleoproteins/genetics/administration &amp; dosage ; Swine ; *CRISPR-Cas Systems ; *CRISPR-Associated Protein 9/genetics/administration &amp; dosage ; Retina/metabolism ; *Genetic Therapy/methods ; Retinal Pigment Epithelium/metabolism ; Disease Models, Animal ; },
abstract = {Genetic medicines, including CRISPR/Cas technologies, extend tremendous promise for addressing unmet medical need in inherited retinal disorders and other indications; however, there remain challenges for the development of therapeutics. Herein, we evaluate genome editing by engineered Cas9 ribonucleoproteins (eRNP) in vivo via subretinal administration using mouse and pig animal models. Subretinal administration of adenine base editor and double strand break-inducing Cas9 nuclease eRNPs mediate genome editing in both species. Editing occurs in retinal pigmented epithelium (RPE) and photoreceptor cells, with favorable tolerability in both species. Using transgenic reporter strains, we determine that editing primarily occurs close to the site of administration, within the bleb region associated with subretinal injection. Our results show that subretinal administration of BE-eRNPs in mice mediates base editing of up to 12% of the total neural retina, with an average rate of 7% observed at the highest dose tested. In contrast, a substantially lower editing efficiency was observed in minipigs; even with direct quantification of only the treated region, a maximum base editing rate of 1.5%, with an average rate of <1%, was observed. Our data highlight the importance of species consideration in preclinical studies for the development of genetic medicines targeting the eye and provide an example of a lack of translation between small and larger animal models in the context of subretinal administration of Cas9 eRNPs.},
}
@article {pmid40553877,
year = {2025},
author = {Saleem, S and Amin, W and Bhatti, F and Majid, M and Fazal, A},
title = {Nucleic acid-based strategies to mitigate stripe rust disease of wheat for achieving global food security - A review.},
journal = {International journal of biological macromolecules},
volume = {},
number = {},
pages = {145353},
doi = {10.1016/j.ijbiomac.2025.145353},
pmid = {40553877},
issn = {1879-0003},
abstract = {Wheat (Triticum aestivum), being a global staple crop, is critical in ensuring food security due to its significant nutritional value. However, it faces numerous challenges from both biotic and abiotic stresses, with fungal diseases being particularly detrimental to yield. Among these, wheat stripe rust, caused by the fungal pathogen Puccinia striiformis, poses a severe threat to wheat. Globally, 5.47 million tons of grains are lost due to the stripe rust pathogen, equivalent to a loss of USD 979 million annually; almost 88 % of the world's wheat production is susceptible to stripe rust. This review accentuates the global extensive distribution of stripe rust, detailing its causes and impact on crop productivity and mitigating approaches following traditional, genomic, and post-genomics. The mitigation approaches to wheat stripe rust have been mainly categorized into primitive (pre-genomic), modern (genomic), and next-generation (post-genomic) approaches. The primitive approaches include traditional breeding, phenotypic selection, and exotic germplasm to introduce resistance leads to early success in disease management. The advanced genomic era, with tools like QTL mapping, GWAS, marker-assisted selection, and high-throughput sequencing to deploy resistance genes, helps in precise mapping and developing high-throughput genotyping for large-scale screening and introgression of multiple resistant genes. The gene-editing approaches, including CRISPR/Cas9, RNAi, and epigenomics, now enable precise gene editing and regulation for durable resistance, together with multi-omics techniques, to identify resistant pathways and biomarkers with enhanced understanding of host-pathogen interactions and resistance mechanisms. Climate change events like shifts in rainfall patterns and rising temperatures expand the rust-prone area and pose more challenges in developing durable rust-resistant cultivars. Furthermore, the review explores using wheat's valuable genetic resources and integrating AI-based technologies to enhance stripe rust resistance by analyzing large datasets, including pathogen evolution and growth stages, allowing for timely interventions of the stripe rust epidemic. The role of multiomics approaches, particularly genomics and transcriptomics, in unraveling the genetic basis of stress tolerance is highlighted. A forward-looking framework is proposed, emphasizing the use of interdisciplinary methodologies, including big data, multi-omics, and AI-driven approaches, that hold immense promise to revolutionize wheat protection with the development of climate-resilient wheat genotypes and ensure real-time disease monitoring and precision-resistant strategies against the evolving rust pathogen.},
}
@article {pmid40553564,
year = {2025},
author = {Kratzmeier, C and Taheri, M and Mei, Z and Lim, I and Khalil, MA and Carter-Cooper, B and Fanaroff, RE and Ong, CS and Schneider, EB and Chang, S and Leyder, E and Li, D and Luzina, IG and Banerjee, A and Krupnick, AS},
title = {Lung adenocarcinoma-derived IFN-γ promotes growth by modulating CD8+ T cell production of CCR5 chemokines.},
journal = {The Journal of clinical investigation},
volume = {},
number = {},
pages = {},
doi = {10.1172/JCI191070},
pmid = {40553564},
issn = {1558-8238},
abstract = {Since the lung is a mucosal barrier organ with a unique immunologic environment, mechanisms of immunoregulation in lung cancer may differ from those of other malignancies. Consistent with this notion, we found that CD8+ T cells play a paradoxical role in facilitating, rather than ameliorating, the growth of multiple lung adenocarcinoma models. These include spontaneous, carcinogen-induced, and transplantable tumor cell line models. Specifically, we found that CD8+ T cells promote homing of CD4+Foxp3+ T regulatory cells to the tumor bed by increasing levels of CCR5 chemokines in the tumor microenvironment in an IFN-γ and TNF-α dependent manner. Contrary to their canonical role, these Th1 cytokines contributed to accelerated growth of murine lung adenocarcinomas while suppressing the growth of other malignancies. Surprisingly, lung cancer cells themselves can serve as a dominant source of IFN-γ, and deletion of this cytokine from cancer cells using CRISPR/Cas-9 decreases tumor growth. Importantly for translational applications, a high level of IFN-γ was also found in human lung cancer patients at both the mRNA and protein level. Our data outlines what we deem a novel and previously undefined lung cancer specific immunoregulatory pathway that may be harnessed to tailor immune based therapy specifically for this malignancy.},
}
@article {pmid40553349,
year = {2025},
author = {Mirchandani, I and Khandhediya, Y and Chauhan, K},
title = {Review on Advancement of AI in Synthetic Biology.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2952},
number = {},
pages = {483-490},
pmid = {40553349},
issn = {1940-6029},
mesh = {*Synthetic Biology/methods ; *Artificial Intelligence ; Gene Editing/methods ; CRISPR-Cas Systems ; Machine Learning ; Humans ; },
abstract = {The way biological systems are built and designed has been revolutionized by synthetic biology. Further enhancements like predictive modeling, optimization and systematic design of complex biological systems, is now possible due to integration of Artificial Intelligence into synthetic biology. This review shares insights on the role of AI in advancement of synthetic biology, including genome editing, metabolic pathway optimization and biological circuit design etc. AI-driven tools contribute to the increased efficiency and precision. Application of deep learning and machine learning has made it possible to make CRISPR-cas9, de novo protein design and gene circuit development more precise. However, there are still some persistent challenges, especially in curating high-quality biological datasets and bridging interdisciplinary gaps between computational and experimental scientists. Future perspectives focus on causal reasoning in AI models, integration of physics based algorithms, and promoting collaboration across disciplines to achieve breakthroughs in both synthetic biology and AI. By joining these fields, the transformative power of synthetic biology and AI can be unlocked and applied in the fields of medicine, biotechnology and environmental sustainability, pioneering a way for a new era of bioengineering.},
}
@article {pmid40553339,
year = {2025},
author = {Pandey, S and Choudhari, JK and Tripathi, A and Singh, A and Antony, A and Chouhan, U},
title = {Artificial Intelligence-Based Genome Editing in CRISPR/Cas9.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2952},
number = {},
pages = {273-282},
pmid = {40553339},
issn = {1940-6029},
mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems ; *Artificial Intelligence ; Humans ; RNA, Guide, CRISPR-Cas Systems/genetics ; Precision Medicine ; Genomics/methods ; },
abstract = {Artificial intelligence (AI) plays a critical role in predicting and improving genome editing methods, including CRISPR/Cas9. Recently, several AI models, such as DeepCRISPR, CRISTA, and Deep High Fidelity (DeepHF), have been utilized to design guide RNAs (gRNAs) for CRISPR-Cas systems. These models assess genomic context, desired mutation type, on-target and off-target scores, and potential off-target locations. AI models help improve various genome editing methods, such as base, prime, and epigenome editing, which allow for precise and intentional changes to DNA sequences without the need for donor DNA templates. Furthermore, integrating AI with genome editing and precision medicine enables the creation of personalized treatments tailored to each individual's unique genetic profile. Examining genomic data enables the identification of mutations, variations, and biomarkers linked to diseases like cancer, diabetes, and Alzheimer's disease. Integrating AI with genome editing can potentially enhance genetic modification techniques' precision, efficiency, and cost-effectiveness. Furthermore, it presents novel prospects for fields like genetics, biomedicine, and healthcare, which could significantly impact human health. However, several challenges still exist, including high costs, inaccurate edits, and effective delivery methods for CRISPR components, improved editing performance, and safety in clinical applications.},
}
@article {pmid40553337,
year = {2025},
author = {Khammampalli, S and Vindal, V},
title = {Artificial Intelligence in CRISPR-Cas Systems: A Review of Tool Applications.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2952},
number = {},
pages = {243-257},
pmid = {40553337},
issn = {1940-6029},
mesh = {*CRISPR-Cas Systems ; *Artificial Intelligence ; *Gene Editing/methods ; Humans ; Machine Learning ; RNA, Guide, CRISPR-Cas Systems/genetics ; RNA Editing ; },
abstract = {Genetic engineering is a method used to alter an organism's DNA, which could entail altering a base pair, removing a section of DNA, or introducing a new DNA segment. Over time, genetic engineering has progressed from basic cloning for research purposes to advanced synthetic biology, leading to new biomedical applications. Targeted genomic editing is one method of cellular reprogramming that aims to change the state of a cell. The invention of CRISPR Cas systems has greatly simplified gene editing. These systems use a unique RNA-guided DNA endonuclease, a protein that can cut DNA and be trained to target new places by changing the sequence of its guide RNA. Integrating CRISPR-Cas systems with artificial intelligence opens new insights into the study of genetic engineering and its applications. Extensive research utilizing deep learning and machine learning has been conducted to predict the outcomes of CRISPR-Cas9 editing. Artificial intelligence also predicts RNA editing events and CRISPR off-target cleavage sites. Scientists often struggle to identify the ideal perturbation for their specific application because of the ample search space and expensive genetic trials. The algorithmic method using artificial intelligence utilizes the cause-and-effect link between variables in a complicated system like genome regulation to determine which perturbation is most effective in each successive round of testing, thereby making artificial intelligence an effective technique in gene editing.},
}
@article {pmid40553335,
year = {2025},
author = {Johnson, SJS},
title = {AI Revolutionizing Cell and Genetic Engineering: Innovations and Applications.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2952},
number = {},
pages = {219-232},
pmid = {40553335},
issn = {1940-6029},
mesh = {Humans ; *Artificial Intelligence ; *Genetic Engineering/methods ; Gene Editing/methods ; CRISPR-Cas Systems ; Precision Medicine/methods ; *Cell Engineering/methods ; Drug Discovery/methods ; Genomics/methods ; },
abstract = {The integration of Artificial Intelligence (AI) into the realms of cell and genetic engineering has ushered in a transformative era of innovation and discovery. This chapter explores the myriad ways in which AI technologies are revolutionizing these fields, from data analysis and drug discovery to genomic sequencing and gene editing. AI algorithms are adept at analyzing vast datasets, uncovering intricate patterns, and predicting biological phenomena with unparalleled accuracy. In drug discovery, AI-driven platforms expedite the identification of potential therapeutics by simulating molecular interactions and predicting their efficacy. Genomic sequencing efforts benefit from AI's ability to interpret genetic variations and their implications for health and disease. Furthermore, AI-guided gene editing techniques, such as CRISPR-Cas9, enable precise and targeted modifications of the genome. Beyond the laboratory, AI facilitates personalized medicine by analyzing genetic data to tailor treatments to individual patients. This chapter underscores the pivotal role of AI in advancing cell and genetic engineering, promising a future of unprecedented scientific breakthroughs and personalized healthcare solutions.},
}
@article {pmid40553283,
year = {2025},
author = {Suk, Y and Apel, E and Custers, S and Miletic, P and Zhai, K and Chokshi, C and Venugopal, C and Moffat, J and Singh, SK},
title = {CRISPR-Cas9-Guided Genetic Manipulation of Patient-Derived Brain Tumor Cells.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2944},
number = {},
pages = {173-184},
pmid = {40553283},
issn = {1940-6029},
mesh = {*CRISPR-Cas Systems ; Humans ; *Brain Neoplasms/genetics/pathology ; *Gene Editing/methods ; Gene Knockout Techniques/methods ; RNA, Guide, CRISPR-Cas Systems/genetics ; Cell Line, Tumor ; },
abstract = {CRISPR-Cas9 technology has revolutionized scientific research and has provided scientists with the ability to change DNA bases specifically and precisely at predetermined sites. The CRISPR-Cas9 knockout (KO) and activation (a) platforms developed by Hart et al. and Sanson et al. allow for RNA-directed genome editing to both decrease or increase gene expression, respectively [1, 2]. In this chapter the two techniques, CRISPR KO and CRISPRa, will be discussed and explained in further detail for optimized application against patient derived brain tumor cells.},
}
@article {pmid40553057,
year = {2025},
author = {Long, W and Li, Q and Jin, T and Lu, Z and Hu, F and Zhang, H and Xu, Y},
title = {Dual detection of hypervirulent genes of Klebsiella pneumoniae using a single CRISPR-Cas12a system modulated using entropy-driven circuits.},
journal = {Analytical methods : advancing methods and applications},
volume = {},
number = {},
pages = {},
doi = {10.1039/d5ay00563a},
pmid = {40553057},
issn = {1759-9679},
abstract = {A set of different CRISPR-based analyses have been extensively used to detect nucleic acids owing to their characteristics of being sensitive, specific, rapid and easy to operate. However, one of its major challenges is application to multiplex detection (https://pubmed.ncbi.nlm.nih.gov/31460243/) using a single CRISPR-Cas system because the detection relies on the indiscriminate trans-cleavage activity of the Cas protein. Here we developed a CRISPR-based dual gene assay system for hypervirulent Klebsiella pneumoniae (hvKp) by integrating two parallel entropy-driven circuits (EDCs) and the trans-cleavage activity of the Cas12a protein towards fluorescent substrate reporters. The EDC reaction released two different fluorescent reporters and the same trigger probe according to different targets. The trigger probe activated the trans-cleavage activity of Cas12a towards reporter probes to achieve the purpose of the dual detection of different genes with a single CRISPR-Cas12a system in one tube. Furthermore, the assay system was designed to detect different mRNA sequences (rmpA and peg-344) of hvKp, requiring no reverse transcription procedure and providing technical support for the identification of hvKp and classical Klebsiella pneumoniae (cKp). The detection limits for rmpA and peg-344 were as low as 0.10 fmol L[-1] and 0.17 fmol L[-1], respectively, within 25 minutes under isothermal conditions. It enabled rapid dual detection of hvKp in a single tube using only a CRISPR-Cas12a system, demonstrating great potential for point-of-care testing due to its high sensitivity, low cost, and robust ability to detect various mRNA biomarkers.},
}
@article {pmid40551276,
year = {2025},
author = {Zhang, Q and Yu, Y and Yin, B and Xu, L and Chen, H and Qiao, R and Chen, A and Zhu, N and Wu, X},
title = {An ultrasensitive and specific CRISPR-Cas13a-mediated point-of-care assay for monkeypox detection and PCR-based clade detection.},
journal = {Infectious diseases of poverty},
volume = {14},
number = {1},
pages = {56},
pmid = {40551276},
issn = {2049-9957},
support = {202308003//Nanjing Science and technology plan project/ ; BE2022669//Social Development Project of Jiangsu Provincial Science and Technology Department/ ; SJCX23_0855//Graduate Research and Innovation Projects of Jiangsu Province/ ; RCMS24008//Talent Lift Program of The Second Hospital of Nanjing/ ; },
mesh = {Sensitivity and Specificity ; Humans ; *CRISPR-Cas Systems ; *Point-of-Care Systems ; *Mpox, Monkeypox/diagnosis/virology ; *Monkeypox virus/isolation &amp; purification/genetics/classification ; *Polymerase Chain Reaction/methods ; *Nucleic Acid Amplification Techniques/methods ; *Molecular Diagnostic Techniques/methods ; },
abstract = {BACKGROUND: The rapid increase in the number of monkeypox cases poses a considerable threat to the international community, necessitating sensitive, fast, and available diagnostic methods. Therefore, the objective of this study was to develop a rapid, sensitive and simple method with high clinical applicability.<br><br>METHODS: We developed a simple, rapid point-of-care assay to detect monkeypox virus (MPXV) using multienzyme isothermal rapid amplification (MIRA) coupled with the clustered regularly interspaced short palindromic repeats (CRISPR)-Cas13a system. The detection system was optimized by synthesizing plasmids, and the detection sensitivity was explored by the continuous dilution of the plasmid. We validated the accuracy of this assay on 202 clinical MPXV samples and 104 interference samples through the kappa test. The visual interpretation of the results was realized by combining the assay with lateral flow strips. In addition, we developed a PCR-based method to identify MPXV Clades I and II, and the accuracy was tested through a kappa test on 202 clinical monkeypox samples and 104 interference samples.<br><br>RESULTS: Our assay achieved an analytical sensitivity of 14.4 copies/ml and high selectivity, as it differentiated MPXV from three other Orthopoxvirus species. The clinical testing results for 202 monkeypox samples and 104 interference samples demonstrated 100% sensitivity and specificity. Compared with quantitative PCR (qPCR), three samples tested as positive using our assay, which showed that the performance of this assay was superior to that of the qPCR assay. Combined with lateral flow strips, its availability and simplicity provide an alternative point-of-care diagnostic method for MPXV testing in remote settings and resource-poor areas. The results of 32 clinical samples showed that lateral flow strips had a high detection sensitivity and could identify samples with Ct value of 39 as positive. The clade identification assay detected as few as 200 copies/ml within 40&#xa0;min and no cross-reaction was observed between Clades I and II. The clinical samples tested were all Clade II, which was consistent with the circulating clade in the Chinese mainland.<br><br>CONCLUSIONS: The MIRA-CRISPR-Cas13a-MPXV system offers a rapid, sensitive and specific approach for monkeypox diagnosis, with significance for&#xa0;monitoring monkeypox epidemics. The clade identification assay based on PCR could accurately distinguish Clade I from Clade II within 40&#xa0;min and can be implemented for high-throughput operation.},
}
@article {pmid40551141,
year = {2025},
author = {Kim, MS and Jeong, DE and Choi, SK},
title = {Harnessing an anti-CRISPR protein for powering CRISPR/Cas9-mediated genome editing in undomesticated Bacillus strains.},
journal = {Microbial cell factories},
volume = {24},
number = {1},
pages = {143},
pmid = {40551141},
issn = {1475-2859},
support = {KGM1192511//Korea Research Institute of Bioscience and Biotechnology/ ; IGM042411//HLB Genex/ ; },
mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; *Bacillus/genetics/metabolism ; *Bacterial Proteins/genetics/metabolism ; Plasmids/genetics ; CRISPR-Associated Protein 9/antagonists &amp; inhibitors ; RNA, Guide, CRISPR-Cas Systems/genetics ; Bacillus subtilis/genetics ; },
abstract = {BACKGROUND: Wild-type Bacillus strains have significant industrial and medical value, but their effective utilization often requires strain improvement. The CRISPR/Cas9 system has become the primary tool for genome editing, allowing precise introduction of desired mutations at specific chromosomal locations. However, the practical application of CRISPR/Cas9 in most wild-type Bacillus strains remains challenging due to cellular toxicity resulting from Cas9/sgRNA activity. Therefore, controlling Cas9 toxicity is essential for the widespread application of the CRISPR/Cas9 system in wild-type Bacillus strains.<br><br>RESULTS: We employed AcrIIA4, an anti-CRISPR protein that inhibits the Cas9/sgRNA ribonucleoprotein complex from interacting with DNA, to mitigate Cas9/sgRNA-mediated toxicity, thereby enabling CRISPR/Cas9-based genome editing in wild-type strains. The newly constructed CRISPR/anti-CRISPR (CAC) plasmids harbor both cas9 and acrIIA4 genes controlled by the Pspac and Pxyl promoters, respectively, along with the repressor genes lacI and xylR. This design allows precise control of Cas9 activity through inducers. Xylose, which induces AcrIIA4 expression, effectively alleviated Cas9/sgRNA-mediated toxicity during transformation. Under xylose induction, the CAC plasmid led to a remarkable 139-fold increase in the transformation efficiency of wild-type Bacillus subtilis compared to a plasmid lacking anti-CRISPR. Meanwhile, IPTG induction promoted Cas9 expression, facilitating efficient genome editing. Upon IPTG induction, the genome editing efficiency in wild-type B. subtilis increased from 0 to 95.8% in transformants carrying the CAC plasmid. Importantly, our findings extend beyond B. subtilis, revealing that the anti-CRISPR protein dramatically enhanced transformation and genome editing efficiencies in Bacillus pumilus. Moreover, we demonstrated that the CAC system successfully enabled the generation of spo0A mutants in Bacillus mojavensis, Bacillus tequilensis, and Paenibacillus polymyxa.<br><br>CONCLUSIONS: In this study, we developed a CAC system that utilizes the anti-CRISPR protein AcrIIA4 to reduce Cas9/sgRNA-mediated toxicity in Bacillus strains. This system enables precise control of AcrIIA4 and Cas9 expression through inducers, significantly enhancing the efficiency of transformation and genome editing in wild-type Bacillus strains. Therefore, the CAC system stands as a powerful tool to facilitate genome editing in diverse wild-type Bacillus species.},
}
@article {pmid40551124,
year = {2025},
author = {Shu, J and Tan, Q and Huang, Z and Zhang, T and Ye, L and Fu, S and Mao, Z},
title = {One-pot one-step detection platform for severe fever with thrombocytopenia syndrome virus via the CRISPR/Cas12a detection system.},
journal = {Virology journal},
volume = {22},
number = {1},
pages = {203},
pmid = {40551124},
issn = {1743-422X},
support = {2025KY435//Medical and Health Research Project of Zhejiang Province/ ; 2022CZBJ088//the Top Talent of Changzhou "The 14th Five-Year Plan" High Level Health Talents Training Project/ ; },
mesh = {*Phlebovirus/genetics/isolation &amp; purification ; *Severe Fever with Thrombocytopenia Syndrome/diagnosis/virology ; Humans ; Sensitivity and Specificity ; *CRISPR-Cas Systems ; *Nucleic Acid Amplification Techniques/methods ; *Molecular Diagnostic Techniques/methods ; Limit of Detection ; CRISPR-Associated Proteins/genetics ; Bacterial Proteins ; Endodeoxyribonucleases ; },
abstract = {Severe fever with thrombocytopenia syndrome virus (SFTSV) is a tick-borne virus that primarily causes SFTS. Although a common testing route is available, a timely, conventional and accurate method for SFTSV detection is urgently needed. In the present study, we established a platform that combines the recombinase polymerase amplification (RPA) assay with the clustered regularly interspaced short palindromic repeats-CRISPR associated proteins (CRISPR/Cas) 12a technique in one step in one pot. The procedure can be completed within 45 min at a constant temperature without a sophisticated instrument. This method targets the S gene of SFTSV, with a detection limit (LoD) of 11.7 copies per reaction and high specificity, without cross reactivity with other pathogens. Furthermore, across 46 test samples, this method achieved 89.13% consistency with the PCR method (41/46). Together, the reaction system developed in the present study provides not only a novel method for SFTSV detection but also an alternative method for detecting RNA viruses.},
}
@article {pmid40550676,
year = {2025},
author = {Zhan, Y and Zheng, L and Shen, J and Hu, Y and Luo, X and Dai, L},
title = {[Development of a miniaturized CRISPR/Cas gene editing tool for human gut Bacteroides].},
journal = {Sheng wu gong cheng xue bao = Chinese journal of biotechnology},
volume = {41},
number = {6},
pages = {2360-2372},
doi = {10.13345/j.cjb.240959},
pmid = {40550676},
issn = {1872-2075},
mesh = {*CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; *Bacteroides/genetics ; Humans ; *Gastrointestinal Microbiome/genetics ; Bacteroides fragilis/genetics ; },
abstract = {: Bacteroides, as one of the most abundant and diverse genera in the human gut, is regarded as a window into the study of gut microbiota-host interactions. Currently, CRISPR/Cas-based gene editing systems targeting Bacteroides have been widely applied, while the large size of Cas nucleases limits their potential application scenarios (such as in situ gut Bacteroides editing based on phage delivery). Therefore, this study aims to develop a compact and highly efficient genetic editing tool in Bacteroides., We developed a miniaturized CRISPR/Cas gene editing system for human gut Bacteroides. First, the editing capabilities of different miniaturized CRISPR/Cas systems, including AsCas12f, CasΦ2, and ISDge10, were evaluated in Bacteroides fragilis. Subsequently, the editing capability of AsCas12f was assessed across various Bacteroides species, and the size of this system was further optimized. The results demonstrated that the CRISPR/AsCas12f genome editing system exhibited the highest editing efficiency in B. fragilis. The CRISPR/AsCas12f system achieved efficient genome editing in B. fragilis, Bacteroides thetaiotaomicron, and Phocaeicola vulgatus. Furthermore, with a repair template of 500 bp homologous arms, the editing efficiency remained as high as 94.7%. In conclusion, CRISPR/AsCas12f can serve as a chassis tool enzyme for the development of Bacteroides-based miniature gene editors and derivative technologies, laying a foundation for the further development of gene editing technology for Bacteroides.},
}
@article {pmid40549893,
year = {2025},
author = {Wei, Y and Yue, T and Wang, Y and Yang, Y},
title = {Fertile androgenetic mice generated by targeted epigenetic editing of imprinting control regions.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {122},
number = {27},
pages = {e2425307122},
doi = {10.1073/pnas.2425307122},
pmid = {40549893},
issn = {1091-6490},
support = {2018YFC1004500//National Key Research and Developmental Program of China/ ; 2017YFC1001300//National Key Research and Developmental Program of China/ ; },
mesh = {Animals ; *Genomic Imprinting/genetics ; Male ; Mice ; DNA Methylation/genetics ; *Gene Editing/methods ; Female ; *Epigenesis, Genetic ; *Fertility/genetics ; Spermatozoa/metabolism ; CRISPR-Cas Systems ; Alleles ; Epigenome Editing ; },
abstract = {Each new mammalian life begins with the fusion of an oocyte and a sperm to produce a fertilized egg containing two sets of genomes, one from the mother and one from the father. Androgenesis, a way for producing offspring solely from male genetic material, is limited in mammals, presumably due to barriers arising from genomic imprinting, an epigenetic mechanism leading to monoallelic gene expression. Here, we report adult mammalian offspring derived from the genetic material of two sperm cells. These mice, which we refer to as androgenetic mice, were produced via targeted DNA methylation editing of seven imprinting control regions (ICRs) through CRISPR-based epigenome engineering. Two sperm cells were injected into an enucleated oocyte to form putatively diploid embryos. Allele-specific epigenetic editing was achieved by injecting guide RNAs with protospacer adjacent motif (PAM) sequences designed to match one allele but not the other. The birth of androgenetic mice that were able to develop to adulthood demonstrates that mammalian androgenesis is achievable by targeted epigenetic remodeling of a few defined ICRs.},
}
@article {pmid40549156,
year = {2025},
author = {Sui, M and Zhou, M and Cui, M and Liu, H and Zhang, X and Hu, N and Li, Y and Wang, B and Yang, G and Gui, P and Zhu, L and Wan, F and Zhang, B},
title = {Novel drug-inducible CRISPRa/i systems for rapid and reversible manipulation of gene transcription.},
journal = {Cellular and molecular life sciences : CMLS},
volume = {82},
number = {1},
pages = {249},
pmid = {40549156},
issn = {1420-9071},
support = {82371443//National Natural Science Foundation of China grant/ ; 2023YFE0117600//National Key R&amp;D Program of China grant/ ; },
mesh = {Humans ; Tamoxifen/pharmacology/analogs &amp; derivatives ; *CRISPR-Cas Systems/genetics ; *Transcription, Genetic/drug effects ; Animals ; Gene Expression Regulation/drug effects ; Mice ; HEK293 Cells ; Receptors, Estrogen/genetics/metabolism ; RNA, Guide, CRISPR-Cas Systems/genetics ; },
abstract = {CRISPR activation and interference (CRISPRa/i) are highly effective tools to regulate transcription by fusing dead Cas9 (dCas9) with transcriptional regulatory factors guided by small guide RNA (sgRNA) in mammalian cells and mice. Still, a controllable gene regulation system is desired to investigate and manipulate dynamic biological processes. Here, we reported flexible drug-responsive CRISPRa/i systems by fusing mutated human estrogen receptor (ERT2) domains, which responded to estrogen analogue tamoxifen or its active metabolite 4-hydroxy-tamoxifen (4OHT), to CRISPRa/i components for transcriptional regulation. Upon 4OHT treatment, the optimal variants, ERT2-ERT2-CRISPRa/i-ERT2 (iCRISPRa/i), showed rapid protein translocation of iCRISPRa/i from cytoplasm to nucleus and subsequent transcriptional response. The inducible transcriptional manipulation could be restored to its original level when 4OHT was withdrawn. Moreover, the efficiencies of gene expression regulation of iCRISPRa/i were comparable to those of non-inducible and doxycycline-inducible counterparts, with a lower leakage and a faster drug response activity. The iCRISPRa/i systems successfully induced phenotypic changes in various cell lines. These results highlight that iCRISPRa/i systems could achieve fast and flexible drug-responsive transcriptional modulation and phenotypic changes, and thus provide better options for gain- and loss-of-function model construction and gene therapy.},
}
@article {pmid40548936,
year = {2025},
author = {Li, Y and Zhao, C and Cao, Y and Chen, X and Tang, Y and Zhou, X and Ingmer, H and Jiao, X and Li, Q},
title = {Oxidative stress elicited by phage infection induces Staphylococcal type III-A CRISPR-Cas system.},
journal = {Nucleic acids research},
volume = {53},
number = {12},
pages = {},
doi = {10.1093/nar/gkaf541},
pmid = {40548936},
issn = {1362-4962},
support = {32430103//National Natural Science Foundation of China/ ; B2302024//National Natural Science Foundation of China/ ; 2023YFD1800503//National Key Research and Development Program of China/ ; 2022YFC2604200//National Key Research and Development Program of China/ ; RZZ202302//Jiangsu Key Laboratory of Zoonosis Major Independent Research/ ; 2024ZB284//Jiangsu Funding Program for Excellent Postdoctoral Talent/ ; 2023B1515120016//Guangdong Basic and Applied Basic Research Foundation/ ; JCYJ20220818100616034//Shenzhen Science and Technology Innovation Commission/ ; KYCX24_3850//Yangzhou University/ ; 32430103//Priority Academic Program Development of Jiangsu Higher Education Institution (PAPD)/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; *Oxidative Stress/genetics ; *Staphylococcus aureus/virology/genetics/metabolism/immunology ; Promoter Regions, Genetic ; Bacterial Proteins/genetics/metabolism ; Gene Expression Regulation, Bacterial ; *Bacteriophages ; *Staphylococcus Phages ; CRISPR-Associated Proteins/genetics/metabolism ; },
abstract = {In prokaryotes, the CRISPR-Cas system provides immunity to invading mobile genetic elements, but its expression is commonly repressed in the absence of phage infection to prevent autoimmunity. How bacteria senses phage infection and activates CRISPR-Cas system are poorly understood. Here, we demonstrate that an essential promoter Pcas, located within the cas1 gene, is the primary promoter driving expression of cas genes encoding the Cas10-Csm interference complex in Staphylococcus aureus type III-A CRISPR-Cas system during phage infection. As a conserved promoter in Staphylococci type III-A CRISPR-Cas system, the Pcas loses its ability to activate cas genes expression when mutated at the C186 site. Importantly, we find that the transcriptional regulator MgrA directly represses type III-A CRISPR-Cas system by interacting with Pcas to prevent autoimmunity. Upon phage infection, MgrA senses oxidative stress and dissociates from the Pcas, alleviating the transcriptional repression and subsequently triggering a robust immunity against phages. Our work provides evidence for the requirement of Pcas within cas1 during type III-A CRISPR-Cas interference stage, and reveals that MgrA-mediated regulation provides an effective mechanism for bacteria to balance avoiding autoimmunityand defending against phages.},
}
@article {pmid40548934,
year = {2025},
author = {Flores-Fernández, CN and Lin, D and Robins, K and O'Callaghan, CA},
title = {UniClo: scarless hierarchical DNA assembly without sequence constraint.},
journal = {Nucleic acids research},
volume = {53},
number = {12},
pages = {},
doi = {10.1093/nar/gkaf548},
pmid = {40548934},
issn = {1362-4962},
support = {BB/X511122/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; //University of Oxford/ ; },
mesh = {DNA Methylation ; *DNA/genetics/metabolism/chemistry ; Plasmids/genetics ; CRISPR-Cas Systems ; *Deoxyribonucleases, Type II Site-Specific/metabolism/genetics ; Genetic Vectors/genetics ; },
abstract = {Type IIS restriction enzyme-mediated DNA assembly is efficient but has sequence constraints and can result in unwanted sequence scars. To overcome these drawbacks, we developed UniClo, a type IIS restriction enzyme-mediated method for universal and flexible DNA assembly. This is achieved through a combination of vector engineering, DNA methylation using recombinant methylases, and steric blockade using CRISPR-dCas9 technology to regulate this methylation. Type IIS restriction enzyme sites within fragments to be assembled are methylated using recombinant methylases, while the fragment-flanking outer sites are protected from methylation by a recombinant dCas9-sgRNA complex. During the subsequent assembly reaction, only the protected flanking sites are cut as only they are unmethylated. Fragments are correctly assembled, despite containing internal sites for the single type IIS restriction enzyme used for the one-pot assembly. The assembled plasmid can be used as a donor plasmid in a subsequent assembly round with the same type IIS restriction enzyme and the assembly vector engineering ensures removal of potential scars by a trimming process. This simplifies assembly design and only three vectors are required for any multi-round assembly. These vectors all use the same pair of overhangs. UniClo provides a simple scarless approach for hierarchical assembly of any sequence and has wide potential application.},
}
@article {pmid40548648,
year = {2025},
author = {Alariqi, M and Ramadan, M and Yu, L and Hui, F and Hussain, A and Zhou, X and Yu, Y and Zhang, X and Jin, S},
title = {Enhancing Specificity, Precision, Accessibility, Flexibility, and Safety to Overcome Traditional CRISPR/Cas Editing Challenges and Shape Future Innovations.},
journal = {Advanced science (Weinheim, Baden-Wurttemberg, Germany)},
volume = {},
number = {},
pages = {e2416331},
doi = {10.1002/advs.202416331},
pmid = {40548648},
issn = {2198-3844},
support = {2023ZD04074//Biological Breeding-Major Projects/ ; 32325039//National Natural Science Fund of China for Distinguished Young Scholars/ ; 2021hszd013//Hubei Hongshan Laboratory/ ; W2433067//National Natural Science Foundation of China for International Young Scientists/ ; 32272128//National Natural Science Foundation of China/ ; },
abstract = {Derived from the bacterial immune system, CRISPR/Cas9 induces DSBs at specific DNA sequences, which are repaired by the cell's endogenous mechanisms, leading to gene insertions, deletions, or substitutions. Despite its transformative potential, several challenges remain in optimizing of CRISPR/Cas systems, including off-target effects, delivery methods, PAM restrictions, and the limitations of traditional editing approaches. This review focuses on the interplay between these challenges and their contributions to gene editing precision, specificity, accessibility, flexibility, and safety. How reducing off-target effects enhances specificity and safety is explored, while discussing the role of HDR-based editing in achieving precise gene modifications, alongside alternative methods such as base editing and prime editing. Improved delivery mechanisms are examined for their impact on accessibility and efficiency, while the reduction of PAM restrictions is highlighted for its contributions to flexibility. Lastly, emerging cleavage-free editing technologies are evaluated as they relate to safety and accessibility. This focused review aims to clarify the connections among these aspects and outline future research directions for advancing CRISPR-based applications.},
}
@article {pmid40548119,
year = {2025},
author = {Kumbhakar, DV and Thakkar, L and Akhand, C and Sharaf, S and Vemuganti, GK},
title = {Nanomaterials targeting cancer stem cells to overcome drug resistance and tumor recurrence.},
journal = {Frontiers in oncology},
volume = {15},
number = {},
pages = {1499283},
pmid = {40548119},
issn = {2234-943X},
abstract = {A cancer stem cell (CSC) is an immortal cell that is capable of self-renewal, continuous proliferation, differentiation into various cancer cell lineages, metastatic dissemination, tumorigenesis, maintaining tumor heterogeneity, and resistance to conventional treatments. Targeted therapies have made huge advances in the past few years, but resistance is still a major roadblock to their success, in addition to their life-threatening side effects. Progressive treatments are now available, including immunotherapies, CRISPR-Cas 9, sonodynamic therapy, chemodynamic therapy, antibody-drug nanoconjugates, cell-based therapies, gene therapy, and ferroptosis-based therapy, which have replaced surgery, chemotherapy, and radiotherapy for cancer treatment. The challenge is to develop targeted treatment strategies that are effective in eradicating CSCs, as they are resistant to anticancer drugs, causing treatment failure, relapse, and recurrence of cancer. An overview of the fundamental characteristics of CSCs, drug resistance, tumor recurrence, and signaling pathways as well as biomarkers associated with their metastatic potential of CSC is elucidated in this review. The regulatory frameworks for manufacturing and conducting clinical trials on cancer therapy are explicated. Furthermore, we summarize a variety of promising nanocarriers (NCs) that have been used directly and/or synergistic therapies coupled with the therapeutic drug of choice for the detection, targeting, and imaging of CSCs to surmount therapeutic resistance and stemness-related signaling pathways and eradicate CSCs, hence alleviating the limitation of conventional therapies. Nanoparticle-mediated ablation therapies (NMATs) are also being argued as a method for burning or freezing cancer cells without undergoing open surgery. Additionally, we discuss the recent clinical trials testing exosomes, CRISPR/Cas9, and nanodrugs, which have already received approval for several new technologies, while others are still in the early stages of testing. The objective of this review is to elucidate the advantages of nanocarriers in conquering cancer drug resistance and to discuss the most recent developments in this field.},
}
@article {pmid40544217,
year = {2025},
author = {Ossio, A and Merino-Mascorro, A and Leon, JS and Heredia, N and Garcia, S},
title = {Detection of Murine Norovirus on Fresh Produce Through a CRISPR/Cas13a RNase-Based Capsid Integrity Assay.},
journal = {Food and environmental virology},
volume = {17},
number = {3},
pages = {35},
pmid = {40544217},
issn = {1867-0342},
support = {2019-67017-29642//National Institute of Food and Agriculture/ ; PROVERICYT//Universidad Autonoma de Nuevo Leon/ ; PROVERICYT//Universidad Autonoma de Nuevo Leon/ ; },
mesh = {*Norovirus/genetics/isolation &amp; purification ; CRISPR-Cas Systems ; *Ribonucleases/genetics/metabolism ; Animals ; *Capsid/chemistry/metabolism ; *Food Contamination/analysis ; Mice ; *Capsid Proteins/genetics/metabolism ; Food Microbiology/methods ; Fruit/virology ; Humans ; },
abstract = {Standard food detection methods do not distinguish between infectious and non-infectious human norovirus leading to uncertainty in the management of a norovirus positive food sample. These methods also require expensive RT-qPCR-based equipment and reagents. In contrast, CRISPR-based, compared to RT-qPCR-based, detection methods are generally less expensive and yield similar sensitivity and specificity. Our goal was to detect norovirus with an intact capsid, a proxy for infectivity, through a CRISPR-Cas13a-based detection method together with an RNase-capsid integrity assay. We termed this assay: Foodborne RNA-virus Enzymatic Sensing for High-throughput on fresh produce (CRISPR FRESH) reflecting its potential to detect infectious or potentially infectious virus particles. Our CRISPR FRESH method detected murine norovirus (MNV-1), with an intact capsid, at a limit of detection of 2.59 log10 gc/25 g (5 gc/rx). This method did not cross-react with other targets (synthetic DNA targets for hepatitis A virus; human norovirus GI, GII; rotavirus). Compared with RT-qPCR, CRISPR FRESH showed an increased sensitivity when detecting low copy numbers of RNase-pre-treated MNV-1 in lettuce and blueberries samples. Viral detection with the RT-qPCR assay is quantifiable while the CRISPR assay is present/absent. This report describes a CRISPR-based detection of potentially infectious viruses in food samples.},
}
@article {pmid40540504,
year = {2025},
author = {Yamashita, K and Muramoto, T},
title = {Efficient endogenous protein labelling in Dictyostelium using CRISPR/Cas9 knock-in and split fluorescent proteins.},
journal = {PloS one},
volume = {20},
number = {6},
pages = {e0326577},
pmid = {40540504},
issn = {1932-6203},
mesh = {*Dictyostelium/genetics/metabolism ; *CRISPR-Cas Systems/genetics ; *Gene Knock-In Techniques/methods ; *Luminescent Proteins/genetics/metabolism ; *Protozoan Proteins/genetics/metabolism ; Green Fluorescent Proteins/genetics/metabolism ; Histones/genetics/metabolism ; },
abstract = {Fluorescent protein tagging is a powerful technique for visualising protein dynamics; however, full-length fluorescent protein knock-in can be inefficient at certain genomic loci, making it challenging to achieve stable and uniform expression. To address this issue, we used CRISPR/Cas9-mediated knock-in strategies with split fluorescent proteins in Dictyostelium discoideum. This approach enabled efficient integration of the short mNeonGreen2 (mNG2) fragment, mNG211, particularly at functionally critical loci such as major histone h2bv3, where full-length tagging was unsuccessful. Our analysis revealed that inserting tandem repeats of mNG211 at the h2bv3 locus progressively impaired cell proliferation, indicating that functional disruption depends on insert size. These findings suggest that using short tags like mNG211 minimises functional interference and facilitates knock-in at sensitive loci. We further optimised the fluorescence intensity by fine-tuning the expression of the long fragment, mNG21-10, and introducing tandem repeats of mNG211. This approach provides a reliable method for precise and stable endogenous protein labelling, facilitating live-cell imaging and functional studies in D. discoideum.},
}
@article {pmid40539512,
year = {2025},
author = {Sung, K and Jung, Y and Kim, N and Kim, YW and Kim, HH and Kim, SK and Bae, S},
title = {A rational engineering strategy for structural dynamics modulation enables target specificity enhancement of the Cas9 nuclease.},
journal = {Nucleic acids research},
volume = {53},
number = {12},
pages = {},
doi = {10.1093/nar/gkaf535},
pmid = {40539512},
issn = {1362-4962},
support = {2018R1A2B2001422//National Research Foundation of Korea/ ; 2021M3A9H3015389//National Research Foundation of Korea/ ; RS-2024-00451880//National Research Foundation of Korea/ ; RS-2024-00455559//National Research Foundation of Korea/ ; SRC-NRF2022R1A5A102641311//National Research Foundation of Korea/ ; RS-2022-NR070713//MSIT/ ; RS-2023-00260968//MSIT/ ; RS-2024-00357556//MSIT/ ; RS-2024-00338871//MSIT/ ; RS-2024-00332601//Korean Fund for Regenerative Medicine (KFRM)/ ; },
mesh = {Substrate Specificity ; *Protein Engineering/methods ; Humans ; *CRISPR-Cas Systems ; *CRISPR-Associated Protein 9/chemistry/genetics/metabolism ; DNA/metabolism/chemistry ; Mutation ; Gene Editing ; Models, Molecular ; *Endonucleases/metabolism/genetics/chemistry ; },
abstract = {Structural dynamics of an enzyme plays a crucial role in enzymatic activity and substrate specificity, yet rational engineering of the dynamics for improved enzymatic properties remains a challenge. Here, we present a new biochemical strategy of intermediate state stabilization that modulates the multistep dynamic mechanisms of enzyme reactions to improve substrate specificity. We employ this strategy to enhance CRISPR-Cas9 nuclease specificity. By incorporating positively charged residues into the noncatalytic REC2 domain of Cas9, we stabilize the REC2-DNA interaction that forms exclusively in a catalytically inactive intermediate conformation of the Cas9 complex. This enables off-target trapping in the inactive conformation and thus reduces off-target cleavage in human cells. Furthermore, we combine the REC2 modification with mutations in previous rational variants, leading to the development of a combinational variant named Correct-Cas9, which connotes "combined with rationally engineered REC-Two" Cas9. Assessed by high-throughput analysis at thousands of target sequences, Correct-Cas9 exhibits increased target specificity compared to its parental variants, demonstrating a synergy between our strategy and previous rational approaches. Our method of intermediate state stabilization, either alone or combined with conventional approaches, could be applied to various nucleic acid-processing enzymes that undergo conformational changes upon target binding, to enhance their target specificity effectively.},
}
@article {pmid40539281,
year = {2025},
author = {Glenthøj, A and Carlsen, SBIS and Hoffmann, M and Haastrup, EK and Andersen, LP and Toft, N and Kornblit, BT and Petersen, JB and Hasle, H and Ifversen, MRS},
title = {[CRISPR as a functional cure for hemoglobinopathies].},
journal = {Ugeskrift for laeger},
volume = {187},
number = {22},
pages = {},
doi = {10.61409/V12240888},
pmid = {40539281},
issn = {1603-6824},
mesh = {Humans ; *Gene Editing/methods ; *Hemoglobinopathies/therapy/genetics ; *Genetic Therapy/methods ; Anemia, Sickle Cell/therapy/genetics ; *CRISPR-Cas Systems ; beta-Thalassemia/therapy/genetics ; Fetal Hemoglobin/genetics/biosynthesis ; },
abstract = {Severe haemoglobinopathies, including sickle cell disease and β-thalassaemia, represent significant global health burdens. CRISPR technology enables precise genetic editing of haematopoietic stem cells, with current therapies focused on boosting fetal haemoglobin production for a functional cure. This review finds that, while promising, ex vivo approaches require advanced facilities and substantial resources, limiting accessibility where the need is highest. Future development of in vivo methods may expand global access, addressing the urgent need for scalable and affordable treatments for these debilitating diseases.},
}
@article {pmid40538650,
year = {2025},
author = {Song, Y and Hu, Q and Han, Y and Liu, H and Huang, Z and Niu, M and Dong, X and Yan, K and Jin, L and Li, H and Sun, Y},
title = {Detection assay of polymyxin resistance coding mcr-1 gene based on CRISPR/Cas13a system.},
journal = {Frontiers in cellular and infection microbiology},
volume = {15},
number = {},
pages = {1553681},
pmid = {40538650},
issn = {2235-2988},
mesh = {*CRISPR-Cas Systems ; *Escherichia coli Proteins/genetics ; *Polymyxins/pharmacology ; *Escherichia coli/genetics/drug effects/isolation &amp; purification ; *Anti-Bacterial Agents/pharmacology ; *Drug Resistance, Bacterial/genetics ; Humans ; Sensitivity and Specificity ; Real-Time Polymerase Chain Reaction/methods ; Microbial Sensitivity Tests ; Escherichia coli Infections/microbiology ; },
abstract = {INTRODUCTION: Polymyxins are reserved as an ultimate defense against multidrug-resistant bacteria. The emergence of the polymyxin resistance gene mcr-1 poses a potential risk for the treatment of severe infections caused by Gram-negative bacteria. Timely detection and monitoring the mcr-1 gene are essential for guiding anti-infective therapy and controlling the spread of polymyxin resistance. Quantitative real-time PCR (qPCR) is one of the common methods for detecting resistance genes. However, qPCR has equipment dependency, and is not feasible in primary healthcare settings. Currently, there remains a lack of a highly sensitive and portable method for detecting the mcr-1 gene.<br><br>METHODS: We established and optimized detection assays of the mcr-1 gene based on CRISPR/Cas13a system and lateral flow strips. The detection method was preliminarily evaluated using clinical isolates from Escherichia coli, compared with qPCR.<br><br>RESULTS: The method for detecting the mcr-1 gene based on the CRISPR/Cas13a system and lateral flow strips was established, with a detection limit of 100 copies/mL. This method demonstrated high analytical specificity, with no cross-reactivity detected in non-mcr-1 and non-resistant strains. Among 36 clinical isolates, the method identified 31 strains as positive for the mcr-1 gene, and had a 100% concordance rate with the results of qPCR.<br><br>CONCLUSIONS: We established a detection method for the polymyxin resistance mcr-1 gene based on the CRISPR/Cas13a system. This method enables visual readouts without instruments, making it potentially applicable to primary healthcare settings and field surveillance.},
}
@article {pmid40537891,
year = {2025},
author = {Gao, Q and Zhang, T and Yuan, Y and Li, G and Li, B and Xiong, C},
title = {Detection of KPC-Producing Carbapenem-Resistant Klebsiella pneumoniae Based on CRISPR Cas12a.},
journal = {Journal of microbiology and biotechnology},
volume = {35},
number = {},
pages = {e2502042},
doi = {10.4014/jmb.2502.02042},
pmid = {40537891},
issn = {1738-8872},
mesh = {*Klebsiella pneumoniae/genetics/drug effects/isolation &amp; purification/enzymology ; *Bacterial Proteins/genetics/metabolism ; *beta-Lactamases/genetics/metabolism ; *Carbapenems/pharmacology ; Sensitivity and Specificity ; *CRISPR-Cas Systems ; Humans ; Klebsiella Infections/microbiology ; Anti-Bacterial Agents/pharmacology ; *Carbapenem-Resistant Enterobacteriaceae/genetics/isolation &amp; purification/enzymology ; *CRISPR-Associated Proteins/genetics ; *Endodeoxyribonucleases/genetics ; Polymerase Chain Reaction/methods ; ROC Curve ; },
abstract = {To develop a detection system for Klebsiella pneumoniae carbapenemase (KPC) and provide a reference for clinical prevention and control of nosocomial infections caused by multidrug-resistant K. pneumoniae. The KPC resistance gene was amplified by PCR. Guided by crRNA, Cas12a specifically identified the resistance gene and activated its trans-cleavage activity. In the detection system, a fluorescence probe was cleaved by activated Cas12a, and the fluorescence signal was measured using a microplate reader. Under optimized conditions, the fluorescence signal appeared within 12 min, peaked at 40 min and completed detection within 60 min. sensitivity: 91.2%, specificity: 84.1%, detection limit: 0.01 ng/μl. The samples were examined by fluorescence-CRISPR Cas12a and PCR. The coincidence rate was 85.9%, Kappa value was 0.8. The ROC curve analysis revealed an AUC of 0.916, with an optimal cutoff value of 1.55, sensitivity of 91.2%, and specificity of 84.1%. The CRISPR Cas12a detection of carbapenem-resistant K. pneumoniae (CRKP) demonstrates high sensitivity, specificity, and broad applicability. This method requires standard molecular biology equipment but does not rely on sequencing-based platforms.},
}
@article {pmid40537846,
year = {2025},
author = {Sarno, F and Jacob, JJ and Eilers, RE and Nebbioso, A and Altucci, L and Rots, MG},
title = {Epigenetic editing and epi-drugs: a combination strategy to simultaneously target KDM4 as a novel anticancer approach.},
journal = {Clinical epigenetics},
volume = {17},
number = {1},
pages = {105},
pmid = {40537846},
issn = {1868-7083},
mesh = {Humans ; *Jumonji Domain-Containing Histone Demethylases/genetics/antagonists &amp; inhibitors ; *Epigenesis, Genetic/drug effects ; DNA Methylation/drug effects ; *Gene Editing/methods ; Cell Line, Tumor ; *Antineoplastic Agents/pharmacology ; Gene Expression Regulation, Neoplastic/drug effects ; *Neoplasms/genetics/drug therapy ; CRISPR-Cas Systems ; Epigenome Editing ; },
abstract = {KDM4-A/B/C, preferentially demethylating di- and tri-methylated lysine 9 on histone H3, are overexpressed in cancers and considered interesting therapeutic targets. Consequently, KDM4 inhibitors have been developed to block their enzymatic activity. However, the potential lack of specificity of such small molecules (epi-drugs) may contribute to dose-limiting toxicities. In the pursuit of more specific interventions, epigenetic editing (epi-editing) has emerged as a powerful tool to modulate gene expression by modifying the epigenetic profile of specific genomic locations. The recently developed CRISPRoff (dCas9 fused to DNMT3A/3L and KRAB), guided by sgRNAs, is successfully used for gene repression by introducing methylation of DNA and (indirectly) of histones at the targeted genomic region. We propose that combining epi-editing (here to prevent the expression of KDM4) with epi-drugs (to inhibit the KDM4 protein activity) may represent a novel path for synergistic anticancer effects through simultaneous inhibition of gene expression and protein activity. Upon validating the downregulation of KDM4A in HEK293T cells through epi-editing, we demonstrated its repression in colon, breast and hepatocellular carcinomas which was effective in preventing (breast, MCF7) or inhibiting (colon, HCT116) cancer cell growth. Anticancer effect was also confirmed for these cell lines using the KDM4 inhibitor QC6352. In parallel, our studies demonstrate a previously unnoticed increase in the expression of KDM4-A/B/C genes following the inhibition of protein activity using the pan-KDM4 inhibitors QC6352 and JIB-04. Importantly, this induction of gene expression was fully prevented or even further inhibited by epi-editing. Then, we assessed the efficacy of our dual-targeted silencing approach in cancer cells and demonstrated that the inhibition in cancer cell growth by epi-drug or epigenetic editing could be further improved by combining the treatments. Building upon these findings, we introduce a novel, potentially synergistic, therapeutic strategy that combines epi-drug administration with epi-editing. This innovative approach aims to reduce drug toxicity and the potential development of resistance by preventing drug-induced upregulation of target enzyme expression, thereby further increasing anticancer effects.},
}
@article {pmid40537532,
year = {2025},
author = {Norota, K and Ishizuka, S and Hirose, M and Sato, Y and Maeki, M and Tokeshi, M and Ibrahim, SM and Harashima, H and Yamada, Y},
title = {Lipid nanoparticle delivery of the CRISPR/Cas9 system directly into the mitochondria of cells carrying m.7778G&gt;T mutation in MtDNA (mt-Atp8).},
journal = {Scientific reports},
volume = {15},
number = {1},
pages = {18717},
pmid = {40537532},
issn = {2045-2322},
support = {HI1813/7-1//Deutsche Forschungsgemeinschaft/ ; 23H00541//Ministry of Education, Culture, Sports, Science and Technology/ ; JPMJFR203X//Japan Science and Technology Corporation/ ; },
mesh = {*CRISPR-Cas Systems ; *DNA, Mitochondrial/genetics ; Humans ; Animals ; *Nanoparticles/chemistry/administration &amp; dosage ; HeLa Cells ; Mice ; *Point Mutation ; *Mitochondria/genetics/metabolism ; *Lipids/chemistry ; Mitochondrial Diseases/genetics/therapy ; DNA Breaks, Double-Stranded ; Ribonucleoproteins/genetics ; Liposomes ; },
abstract = {Mitochondrial genome mutations are associated with various diseases and gene therapy targeted to mitochondria has the potential to effectively treat such diseases. Here, we targeted a point mutation in mitochondrial DNA (mtDNA) that can cause mitochondrial diseases via delivery of the clustered, regularly interspaced, short palindromic repeats/Cas9 (CRISPR/Cas9) system to mitochondria using an innovative lipid nanoparticle (LNP) delivery system. To overcome the major barrier of the mitochondrial membrane structure, we investigated a strategy to deliver ribonucleoprotein (RNP) directly to mitochondria via membrane fusion using MITO-Porter, a mitochondria-targeting lipid nanoparticle. First, we constructed RNP-MITO-Porter, in which an RNP was loaded into MITO-Porter using a microfluidic device. Sequence-specific double-strand breaks were confirmed when the constructed RNP-MITO-Porter was applied to isolated mitochondria. Next, the RNP-MITO-Porter was applied to HeLa cells, and a portion of the RNP-MITO-Porter was colocalized with mitochondria and caused sequence-specific double-strand breaks in mtDNA. Finally, RNP-MITO-Porter was successfully delivered to mitochondria of cells derived from a mouse carrying a point mutation (m.7778G > T) in mtDNA (mt-Atp8) (LMSF-N-MTFVB cells), and created double-strand breaks at the target sequence. RNP-MITO-Porter is expected to contribute significantly to the clinical application of mitochondrion-targeted gene therapy.},
}
@article {pmid40480225,
year = {2025},
author = {Zou, S and Sun, Y and Tang, W},
title = {Charting the development and engineering of CRISPR base editors: lessons and inspirations.},
journal = {Cell chemical biology},
volume = {32},
number = {6},
pages = {789-808},
doi = {10.1016/j.chembiol.2025.05.003},
pmid = {40480225},
issn = {2451-9448},
mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; Humans ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Cytosine/metabolism/chemistry ; Adenine/chemistry/metabolism ; },
abstract = {CRISPR base editors (BEs) have introduced a new chapter in precise genome editing. The brief but fruitful history of BE development documents many case studies that not only lay the foundation of base-editing technology but are also instrumental to future protein engineering efforts. In this review, we summarize the development and engineering of various BEs with a focus on recent progress. These include traditional cytosine and adenine base editors (CBEs and ABEs), novel TadA-derived CBEs, transversion BEs, dual BEs, and CRISPR-free BEs. We discuss each aspect of the workflow and highlight the successes and challenges encountered in the engineering process.},
}
@article {pmid40456614,
year = {2025},
author = {Zetterdahl, OG and Crowe, JA and Reyhani, S and Güra, MA and Labastida-Botey, O and Girard, AS and Froese, DS and Ahlenius, H and Canals, I},
title = {Generation of iPSC Lines with Tagged α-Synuclein for Visualization of Endogenous Protein in Human Cellular Models of Neurodegenerative Disorders.},
journal = {eNeuro},
volume = {12},
number = {6},
pages = {},
doi = {10.1523/ENEURO.0093-25.2025},
pmid = {40456614},
issn = {2373-2822},
mesh = {Humans ; *alpha-Synuclein/metabolism/genetics ; *Induced Pluripotent Stem Cells/metabolism ; *Neurodegenerative Diseases/metabolism/pathology ; Neurons/metabolism ; Cell Line ; CRISPR-Cas Systems ; Gene Editing ; Cell Differentiation ; },
abstract = {α-Synuclein is a synaptic protein that accumulates primarily in synucleinopathies and secondarily in certain lysosomal storage disorders. However, its physiological roles in health and disease are not fully understood. In part, this has been hampered by the inability to visualize α-synuclein and its cellular localization, due to the lack of specific antibodies and faithful reporters. Here, we used CRISPR/Cas9-based genome editing to generate human-induced pluripotent stem cell (iPSC) lines in which the α-synuclein (SNCA) gene has been tagged with the short HA peptide either at the N-terminus or C-terminus or with the fluorescent protein mCherry at the C-terminus of the protein. These diverse strategies revealed the C-terminus HA-tag as the best option. C-Terminus HA-tagged α-synuclein had unchanged protein expression and did not generate degradation by-products. Importantly, we show that following differentiation to neurons, the C-terminus HA-tagged iPSC line had unaffected electrophysiological properties and could be used to visualize accumulation of α-synuclein upon inhibition of lysosomal function and under physiological protein levels. It is our expectation that this line and tagging approach will be very useful in further studies examining α-synuclein aggregation and its role in cellular dysfunction and neurodegeneration.},
}
@article {pmid40349174,
year = {2025},
author = {Cui, Z and Huang, F and Fang, K and Yan, J and Zhang, Y and Kang, DD and Zhou, Y and Zhao, Y and Everitt, JI and Hankey, W and Armstrong, AJ and Huang, J and Wang, H and Jin, VX and Dong, Y and Wang, Q},
title = {SCORT-Cas13d Nanotherapy Precisely Targets the 'Undruggable' Transcription Factor HoxB13 in Metastatic Prostate Cancer In Vivo.},
journal = {Advanced science (Weinheim, Baden-Wurttemberg, Germany)},
volume = {12},
number = {23},
pages = {e2417605},
doi = {10.1002/advs.202417605},
pmid = {40349174},
issn = {2198-3844},
support = {R01CA287510/CA/NCI NIH HHS/United States ; R35GM144117/GM/NIGMS NIH HHS/United States ; //Department of Pathology/ ; //Duke University School of Medicine/ ; 2023-TRG-0006//North Carolina Biotechnology Center/ ; R01CA287510/CA/NCI NIH HHS/United States ; R35GM144117/GM/NIGMS NIH HHS/United States ; },
mesh = {Male ; *Homeodomain Proteins/genetics/metabolism ; *Prostatic Neoplasms/genetics/pathology/metabolism/therapy ; Animals ; Mice ; Humans ; Cell Line, Tumor ; *Nanoparticles ; CRISPR-Cas Systems/genetics ; Neoplasm Metastasis ; Cell Proliferation ; },
abstract = {Metastatic cancer, the primary cause of cancer mortality, frequently exhibits heightened dependence on certain transcription factors (TFs), which serve as master regulators of oncogenic signaling yet are often untargetable by small molecules. Selective Cell in ORgan Targeting (SCORT) nanoparticles are developed for precise CRISPR/Cas13d mRNA and gRNA delivery to metastatic cancer cells in vivo, aiming to knock down the undruggable oncogenic TF HoxB13. In prostate cancer liver metastasis models driven by HoxB13, repeated systemic SCORT-Cas13d-gHoxB13 treatment significantly decreases HoxB13 expression, reduces metastasis, and extends mouse survival. Prolonged treatment shows no significant impact on major organ function, histology or immune markers. Mechanistically, SCORT-Cas13d-gHoxB13 treatment suppresses metastatic tumor proliferation and angiogenesis while promoting apoptosis by regulating multiple gene pathways. Unexpectedly, it inhibits the non-canonical, EMT-independent oncogenic function of Snail. These findings suggest that SCORT-Cas13d-gHoxB13 can effectively and safely target the undruggable HoxB13 in metastatic prostate cancer, positioning CRISPR/Cas13d as a potential treatment.},
}
@article {pmid40325913,
year = {2025},
author = {Sugita, K and Kurata, M},
title = {Identification of Target Genes Using Innovative Screening Systems.},
journal = {Pathology international},
volume = {75},
number = {6},
pages = {257-266},
pmid = {40325913},
issn = {1440-1827},
mesh = {Humans ; *Neoplasms/genetics ; *Genetic Testing/methods ; Oncogenes/genetics ; High-Throughput Nucleotide Sequencing ; Single-Cell Analysis/methods ; CRISPR-Cas Systems ; Mutation ; Animals ; },
abstract = {Advances in cancer biology have been achieved by the identification of oncogenes and tumor suppressor genes through the remarkable progression of next-generation sequencing. New techniques, such as single-cell analysis, help uncover cancer progression and heterogeneity. Reverse genetic screenings, including methods like random mutagenesis via retroviruses, transposons, RNA interference, and CRISPR, are useful for exploring gene functions and their roles in cancer. Especially in random mutagenesis, CRISPR screening and its modifications have recently emerged as powerful tools due to their comprehensiveness and simplicity in inducing genetic mutations. Initially, CRISPR screening focused on analyzing biological phenotypes in a cell population. It has since evolved to incorporate advanced techniques, such as combining single-cell and spatial analyses. These developments enable the investigation of cell-cell and spatial interactions, which more closely mimic In Vivo microenvironments, offering deeper insights into complex biological processes. These approaches allow for the identification of essential genes involved in cancer survival, drug resistance, and tumorigenesis. Together, these technologies are advancing cancer research and therapeutic development.},
}
@article {pmid40301572,
year = {2025},
author = {Zamora-Dorta, M and Laine-Menéndez, S and Abia, D and González-García, P and López, LC and Fernández-Montes, P and Calvo, E and Vázquez, J and Enríquez, JA and Balsa, E},
title = {Time-resolved mitochondrial screen identifies regulatory components of oxidative metabolism.},
journal = {EMBO reports},
volume = {26},
number = {12},
pages = {3045-3074},
pmid = {40301572},
issn = {1469-3178},
support = {PID2019-110766GA-I00//Ministerio de Ciencia, Innovaci&#xf3;n y Universidades (MCIU)/ ; PID2022-137404OB-I00//Ministerio de Ciencia, Innovaci&#xf3;n y Universidades (MCIU)/ ; PID2021-1279880B//Ministerio de Ciencia, Innovaci&#xf3;n y Universidades (MCIU)/ ; FPU21/06416//Ministerio de Ciencia, Innovaci&#xf3;n y Universidades (MCIU)/ ; CEX2021-001154-S//Ministerio de Ciencia, Innovaci&#xf3;n y Universidades (MCIU)/ ; CEX2020-001041-S//Ministerio de Ciencia, Innovaci&#xf3;n y Universidades (MCIU)/ ; Margarita Salas-UAM postdoctoral fellowship CA4/RSUE/2022-00037//Ministerio de Ciencia, Innovaci&#xf3;n y Universidades (MCIU)/ ; FPI-UAM PhD fellowship//Ministerio de Ciencia, Innovaci&#xf3;n y Universidades (MCIU)/ ; ERC-2020-STG grant agreement n{degree sign}
948478//EC | ERC | HORIZON EUROPE European Research Council (ERC)/ ; PR_EX_2022-01//CRIS Cancer Foundation (CRIS Foundation)/ ; LCF/PR/H23/52430010//'la Caixa' Foundation ('la Caixa')/ ; PRTR//EC | NextGenerationEU (NGEU)/ ; CB16/10/00282//Centro de Investigaci&#xf3;n Biom&#xe9;dica en Red Fragilidad y Envejecimiento Saludable (CIBERFES)/ ; },
mesh = {Humans ; *Mitochondria/metabolism/genetics ; *Oxidative Phosphorylation ; CRISPR-Cas Systems ; Mitochondrial Proteins/metabolism/genetics ; Oxidation-Reduction ; Amino Acids, Branched-Chain/metabolism ; Mitochondrial Diseases/genetics/metabolism ; },
abstract = {Defects in mitochondrial oxidative metabolism underlie many genetic disorders with limited treatment options. The incomplete annotation of mitochondrial proteins highlights the need for a comprehensive gene inventory, particularly for Oxidative Phosphorylation (OXPHOS). To address this, we developed a CRISPR/Cas9 loss-of-function library targeting nuclear-encoded mitochondrial genes and conducted galactose-based screenings to identify novel regulators of mitochondrial function. Our study generates a gene catalog essential for mitochondrial metabolism and maps a dynamic network of mitochondrial pathways, focusing on OXPHOS complexes. Computational analysis identifies RTN4IP1 and ECHS1 as key OXPHOS genes linked to mitochondrial diseases in humans. RTN4IP1 is found to be crucial for mitochondrial respiration, with complexome profiling revealing its role as an assembly factor required for the complete assembly of complex I. Furthermore, we discovered that ECHS1 controls oxidative metabolism independently of its canonical function in fatty acid oxidation. Its deletion impairs branched-chain amino acids (BCAA) catabolism, disrupting lipoic acid-dependent enzymes such as pyruvate dehydrogenase (PDH). This deleterious phenotype can be rescued by restricting valine intake or catabolism in ECHS1-deficient cells.},
}
@article {pmid40268165,
year = {2025},
author = {Jerke, U and Eulenberg-Gustavus, C and Wagner, DL and Schreiber, A and Kettritz, R},
title = {CRISPR-Cas9 mediated proteinase 3 autoantigen deletion as a treatment strategy for anti-neutrophil cytoplasmic autoantibody-associated vasculitis.},
journal = {Kidney international},
volume = {108},
number = {1},
pages = {145-149},
doi = {10.1016/j.kint.2025.03.020},
pmid = {40268165},
issn = {1523-1755},
mesh = {Humans ; *Myeloblastin/genetics/immunology/metabolism ; *CRISPR-Cas Systems ; *Autoantigens/genetics/immunology ; Neutrophils/immunology ; *Gene Editing/methods ; *Anti-Neutrophil Cytoplasmic Antibody-Associated Vasculitis/therapy/immunology/genetics ; Hematopoietic Stem Cells/immunology ; Cell Differentiation ; Proof of Concept Study ; Antibodies, Antineutrophil Cytoplasmic/immunology ; RNA, Guide, CRISPR-Cas Systems/genetics ; Cells, Cultured ; *Genetic Therapy/methods ; Leukocyte Elastase ; },
abstract = {INTRODUCTION: Proteinase 3 (PR3) is a major autoantigen in patients with anti-neutrophil cytoplasmic autoantibody (ANCA)-associated vasculitis (AAV). Here, we performed a proof-of-principle study using ex vivo CRISPR-Cas9 guided gene editing to eliminate the PR3 autoantigen as an alternative to suppressing the autoimmune response to PR3.<br><br>METHODS: A ribonucleoprotein (RNP) complex of Cas9 protein and a PR3-specific single guide-RNA was transfected into human CD34[+] hematopoietic stem and progenitor cells (HSPC) by electroporation. Effects on PR3 protein abundance, neutrophil differentiation, and ANCA-dependent and -independent neutrophil responses were assessed.<br><br>RESULTS: Gene editing introduced a frame shift in exon 2 of PRTN3. Consequently, PR3 protein was efficiently reduced in neutrophil-differentiated HSPCs as demonstrated by immunoblotting, ELISA, microscopy, and the complete absence of PR3-specific proteolytic activity. Human neutrophil elastase served as control and was not affected. PR3-deleted (PR3[KO])- and PR3 wild-type (PR3[WT])-HSPCs showed similar neutrophil differentiation. Importantly, general neutrophil defense functions to non-ANCA receptor-independent and -dependent stimuli were similar in PR3[KO]- and PR3[WT]-neutrophils as was constitutive apoptosis. Flow cytometry showed that cell membrane-PR3 was significantly reduced on PR3[KO]-neutrophils and consequent neutrophil activation to either monoclonal antibodies to PR3 or human PR3-ANCA was attenuated. In contrast, myeloperoxidase-ANCA stimulation was not affected.<br><br>CONCLUSIONS: We show the feasibility and efficacy of depleting the PR3 autoantigen in human CD34[+] HSPCs using CRISPR-Cas9. Depleting the PR3 autoantigen instead of suppressing the autoimmune response to PR3 could potentially lead to drug-free remission, particularly in patients with refractory or relapsing disease.},
}
@article {pmid40172759,
year = {2025},
author = {Yang, H and Ji, X and Zhong, H and Yang, X and Hu, D and Cai, G and Wu, Z},
title = {CRISPR screening identifies protein methylation and ubiquitination modifications that modulate aflatoxin B1 cytotoxicity.},
journal = {Science China. Life sciences},
volume = {68},
number = {7},
pages = {2121-2136},
pmid = {40172759},
issn = {1869-1889},
mesh = {*Aflatoxin B1/toxicity ; *Ubiquitination/drug effects ; Humans ; Protein Processing, Post-Translational/drug effects ; Methylation/drug effects ; *Cystathionine beta-Synthase/metabolism/genetics ; S-Adenosylmethionine/metabolism ; CRISPR-Cas Systems ; Mitochondria/metabolism/drug effects ; Cell Survival/drug effects ; Gene Knockout Techniques ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Cell Death/drug effects ; },
abstract = {Aflatoxin B1 (AFB1) is one of the most potent mycotoxins affecting human health and animal production. To deeply understand the host-toxin interaction, we performed CRISPR screening and identified cystathionine β-synthase (CBS) as a critical host gene affecting AFB1 cytotoxicity. Mechanistic studies revealed that CBS affects AFB1-induced cell death by regulating the abundance of protein post-translational modifications (PTMs) in host cells. First, AFB1 disrupted the transfer of S-adenosylmethionine (SAM) from the cytoplasm to the mitochondria, thereby reducing the intra-mitochondrial protein methylation level. Deficient intra-mitochondrial protein methylation impaired mitochondrial function and caused cell death. CBS knockout (KO) can enhance SAM generation and mobilization to restore intra-mitochondrial SAM levels by rescuing the perturbed methionine cycle after AFB1 exposure, thereby alleviating AFB1-induced cell death. Second, AFB1 decreased global protein ubiquitination levels by affecting gene expression of ubiquitin-modified enzymes. CBS-KO and pharmaceutical treatment correcting gene expression of ubiquitin-modified enzymes can rescue AFB1-induced cell death. We also investigated two PTM-regulating small molecules, SAM and PR-619, which can increase cell viability in AFB1-exposed cells.},
}
@article {pmid40100189,
year = {2025},
author = {Džafo, E and Hafezi, M and Attianese, GMPG and Reichenbach, P and Grillet, S and Garcia, H and Cribioli, E and Voize, C and Tissot, S and de Silly, RV and Coukos, G and Scholten, K and Irving, M and Gentner, B},
title = {DNA-dependent protein kinase inhibitors PI-103 and samotolisib augment CRISPR/Cas9 knock-in efficiency in human T cells.},
journal = {Cytotherapy},
volume = {27},
number = {6},
pages = {766-773},
doi = {10.1016/j.jcyt.2025.02.001},
pmid = {40100189},
issn = {1477-2566},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *T-Lymphocytes/drug effects/metabolism/immunology ; *DNA-Activated Protein Kinase/antagonists &amp; inhibitors ; *Gene Knock-In Techniques/methods ; *Protein Kinase Inhibitors/pharmacology ; *Pyrimidines/pharmacology ; },
abstract = {The adoptive transfer of autologous peripheral blood T cells gene-modified to express preselected, tumor antigen-specific T-cell receptors (TCRs) is a promising treatment for solid cancers. While gene-transfer by viral transduction is highly efficient, the insertional site is not targeted and persistence of the T cells is oftentimes limited. In contrast, site-specific integration of the TCR into the TCR α chain (TRAC) locus by CRISPR/Cas9 has been shown to enable more consistent and physiologic levels of exogenous TCR expression coupled with superior persistence and tumor control in preclinical studies. Here, we sought to improve the efficiency of CRISPR/Cas9 mediated TCR knock-in (KI) into the TRAC locus of primary human T cells. In addition to the previously reported DNA-dependent protein kinase (DNA-PK) inhibitor M3814, we demonstrated that PI-103 and samotolisib markedly increase KI efficiency in a process that is good manufacturing process (GMP)-compatible. Importantly, samotolisib enabled the generation of a potent T-cell product, having no negative impact on T-cell viability, phenotype, expansion, effector function, and tumor control. Overall, we conclude that our GMP-compatible CRISPR/Cas9 protocol comprising samotolisib to augment TCR KI efficiency is suitable for the generation of genetically modified T cells for clinical use.},
}
@article {pmid40035969,
year = {2025},
author = {Feng, Y and Liu, G and Li, H and Cheng, L},
title = {The landscape of cell lineage tracing.},
journal = {Science China. Life sciences},
volume = {68},
number = {7},
pages = {1941-1963},
pmid = {40035969},
issn = {1869-1889},
mesh = {*Cell Lineage/genetics ; Humans ; Animals ; *Cell Tracking/methods ; CRISPR-Cas Systems ; Cell Differentiation ; },
abstract = {Cell fate changes play a crucial role in the processes of natural development, disease progression, and the efficacy of therapeutic interventions. The definition of the various types of cell fate changes, including cell expansion, differentiation, transdifferentiation, dedifferentiation, reprogramming, and state transitions, represents a complex and evolving field of research known as cell lineage tracing. This review will systematically introduce the research history and progress in this field, which can be broadly divided into two parts: prospective tracing and retrospective tracing. The initial section encompasses an array of methodologies pertaining to isotope labeling, transient fluorescent tracers, non-fluorescent transient tracers, non-fluorescent genetic markers, fluorescent protein, genetic marker delivery, genetic recombination, exogenous DNA barcodes, CRISPR-Cas9 mediated DNA barcodes, and base editor-mediated DNA barcodes. The second part of the review covers genetic mosaicism, genomic DNA alteration, TCR/BCR, DNA methylation, and mitochondrial DNA mutation. In the final section, we will address the principal challenges and prospective avenues of enquiry in the field of cell lineage tracing, with a particular focus on the sequencing techniques and mathematical models pertinent to single-cell genetic lineage tracing, and the value of pursuing a more comprehensive investigation at both the spatial and temporal levels in the study of cell lineage tracing.},
}
@article {pmid39828625,
year = {2025},
author = {Yang, J and Xu, X and Yang, L and Tian, Y and Wang, J and Han, D},
title = {Dynamic Genomic Imaging and Tracking in Living Cells by a DNA Origami-Based CRISPR‒dCas9 System.},
journal = {Small methods},
volume = {9},
number = {6},
pages = {e2401559},
doi = {10.1002/smtd.202401559},
pmid = {39828625},
issn = {2366-9608},
support = {2021YFA0909400//National Key Research and Development Program of China/ ; 2020YFA0211100//National Key Research and Development Program of China/ ; 22225402//National Natural Science Foundation of China/ ; 22204099//National Natural Science Foundation of China/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; Humans ; *DNA/chemistry/genetics ; *Genomics/methods ; Nanostructures/chemistry ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Green Fluorescent Proteins/genetics/metabolism ; },
abstract = {The clustered regularly interspaced short palindromic repeat (CRISPR)-associated system has displayed promise in visualizing the dynamics of target loci in living cells, which is important for studying genome regulation. However, developing a cell-friendly and rapid transfection method for achieving dynamic and long-term genomic imaging in living cells with high specificity and accuracy is still challenging. Herein, a robust and versatile method is presented that employs a barrel-shaped DNA nanostructure (TUBE) modified with aptamers for loading, protecting, and delivering CRISPR-Cas9 to visualize specific genomic loci in living cells. This approach enables dynamic tracking of target genomic regions (Chr3q29, a repetitive region of chromosome 3) throughout the mitotic process and captures variations in their spatial distribution and quantity accurately. Distinct dynamic behaviors between the Chr3q29 and telomeres are observed, which are linked to their unique chromosomal positions and levels of mobility. High-resolution multicolor labeling of the target genes is achieved, with a high degree of colocalization between the enhanced green fluorescent protein and cyanine-5 channels, facilitating precise imaging of target loci. This method not only supports dynamic genomic imaging but also enables multiplexed tracking, providing a powerful visualization tool for studying cellular processes and genetic interactions in real time within living cells.},
}
@article {pmid39718905,
year = {2025},
author = {Yao, ZY and Yu, MJ and Li, QQ and Gong, JS and Zhang, P and Jiang, JY and Su, C and Xu, G and Jia, BY and Xu, ZH and Shi, JS},
title = {Unlocking Green Biomanufacturing Potential: Superior Heterologous Gene Expression with a T7 Integration Overexpression System in Bacillus subtilis.},
journal = {ACS synthetic biology},
volume = {14},
number = {6},
pages = {1977-1987},
doi = {10.1021/acssynbio.4c00694},
pmid = {39718905},
issn = {2161-5063},
mesh = {*Bacillus subtilis/genetics/metabolism ; CRISPR-Cas Systems/genetics ; *DNA-Directed RNA Polymerases/genetics/metabolism ; *Viral Proteins/genetics/metabolism ; Gene Expression Regulation, Bacterial ; Gene Expression ; },
abstract = {Industrial biotechnology employs cells for producing valuable products and serving as biocatalysts sustainably, addressing resource, energy, and environmental issues. Bacillus subtilis is a preferred host for creating microbial chassis cells and producing industrial enzymes and functional nutritional products. In this study, a dual-module T7 integration expression system in B. subtilis was established. The first module, driven by the T7 RNA polymerase, was integrated into the genome via the CRISPR/Cas9 system. Another module responsible for expression control was systematically integrated into 28 discrete chromosomal loci and the impact of different genomic positions on gene expression was explored, resulting in a high-intensity integrated expression system. Furthermore, by modifying the LacI repressor factor for biological regulation, we achieved a strong expression intensity without the inducer addition. This system was successfully used to express phospholipase D and hyaluronic acid lyase, resulting in extracellular enzyme activities of 339.12 U/mL and 2.60 × 10[4] U/mL, respectively. Additionally, by exclusively targeting the HA gene cluster for expression, a production yield of 6.86 g/L was achieved on a 5 L fermentation scale. The system eliminates the use of antibiotics and inducers, offering a controllable, efficient, and promising gene expression regulation tool in B. subtilis, enhancing its potential for biomanufacturing applications.},
}
@article {pmid39236976,
year = {2025},
author = {Hou, ZH and Gao, Y and Zheng, JC and Zhao, MJ and Liu, Y and Cui, XY and Li, ZY and Wei, JT and Yu, TF and Zheng, L and Jiao, YC and Yang, SH and Hao, JM and Chen, J and Zhou, YB and Chen, M and Qiu, L and Ma, YZ and Xu, ZS},
title = {GmBSK1-GmGSK1-GmBES1.5 regulatory module controls heat tolerance in soybean.},
journal = {Journal of advanced research},
volume = {73},
number = {},
pages = {187-198},
doi = {10.1016/j.jare.2024.09.004},
pmid = {39236976},
issn = {2090-1224},
mesh = {*Glycine max/genetics/physiology/metabolism ; Plants, Genetically Modified/genetics ; Gene Expression Regulation, Plant ; *Thermotolerance/genetics ; CRISPR-Cas Systems ; *Plant Proteins/genetics/metabolism ; *Heat-Shock Response/genetics ; Reactive Oxygen Species/metabolism ; Signal Transduction/genetics ; Brassinosteroids/metabolism ; },
abstract = {INTRODUCTION: Heat stress poses a severe threat to the growth and production of soybean (Glycine max). Brassinosteroids (BRs) actively participate in plant responses to abiotic stresses, however, the role of BR signaling pathway genes in response to heat stress in soybean remains poorly understood.<br><br>OBJECTIVES: In this study, we investigate the regulatory mechanisms of GmBSK1 and GmBES1.5 in response to heat stress and the physiological characteristics and yield performance under heat stress conditions.<br><br>METHODS: Transgenic technology and CRISPR/Cas9 technology were used to generated GmBSK1-OE, GmBES1.5-OE and gmbsk1 transgenic soybean plants, and transcriptome analysis, LUC activity assay and EMSA assay were carried out to elucidate the potential molecular mechanism underlying GmBSK1-GmBES1.5-mediated heat stress tolerance in soybean.<br><br>RESULTS: CRISPR/Cas9-generated gmbsk1 knockout mutants exhibited increased sensitivity to heat stress due to a reduction in their ability to scavenge reactive oxygen species (ROS). The expression of GmBES1.5 was up-regulated in GmBSK1-OE plants under heat stress conditions, and it directly binds to the E-box motif present in the promoters of abiotic stress-related genes, thereby enhancing heat stress tolerance in soybean plants. Furthermore, we identified an interaction between GmGSK1 and GmBES1.5, while GmGSK1 inhibits the transcriptional activity of GmBES1.5. Interestingly, the interaction between GmBSK1 and GmGSK1 promotes the localization of GmGSK1 to the plasma membrane and releases the transcriptional activity of GmBES1.5.<br><br>CONCLUSION: Our findings suggest that both GmBSK1 and GmBES1.5 play crucial roles in conferring heat stress tolerance, highlighting a potential strategy for breeding heat-tolerant soybean crops involving the regulatory module consisting of GmBSK1-GmGSK1-GmBES1.5.},
}
@article {pmid39097089,
year = {2025},
author = {Tan, S and Yuan, C and Zhu, Y and Chang, S and Li, Q and Ding, J and Gao, X and Tian, R and Han, Z and Hu, Z},
title = {Glutathione hybrid poly (beta-amino ester)-plasmid nanoparticles for enhancing gene delivery and biosafety.},
journal = {Journal of advanced research},
volume = {73},
number = {},
pages = {697-711},
doi = {10.1016/j.jare.2024.07.038},
pmid = {39097089},
issn = {2090-1224},
mesh = {*Glutathione/chemistry ; *Plasmids/chemistry/genetics ; Animals ; *Nanoparticles/chemistry ; Humans ; Mice ; Reactive Oxygen Species/metabolism ; *Gene Transfer Techniques ; *Polymers/chemistry ; Gene Editing/methods ; Genetic Therapy/methods ; Transfection/methods ; CRISPR-Cas Systems ; },
abstract = {INTRODUCTION: CRISPR/Cas9 gene editing technology has significantly advanced gene therapy, with gene vectors being one of the key factors for its success. Poly (beta-amino ester) (PBAE), a distinguished non-viral cationic gene vector, is known to elevate intracellular reactive oxygen species (ROS) levels, which may cause cytotoxicity and, consequently, impact gene transfection efficacy (T.E.).<br><br>OBJECTIVES: To develop a simple but efficient strategy to improve the gene delivery ability and biosafety of PBAE both in vivo and in vitro.<br><br>METHODS: We used glutathione (GSH), a clinically utilized drug with capability to modulating intracellular ROS level, to prepare a hybrid system with PBAE-plasmid nanoparticles (NPs). This system was characterized by flow cytometry, RNA-seq, Polymerase Chain Reaction (PCR) and Sanger sequencing in vitro, and its safety and efficacy in vivo was evaluated by imaging, PCR, Sanger sequencing and histology analysis.<br><br>RESULTS: The particle size of GSH-PBAE-plasmid NPs were 168.31&#xa0;nm with a &#x3b6;-potential of 15.21&#xa0;mV. An enhancement in T.E. and gene editing efficiency, ranging from 10&#xa0;% to 100&#xa0;%, was observed compared to GSH-free PBAE-plasmid NPs in various cell lines. In vitro results proved that GSH-PBAE-plasmid NPs reduced intracellular ROS levels by 25&#xa0;%-40&#xa0;%, decreased the total number of upregulated/downregulated genes from 4,952 to 789, and significantly avoided the disturbance in gene expression related to cellular oxidative stress-response and cell growth regulation signaling pathway compared to PBAE-plasmid NPs. They also demonstrated lower impact on the cell cycle, slighter hemolysis, and higher cell viability after gene transfection. Furthermore, GSH hybrid PBAE-plasmid NPs exhibited superior safety and improved tumor suppression ability in an Epstein-Barr virus (EBV)-infected murine tumor model, via targeting cleavage the EBV related oncogene by delivering CRISPR/Cas9 gene editing system and down-regulating the expression levels. This simple but effective strategy is expected to promote clinical applications of non-viral vector gene delivery.},
}
@article {pmid38084666,
year = {2025},
author = {Ogawa, Y and Lu, Y and Kiyozumi, D and Chang, HY and Ikawa, M},
title = {CRISPR/Cas9-mediated genome editing reveals seven testis-enriched transmembrane glycoproteins dispensable for male fertility in mice.},
journal = {Andrology},
volume = {13},
number = {5},
pages = {1251-1260},
pmid = {38084666},
issn = {2047-2927},
support = {R01 HD088412/HD/NICHD NIH HHS/United States ; JP22K15103//Japan Society for the Promotion of Science/ ; JP21H02487//Japan Society for the Promotion of Science/ ; JP21H00231//Japan Society for the Promotion of Science/ ; JP21K19263//Japan Society for the Promotion of Science/ ; JP21H05033//Japan Society for the Promotion of Science/ ; JPMJPR2143//Japan Science and Technology Agency/ ; R01HD088412//Eunice Kennedy Shriver National Institute of Child Health and Human Development/ ; INV-001902/GATES/Gates Foundation/United States ; },
mesh = {Animals ; Male ; *Testis/metabolism ; *Fertility/genetics ; Mice ; *CRISPR-Cas Systems ; Mice, Knockout ; *Gene Editing ; *Membrane Glycoproteins/genetics/metabolism ; Female ; Sperm Motility/genetics ; Spermatozoa/metabolism ; Spermatogenesis/genetics ; },
abstract = {BACKGROUND: Mammalian fertilization is mediated by multiple sperm acrosomal proteins, many of which are testis-enriched transmembrane glycoproteins expressed during spermiogenesis (e.g., Izumo sperm-egg fusion 1, Sperm acrosome associated 6, and Transmembrane protein 95).<br><br>METHODS: We hypothesized that proteins with these features might have a role in sperm-egg interaction and thus carried out an in-silico screen based on multiple public databases. We generated knockout mouse lines lacking seven candidate proteins by the CRISPR/Cas9 system and conducted detailed analyses on the fecundity of the knockout males, as well as their testis appearance and weight, testis and epididymis histology, and sperm motility and morphology.<br><br>RESULTS: Through the in-silico screen, we identified 4932438H23Rik, A disintegrin and metalloproteinase domain-containing protein 29, SAYSvFN domain-containing protein 1, Sel-1 suppressor of lin-12-like 2 (C. elegans), Testis-expressed protein 2, Transmembrane and immunoglobulin domain-containing 3, and Zinc and ring finger 4. Phenotypic analyses unveiled that the knockout males showed normal testis gross appearance, normal testis and epididymis histology, and normal sperm morphology and motility. Fertility tests further indicated that the knockout male mice could sire pups with normal litter sizes when paired with wild-type females.<br><br>DISCUSSION AND CONCLUSION: These findings suggest that these seven proteins are individually dispensable for male reproduction and fertilization. Future studies are warranted to devise advanced in-silico screening approaches that permit effective identification of gamete fusion-required sperm proteins.},
}
@article {pmid40545778,
year = {2025},
author = {Li, Y and Jin, L and Li, W and Wang, K and Su, H and Mao, H and Chen, W and Lan, C and Li, Q and Kaufmann, K and Yan, W},
title = {Modulation of an evolutionarily conserved epigenetic regulon controlling abscisic acid catabolism enhances drought tolerance in wheat.},
journal = {The New phytologist},
volume = {},
number = {},
pages = {},
doi = {10.1111/nph.70302},
pmid = {40545778},
issn = {1469-8137},
support = {32272127//National Natural Science Foundation of China/ ; },
abstract = {Drought stress significantly reduces crop yield by triggering abscisic acid (ABA) accumulation in plants. It involves the suppression of CYP707A genes, which encode enzymes that catalyze ABA. However, little is known about epigenetic control in the CYP707A gene-mediated drought stress response in wheat. In this study, we reported that TaCYP707A-6A/6B/6D but not TaCYP707A-5A/5B/5D participates in drought response in common wheat. Disruption of TaCYP707A-6B showed enhanced drought tolerance but also decreased fertility. Expression of TaCYP707A-6B is negatively associated with H3K27me3 level. An evolutionarily conserved CTCTGYTY motif cluster (binding site for a Jumonji H3K27me3 demethylase) was found in the intron of TaCYP707A-6B as well as the intron of CYP707A homologs in other plant species. Blocking the CTCTGYTY motif by dead Cas9 (dCas9) maintained a high level of H3K27me3 on the CYP707A gene, while decreasing its expression level leading to enhanced drought tolerance in both wheat and Arabidopsis. In particular, the mutant in which the intron bound by H3K27me3 demethylase was cut out without change of splicing pattern showed enhanced drought tolerance. Therefore, our study provides a novel approach to improve plant drought tolerance by manipulating an evolutionarily conserved cis-element bound by histone demethylases in the intron of CYP707A genes.},
}
@article {pmid40545632,
year = {2025},
author = {Swinnen, G and Lizé, E and Loera Sánchez, M and Stolz, S and Soyk, S},
title = {Application of a GRF-GIF chimera enhances plant regeneration for genome editing in tomato.},
journal = {Plant biotechnology journal},
volume = {},
number = {},
pages = {},
doi = {10.1111/pbi.70212},
pmid = {40545632},
issn = {1467-7652},
support = {802008//European Research Council/International ; 310030_212218//Schweizerischer Nationalfonds zur F&#xf6;rderung der Wissenschaftlichen Forschung/ ; },
abstract = {Genome editing has become a routine tool for functionally characterizing plant and animal genomes. However, stable genome editing in plants remains limited by the time- and labour-intensive process of generating transgenic plants, as well as by the efficient isolation of desired heritable edits. In this study, we evaluated the impact of the morphogenic regulator GRF-GIF on plant regeneration and genome editing outcomes in tomato. We demonstrate that expressing a tomato GRF-GIF chimera reliably accelerates the onset of shoot regeneration from callus tissue culture by approximately one month and nearly doubles the number of recovered transgenic plants. Consequently, the GRF-GIF chimera enables the recovery of a broader range of edited haplotypes and simplifies the isolation of mutants harbouring heritable edits, but without markedly interfering with plant growth and development. Based on these findings, we outline strategies that employ basic or advanced diagnostic pipelines for efficient isolation of single- and higher-order mutants in tomato. Our work represents a technical advantage for tomato transformation and genome editing, with potential applications across other Solanaceae species.},
}
@article {pmid40545185,
year = {2025},
author = {Mondal, AR and Misra, A},
title = {'Emerging cell-specific therapies in cardiovascular disease'.},
journal = {Vascular pharmacology},
volume = {},
number = {},
pages = {107516},
doi = {10.1016/j.vph.2025.107516},
pmid = {40545185},
issn = {1879-3649},
abstract = {Atherosclerosis is a leading cause of cardiovascular morbidity and mortality worldwide, driven by complex interactions among various plaque cell types, including endothelial cells, macrophages, and smooth muscle cells. Traditional therapies targeting systemic risk factors such as cholesterol and blood pressure fail to directly address the underlying mechanisms governing plaque formation and progression. Recent advances in cell-specific therapies offer new avenues for targeting the cellular and molecular processes driving atherosclerosis. This Review explores innovative strategies including nanoparticles, viral vectors and CRISPR-Cas9 technology, which have the potential to modulate gene expression and behaviour within plaques cells to alleviate disease. By focusing on the specific roles of key cell types in atherosclerosis, these emerging therapies promise to provide more precise, effective, and personalised treatment options without inducing off-target effects. Moreover, insights gained from successful applications of these technologies in oncology are considered for potential repurposing in atherosclerosis-related disease. As these cell-specific approaches advance through preclinical and clinical development, they may significantly enhance our ability to treat atherosclerosis at its cellular roots, offering new hope for reducing the burden of cardiovascular disease.},
}
@article {pmid40544537,
year = {2025},
author = {Sinha, S and Upadhyay, LSB},
title = {Understanding antimicrobial resistance (AMR) mechanisms and advancements in AMR diagnostics.},
journal = {Diagnostic microbiology and infectious disease},
volume = {113},
number = {2},
pages = {116949},
doi = {10.1016/j.diagmicrobio.2025.116949},
pmid = {40544537},
issn = {1879-0070},
abstract = {The overuse and abuse of antibiotics, which results in the evolution of resistant microorganisms, is the primary cause of the global health catastrophe known as antimicrobial resistance (AMR). The enzymatic breakdown of antibiotics, target site modification, efflux pump overexpression, and the formation of biofilm are some of the mechanisms responsible for acquiring antimicrobial resistance (AMR). These mechanisms enable bacteria to evade or neutralize the effects of antimicrobial agents, complicating treatment options and increasing mortality rates. The rapid dissemination of resistance genes via horizontal gene transfer further exacerbates the problem, necessitating urgent intervention. Advanced AMR diagnostics are transforming the fight against antimicrobial resistance. Biosensors enable rapid, point-of-care detection; Cluster regularly interspaced short palindromic repeat (CRISPR) technologies offer precise identification of resistance genes; and mass spectrometry provides fast, accurate profiling. Automated systems streamline workflows and boost throughput, while flow cytometry delivers real-time, single-cell analysis of phenotypic resistance. Together, these innovations accelerate detection and support targeted antimicrobial stewardship, essential for combating the global AMR threat. This review covers the mechanisms underlying antimicrobial resistance (AMR) and recent advancements in AMR diagnostic technologies.},
}
@article {pmid40544408,
year = {2025},
author = {Yu, D and Xie, Z and Zhang, Y and Xie, Z and Fan, Q and Luo, S and Xie, L and Li, M and Zeng, T and Zhang, M and Li, X and Wei, Y and Wu, A and Wan, L},
title = {A dual fluorescence channel RAA-based CRISPR-Cas12a/Cas13a system for highly sensitive detection of Gyrovirus galga1 and Gyrovirus homsa1.},
journal = {Virulence},
volume = {16},
number = {1},
pages = {2521012},
doi = {10.1080/21505594.2025.2521012},
pmid = {40544408},
issn = {2150-5608},
abstract = {Gyrovirus galga1 (GyG1) and Gyrovirus homsa1 (GyH1) are the second and third most common gyroviruses identified, respectively, after chicken anaemia virus. They were first reported in 2011 and are currently prevalent worldwide. However, limited research on these pathogens and a lack of prevention and control strategies have necessitated the establishment of a rapid diagnostic technique to address new challenges in infectious diseases. Recombinase acid amplification (RAA) combined with CRISPR - Cas12a or CRISPR - Cas13a technology has major advantages for highly sensitive and rapid diagnosis. Specific targets can activate CRISPR-Cas trans-cleavage activity, resulting in non-specific cleavage of single-stranded DNA by the CRISPR - Cas12a complex and RNA cleavage by the CRISPR - Cas13a complex. In this study, for the first time, we combined RAA-based CRISPR - Cas12a and CRISPR - Cas13a systems for simultaneous differential diagnosis of GyG1 and GyH1 infection. The results showed that dual fluorescence channel RAA-based CRISPR - Cas12a/Cas13a technology could detect GyG1 and GyH1 within one hour, with a minimum detection limit of 1.5 copies of the target DNA standard/µL and no cross-reactivity with other avian pathogens. In addition, this method could be used for clinical detection, with the results exhibiting high consistency with those obtained by qPCR. These findings demonstrate that our RAA-based CRISPR - Cas12a/Cas13a dual-channel detection system can detect two different subtypes of gyrovirus in a sample with good specificity and high sensitivity, improving the detection efficiency and providing a new technique for the study of viral infection dynamics.},
}
@article {pmid40544221,
year = {2025},
author = {Ajibode, ET and Bender, AR and Yehl, K},
title = {Profiling crRNA architectures for enhanced Cas12 biosensing.},
journal = {Communications biology},
volume = {8},
number = {1},
pages = {947},
pmid = {40544221},
issn = {2399-3642},
abstract = {CRISPR-Cas diagnostics are revolutionizing point-of-care molecular testing due to the programmability, simplicity, and sensitivity of Cas systems with trans-cleavage activity. CRISPR-Cas12 assays are promising for detecting single nucleotide polymorphisms (SNPs). However, reports vary widely describing Cas12 SNP sensitivity, and an underlying mechanism is lacking. We systematically varied crRNA length and valency to investigate the role of crRNA architectures on Cas12 biosensing in the context of speed-of-detection, sensitivity, and selectivity. Our results demonstrate that crRNAs complementary to 20 base pairs of the target DNA is optimal for rapid and sensitive detection, while a complementary length of 15 base pairs is ideal for robust SNP detection. Additionally, we uncovered a unique periodicity in SNP sensitivity based on nucleotide position and developed a structural model explaining what drives Cas12 SNP sensitivity. Lastly, we showed that bivalent CRISPR-Cas sensors have synergistic and enhanced activity that is distance dependent.},
}
@article {pmid40542569,
year = {2025},
author = {Han, R and Gao, X and Qi, Y and Lu, X and Wang, X and Tang, X},
title = {Spacer-Complementary Single-Stranded DNA Oligonucleotides Can Serve as Target-Specific Inhibitors in CRISPR/Cas9 Systems.},
journal = {Cell biochemistry and function},
volume = {43},
number = {6},
pages = {e70088},
doi = {10.1002/cbf.70088},
pmid = {40542569},
issn = {1099-0844},
support = {//This research was supported by Guangxi Natural Science Foundation Project (2025GXNSFAA069363, 2023GXNSFAA026260)./ ; },
abstract = {The continuous expression of the CRISPR/Cas system in organisms can lead to various potential issues. Some anti-CRISPR strategies have been developed to achieve precise control over CRISPR/Cas, yet these strategies are predominantly protein-based, with the most commonly used anti-CRISPR (Acr) proteins lacking sufficient target specificity. However, in this study, we designed a single-stranded DNA (ssDNA) inhibitor that was complementary to the spacer region on the guide RNA, operating at the nucleic acid level. We demonstrated that this method effectively inhibits the cleavage activity of Cas9-sgRNA ribonucleoprotein (RNP) in a target-specific manner through in vitro cleavage experiments. Furthermore, we explored the binding position and effective length of this inhibitory ssDNA, finding that its inhibitory effect was significantly reduced when the length of continuous complementarity with the 5' end of the spacer was less than 7nt. The truncated ssDNA also showed potential in reducing off-target effects. Moreover, we applied nucleic acid inhibitors to embryos via microinjection, and gene editing activity was significantly reduced, as evidenced by a decrease in the mosaicism rate of mouse embryos undergoing normal gene editing from (84.4 ± 4.4) % to 0%. Our study introduces a convenient and target-specific nucleic acid inhibitor capable of achieving precise regulation of gene editing.},
}
@article {pmid40540289,
year = {2025},
author = {Han, JH and Lee, SY and Park, HH},
title = {AcrVIA6 Is a Monomeric DNA-Binding Protein That Does Not Directly Inhibit Cas13a.},
journal = {FASEB journal : official publication of the Federation of American Societies for Experimental Biology},
volume = {39},
number = {12},
pages = {e70753},
pmid = {40540289},
issn = {1530-6860},
support = {RS-(2025-02316334)//National Research Foundation of Korea (NRF)/ ; },
abstract = {The CRISPR-Cas system is a crucial adaptive immune mechanism in prokaryotes, providing defense against invading genetic elements. Among various CRISPR-Cas systems, the type VI-A system, employing RNA-guided RNase Cas13a, has been extensively studied for its ability to target and degrade single-stranded RNA. Anti-CRISPR (Acr) proteins have evolved as natural inhibitors of these systems, with AcrVIA proteins specifically targeting the Cas13a enzyme. However, there is currently conflicting debate regarding the anti-CRISPR function of AcrVIA6. This study reveals that AcrVIA6 functions as a DNA-binding protein rather than a Cas13a inhibitor, as it does not block RNA cleavage. These findings challenge its role in CRISPR-Cas regulation.},
}
@article {pmid40540136,
year = {2025},
author = {Ponnurangan, V and Namachivayam, R and Pradeep, RKM and Jesudoss, D and Eswaran, K and Loganathan, A and Kumar, KK and Vaikuntavasan, P and Maduraimuthu, D and Shanmugam, V},
title = {Biotechnological breakthroughs in rice disease management: an overview from transgenics to CRISPR.},
journal = {Molecular biology reports},
volume = {52},
number = {1},
pages = {616},
pmid = {40540136},
issn = {1573-4978},
support = {BT/PR40456/ AGIII/103/1248/2020//Department of Biotechnology, New Delhi, India/ ; },
abstract = {Rice production faces persistent challenges due to a plethora of infectious agents, which cause substantial economic losses and pose significant threats to food security. Effective management of rice diseases is hindered by the lack of resistant sources and the slow pace of conventional breeding, which often fails to keep up with the rapid evolution of pathogens. Although transgenic approaches have contributed to disease resistance, they are limited by regulatory hurdles and public concern over the use of genetically modified organisms. Emerging genome-editing technologies, particularly CRISPR/Cas systems, offer promising alternatives by enabling the precise modification of native genes involved in plant susceptibility. CRISPR/Cas9-mediated inactivation of negative defense regulators, such as SWEET and ERF family genes, conferred broad-spectrum disease resistance. This review discusses the use of CRISPR/Cas technologies in developing disease-resistant rice varieties, along with emerging approaches and databases relevant to genome editing in plant research. Furthermore, it also highlights recent developments in CRISPR-based diagnostics for pathogen detection and the regulatory frameworks for the commercialization of edited crops. Overall, it underscores the transformative potential of CRISPR technologies in developing elite, climate-resilient rice cultivars, thereby supporting sustainable disease management and global food security.},
}
@article {pmid40539514,
year = {2025},
author = {Li, Y and Li, J and Pei, X and Wei, J and Gan, J and Lin, J},
title = {Catalytic-state structure of Candidatus Hydrogenedentes Cas12b revealed by cryo-EM studies.},
journal = {Nucleic acids research},
volume = {53},
number = {12},
pages = {},
doi = {10.1093/nar/gkaf519},
pmid = {40539514},
issn = {1362-4962},
support = {2017YFA0504602//National Key Research and Development Program of China/ ; 32130063//National Key Research and Development Program of China/ ; 31770784//National Natural Science Foundation of China/ ; 32371252//National Natural Science Foundation of China/ ; 2021-01-07-00-07-E00074//Shanghai Municipal Education Commission/ ; SN-ZJU-SIAS009//Zhejiang University/ ; 31770784//National Natural Science Foundation of China/ ; 32371252//National Natural Science Foundation of China/ ; },
abstract = {The CRISPR-Cas (clustered regularly interspaced short palindromic repeats and CRISPR-associated protein) systems are adaptive immune mechanisms that play critical roles in defending archaea and bacteria against invading entities. These systems can be divided into two classes, with class 2 comprising three types (II, V, and VI). Because of their ability to cleave double-stranded DNA, many class 2 CRISPR-Cas proteins have been harnessed as genome editing tools. Unlike the well-studied type II Cas9 proteins, the structural studies of the type V-B Cas12b proteins are limited, hindering their engineering and broader application. Here, we report four complex structures of ChCas12b, which reveal many unique structural features. The folding of the single guide RNA (sgRNA) of ChCas12b is distinct from that of AacCas12b and BthCas12b. Notably, many of these unique features are involved in ChCas12b-sgRNA interaction, suggesting that they are co-evolved. While ChCas12b shares a conserved two-cation-assisted catalytic mechanism with its homologs, it recognizes a longer guide:target heteroduplex, potentially offering higher fidelity in DNA editing. Altogether, our studies suggested that Cas12b family proteins exhibit significant diversity in their folding, sgRNA and target DNA binding. In the future, it is worth characterizing more representative proteins to identify CRISPR-Cas proteins with higher gene editing ability and fidelity.},
}
@article {pmid40539280,
year = {2025},
author = {Bak, RO and Holm, M and Møller, B and Mikkelsen, JG and Mogensen, TH},
title = {[CRISPR/Cas gene editing of haematopoietic stem cells for curing primary immunodeficiency].},
journal = {Ugeskrift for laeger},
volume = {187},
number = {22},
pages = {},
doi = {10.61409/V02250083},
pmid = {40539280},
issn = {1603-6824},
abstract = {Primary immunodeficiencies are rare monogenic inborn errors of immunity and can involve any combination of infection, autoimmunity, inflammation, and malignancy. While increased use of whole genome sequencing has vastly improved diagnosis, curative treatment options beyond haematopoietic stem cell transplantation are still lacking behind. In this review, we present and discuss the promising avenues of CRISPR/Cas gene editing of patient stem cells for curing these diseases through homology-directed repair, base- or prime editing and delivery by nanoparticles or viral derivatives. However, technological, regulatory, and economic challenges exist on the road to safe and broad implementation of this technology for personalized medicine in the clinic.},
}
@article {pmid40538711,
year = {2025},
author = {Lathakumari, RH and Vajravelu, LK and Gopinathan, A and Vimala, PB and Panneerselvam, V and Ravi, SSS and Thulukanam, J},
title = {The gut virome and human health: From diversity to personalized medicine.},
journal = {Engineering microbiology},
volume = {5},
number = {1},
pages = {100191},
pmid = {40538711},
issn = {2667-3703},
abstract = {The human gut virome plays a crucial role in the gut and overall health; its diversity and regulatory functions influence bacterial populations, metabolism, and immune responses. Bacteriophages (phages) and eukaryotic viruses within the gut microbiome contribute to these processes, and recent advancements in sequencing technologies and bioinformatics have greatly expanded our understanding of the gut virome. These advances have led to the development of phage-based therapeutics, diagnostics, and artificial intelligence-driven precision medicine. The emerging field of phageomics shows promise for delivering personalized phage therapies that combat antimicrobial resistance by specifically targeting pathogenic bacteria while preserving beneficial microbes. Moreover, CRISPR-Cas systems delivered via phages have shown success in selectively targeting antibiotic resistance genes and enhancing treatment effectiveness. Phage-based diagnostics are highly sensitive in detecting bacterial pathogens, offering significant benefits for human health and zoonotic disease surveillance. This synthesis of the current knowledge highlights the pivotal role of the gut virome in regulating microbial communities and its transformative potential in personalized medicine, emphasizing its importance in advancing therapeutic and diagnostic strategies for improving health outcomes.},
}
@article {pmid40536889,
year = {2025},
author = {Wall, LA and Wall, D},
title = {Acquired CRISPR spacers and rhamnose-glucose polysaccharide defects confer resistance to Streptococcus mutans phage ɸAPCM01.},
journal = {Microbiology (Reading, England)},
volume = {171},
number = {6},
pages = {},
pmid = {40536889},
issn = {1465-2080},
mesh = {*Streptococcus mutans/virology/genetics/metabolism ; *Rhamnose/metabolism/genetics ; Biofilms/growth &amp; development ; *Streptococcus Phages/genetics/physiology ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; *Glucose/metabolism ; Mutation ; *Polysaccharides/metabolism ; Bacterial Proteins/genetics/metabolism ; *Polysaccharides, Bacterial/genetics ; },
abstract = {Streptococcus mutans is commonly associated with the development of dental caries worldwide. Due to their specificity for S. mutans, phage represents a promising avenue for future targeted therapeutic strategies. In this study, we investigated how phage resistance develops in S. mutans. As a model phage, we used ɸAPCM01, which is known to infect a serotype e strain. We isolated and sequenced the genomes of 15 spontaneous resistant mutants and found that 10 had acquired novel clustered regularly interspaced short palindromic repeats (CRIPSR) spacers targeting the phage, with a total of 18 new spacers identified. Additionally, eight strains contained mutations in rhamnose-glucose polysaccharide biosynthetic genes, three of which also acquired spacers. Only the rgp mutants exhibited defects in phage adsorption, supporting the role of these cell surface glycans as the phage receptor. Mutations in rgpF and the newly identified gene rgpX led to severe cell division defects and impaired biofilm formation, the latter of which was also shared by an rgpD mutant. Thus, rgp mutations confer phage resistance but impose severe fitness costs, limiting pathogenic potential. Surprisingly, we found that ɸAPCM01 was capable of binding to and injecting its genome into UA159, a model serotype c strain. However, UA159 was resistant to infection due to an unknown post-entry defence mechanism. Consequently, ɸAPCM01 has the potential to infect both major serotypes associated with dental caries.},
}
@article {pmid40536816,
year = {2025},
author = {Liao, X and Liu, Q and Chuai, G},
title = {PrimeNet: rational design of Prime editing pegRNAs by deep learning.},
journal = {Briefings in bioinformatics},
volume = {26},
number = {3},
pages = {},
doi = {10.1093/bib/bbaf293},
pmid = {40536816},
issn = {1477-4054},
support = {T24250193//National Natural Science Foundation of China/ ; 32341008//National Natural Science Foundation of China/ ; 62002265//National Natural Science Foundation of China/ ; 2021YFF1201200//National Key Research and Development Program of China/ ; 2021YFF1200900//National Key Research and Development Program of China/ ; //Shanghai Pilot Program for Basic Research/ ; //Shanghai Science and Technology Innovation Action Plan-Key Specialization in Computational Biology/ ; //Shanghai Shuguang Scholars Project/ ; //Shanghai Excellent Academic Leader Project/ ; 2021SHZDZX0100//Shanghai Municipal Science and Technology Major Project/ ; //Fundamental Research Funds for the Central Universities/ ; 2025080107//Tongji University "Medicine + X" Cross Research Program/ ; },
mesh = {Humans ; *Gene Editing/methods ; *Deep Learning ; HEK293 Cells ; K562 Cells ; CRISPR-Cas Systems ; DNA Methylation ; Epigenesis, Genetic ; },
abstract = {The rapid development of gene editing technology has revolutionized life science research and biotechnology applications. Prime editing, a precise gene editing tool, has shown promise in various applications, including disease research and therapeutic interventions. However, its suboptimal editing efficiency for extensive fragments and lack of predictive models have hindered its widespread adoption. Existing models exhibit low prediction accuracy and limitations, such as neglecting epigenetic factors that impact gene editing effects. To address these challenges, we developed PrimeNet, a novel prediction model that integrates significant epigenetic factors, including chromatin accessibility and DNA methylation. By incorporating data from multiple cell lines and introducing multiscale convolution and attention mechanisms, PrimeNet enhances the accuracy of predictions and generalization performance. Our results show that PrimeNet achieves a Spearman correlation coefficient of 0.94 and 0.82 on two datasets originated from HEK293T and K562 cell lines, respectively, outperforming existing models. This novel model has the potential to guide experimental design, enhance the success rate of gene editing, and reduce unnecessary experimental costs, thereby advancing the application of gene editing technology in genetic disease treatment and related fields.},
}
@article {pmid40536049,
year = {2025},
author = {Ullah, M and Naeem, M and Andoh, V and Khan, MN and Han, J and Rizwan, M and Hussain, N and Saeed, M and Chen, Y and Chen, H},
title = {Lactic Acid Bacteria as Biofactories: Mechanistic Insights, Engineering Strategies, and Future Horizons for Heterologous Enzyme Expression.},
journal = {Journal of agricultural and food chemistry},
volume = {},
number = {},
pages = {},
doi = {10.1021/acs.jafc.5c02311},
pmid = {40536049},
issn = {1520-5118},
abstract = {Lactic acid bacteria (LAB) constitute a genetically heterogeneous group that is uniquely capable of converting soluble carbohydrates into lactic acid. Such LAB, with a long history of safe consumption in fermented foods, are considered food-grade microorganisms and are highly sought after for a variety of biotechnological applications. Due to their unique properties, LAB can be genetically engineered to produce industrially significant enzymes. LAB act as an expression host for these enzymes by combining already existing engineering systems with techniques such as CRISPR-Cas. This review outlines the progress achieved to date on genome manipulation methods for LAB engineering and future perspectives of genetic tools. These strategies contribute greatly to fully unleashing the potential of LAB, and we further elaborate on how genome editing tools can enhance the capacity of heterologous expression in LAB.},
}
@article {pmid40533429,
year = {2025},
author = {Avaro, AS and Griffiths, AD and Santiago, JG},
title = {Degradation of Reporter Molecules Imposes a Fundamental Limit of Detection on CRISPR Diagnostics.},
journal = {Analytical chemistry},
volume = {},
number = {},
pages = {},
doi = {10.1021/acs.analchem.5c01060},
pmid = {40533429},
issn = {1520-6882},
abstract = {The sensitivity of CRISPR-Cas systems used for molecular diagnostics remains a major bottleneck in the adoption of this technology. The vast majority of CRISPR-based assays use dually labeled, single-stranded reporters and fluorescence signal readouts to infer enzymatic activity and the presence (or absence) of target nucleic acid. The limit of detection of such assays is set by the kinetics of the Cas enzymes and a slow yet measurable increase in the fluorescence signal. We demonstrate here that the background signal and limits of detection of most assays are very likely limited by the degradation of reporter molecules. This degradation is dynamic and is not associated with enzymatic activity. We present theory and experiments to design and calibrate CRISPR assays. We introduce a new kinetic framework to account for the degradation of reporter molecules and derive a fundamental limit of detection for CRISPR-based assays. Our data show that Michaelis-Menten kinetics alone are insufficient to describe reporter (substrate) cleavage rates. The framework and techniques presented here should help reduce the frequency and magnitude of errors currently routinely made in quantifying CRISPR kinetics and interpreting CRISPR diagnostic fluorescence signals.},
}
@article {pmid40531937,
year = {2025},
author = {Poudel, B and Sathe, A and Bede, JC and Kushalappa, AC},
title = {Editing metacaspase (StMC7) gene enhances late blight resistance in Russet Burbank potato.},
journal = {PloS one},
volume = {20},
number = {6},
pages = {e0325702},
pmid = {40531937},
issn = {1932-6203},
mesh = {*Solanum tuberosum/genetics/microbiology ; *Disease Resistance/genetics ; *Plant Diseases/microbiology/genetics ; Phytophthora infestans/pathogenicity ; CRISPR-Cas Systems ; *Gene Editing ; *Plant Proteins/genetics ; *Caspases/genetics ; Gene Expression Regulation, Plant ; Gene Silencing ; Alternaria/pathogenicity ; Plants, Genetically Modified ; },
abstract = {Plants induce hypersensitive response programmed cell death (HR-PCD), upon biotrophic pathogen infection, to contain the pathogen to the point of infection. Apoptotic-like PCD (AL-PCD) has been reported upon prolonged hemibiotrophic and necrotrophic pathogen infection in potato, to feed on the dead cells for their growth. In potato, silencing of the gene StHRC lead to the suppression of AL-PCD, thus increasing resistance to blights in potato. This was also associated with a significant reduction in the expression of the metacaspase gene StMC7. Accordingly, the gene StMC7 was silenced in potato cultivar 'Russet Burbank' using CRISPR-Cas9 to improve disease resistance against late blight of potato caused by Phytophthora infestans. Following pathogen infection, the disease severity, pathogen biomass and StMC7 gene expression was lower in Stmc7 mutants as compared to wild type. Disease severity was also decreased in Alternaria solani inoculated Stmc7 mutants, compared to the wild type, suggesting possible multiple disease resistance in the Stmc7 knockdown mutants. This confirms that the silencing of StMC7 improves late blight disease resistance in potato.},
}
@article {pmid40531391,
year = {2025},
author = {Wang, Y and Li, H and Luo, S and Zhong, M and Liu, J and Li, B},
title = {Research Progress on Signal Conversion Based on Aptamer Combined CRISPR/Cas System in Biosensors.},
journal = {Molecular diagnosis &amp; therapy},
volume = {},
number = {},
pages = {},
pmid = {40531391},
issn = {1179-2000},
support = {2025YFHZ0329//Sichuan Province Science and Technology Support Program/ ; 2024JYJ014//Sichuan Province Science and Technology Support Program/ ; 24CGZH10//Sichuan Province Science and Technology Support Program/ ; },
abstract = {The CRISPR/Cas system has been extensively used in the fields of biology, food safety, and environmental monitoring. This is in part because its can to be used in combination with isothermal amplification-mediated signal amplification technology along with its extraordinary trans-cleavage ability, which has initiated a new era of biosensing applications. The popularity of functional nucleic acids has enabled aptamers to convert non-nucleic acid substances into programmable nucleic acid sequences through methods such as direct detection, lock activation, sandwich design, induction of conformations, and split aptamers. Additionally, CRISPR/Cas systems have been extended beyond nucleic acid detection to include ions, small molecules, proteins, cells, bacteria, viruses, and other non-nucleic acid-based target substances. This article provides a brief overview of the mechanisms of action of four Cas proteins, the generation of aptamers, and their combined applications. Moreover, we focus on the research progress of biosensors based on aptamer-based signal conversion combined with the CRISPR/Cas system.},
}
@article {pmid40531235,
year = {2025},
author = {Wang, Y and Yang, H and Wang, K and Zhu, W and Wang, X},
title = {Establishment of a method for the rapid detection of carbapenem-resistant Pseudomonas aeruginosa based on bla_NDM with one-tube RPA-CRISPR/Cas12a visualization.},
journal = {Archives of microbiology},
volume = {207},
number = {8},
pages = {179},
pmid = {40531235},
issn = {1432-072X},
support = {No. LYG06521202376//the Lianyungang City Sixth 521 High-level Talent Training Project Scientific Research Project/ ; No. Z2022070//the 2022 Medical Research guiding Project of Jiangsu Provincial Health Commission/ ; No. SJCX23_1838//the 2023 Jiangsu Graduate Research Innovation Plan project/ ; },
mesh = {*Pseudomonas aeruginosa/genetics/drug effects/isolation &amp; purification/enzymology ; *Carbapenems/pharmacology ; *Pseudomonas Infections/microbiology/diagnosis ; Humans ; *Nucleic Acid Amplification Techniques/methods ; CRISPR-Cas Systems ; Anti-Bacterial Agents/pharmacology ; *beta-Lactamases/genetics ; Bacterial Proteins/genetics ; China ; Drug Resistance, Bacterial/genetics ; Microbial Sensitivity Tests ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {Pseudomonas aeruginosa (PA), which is a common Gram-negative bacterium, can become carbapenem-resistant Pseudomonas aeruginosa (CRPA) upon the development of antibiotic resistance, making clinical treatment difficult. CRPA with antibiotic resistance genes (ARGs), such as bla_NDM and bla_KPC, is common in China. The development of tests for ARGs would facilitate the more rapid identification of CRPA in China. Isothermal amplification research has improved, but limitations remain, including a lack of specialized equipment, the difficulty of developing sophisticated primers, and aerosol pollution. Thus, clinical examination methods must improve. We successfully integrated RPA with CRISPR/Cas12a, and we identified bla_NDM as our institution's primary CRPA. RPA-CRISPR/Cas12a could accurately detect target DNA within 40 min without cross-reacting with other bacteria. The results showed high concordance with conventional culture-based methods, including 100% agreement in clinical sample validation. The method reliably identified standard PA strains and 29 clinical isolates, detecting PA at concentrations as low as 10[-1] CFU. In addition, the closed-tube format effectively minimized the risk of aerosol contamination. This platform offers a rapid and user-friendly tool for identifying bla_NDM-positive CRPA, and this tool is particularly suitable for early screening and clinical management in resource-limited settings. The simplicity and accuracy of this approach make it a promising option for infection control and public health surveillance.},
}
@article {pmid40530883,
year = {2025},
author = {Ganote, CL and Caesar, L and Rice, DW and Whitaker, RJ and Newton, ILG},
title = {Evolutionary trends in Bombella apis CRISPR-Cas systems.},
journal = {mSystems},
volume = {},
number = {},
pages = {e0016625},
doi = {10.1128/msystems.00166-25},
pmid = {40530883},
issn = {2379-5077},
abstract = {UNLABELLED: Bacteria and archaea employ a rudimentary immune system, CRISPR-Cas, to protect against foreign genetic elements such as bacteriophage. CRISPR-Cas systems are found in Bombella apis. B. apis is an important honey bee symbiont, found primarily in larvae, queens, and hive compartments. B. apis is found in the worker bee gut but is not considered a core member of the bee microbiome and has therefore been understudied with regard to its importance in the honey bee colony. However, B. apis appears to play beneficial roles in the colony, by protecting developing brood from fungal pathogens and by bolstering their development under nutritional stress. Previously, we identified CRISPR-Cas systems as being acquired by B. apis in its transition to bee association, as they are absent in a sister clade. Here, we assess the variation and distribution of CRISPR-Cas types across B. apis strains. We found multiple CRISPR-Cas types, some of which have multiple arrays, within the same B. apis genomes and also in the honey bee queen gut metagenomes. We analyzed the spacers between strains to identify the history of mobile element interaction for each B. apis strain. Finally, we predict interactions between viral sequences and CRISPR systems from different honey bee microbiome members. Our analyses show that the B. apis CRISPR-Cas systems are dynamic; that microbes in the same niche have unique spacers, which supports the functionality of these CRISPR-Cas systems; and that acquisition of new spacers may be occurring in multiple locations in the genome, allowing for a flexible antiviral arsenal for the microbe.<br><br>IMPORTANCE: Honey bee worker gut microbes have been implicated in everything from protection from pathogens to breakdown of complex polysaccharides in the diet. However, there are multiple niches within a honey bee colony that host different groups of microbes, including the acetic acid bacterium Bombella apis. B. apis is found in the colony food stores, in association with brood, in worker hypopharyngeal glands, and in the queen's digestive tract. The roles that B. apis may serve in these environments are just beginning to be discovered and include the production of a potent antifungal that protects developing bees and supplementation of dietary lysine to young larvae, bolstering their nutrition. Niche specificity in B. apis may be affected by the pressures of bacteriophage and other mobile elements, which may target different strains in each specific bee environment. Studying the interplay between B. apis and its mobile genetic elements (MGEs) may help us better understand microbial community dynamics within the colony and the potential ramifications for the honey bee host.},
}
@article {pmid40530826,
year = {2025},
author = {Carolak, E and Czajkowska, J and Stypułkowska, A and Waszczuk, W and Dutkiewicz, A and Grzymajlo, K},
title = {Being a better version of yourself: genetically engineered probiotic bacteria as host defense enhancers in the control of intestinal pathogens.},
journal = {Gut microbes},
volume = {17},
number = {1},
pages = {2519696},
doi = {10.1080/19490976.2025.2519696},
pmid = {40530826},
issn = {1949-0984},
mesh = {*Probiotics ; Humans ; *Gastrointestinal Microbiome ; Genetic Engineering ; Animals ; *Microorganisms, Genetically-Modified/genetics ; *Bacteria/genetics ; },
abstract = {Intestinal pathogens pose a significant global health burden, and traditional antibiotic treatments often disrupt the beneficial gut microbiota that plays a crucial role in maintaining host health through pathogen prevention and immune regulation. Although probiotics have emerged as promising therapeutic agents, their efficacy is limited by strain-dependent variations, survival challenges in the gastrointestinal tract, and inconsistent immune responses. Recent advances in genetic engineering, particularly CRISPR-Cas systems and their combinations with complementary technologies, such as Cre-lox and RecE/T, have enabled the precise modification of probiotic strains to enhance their therapeutic potential. These enhanced probiotics demonstrate improved functionality through multiple mechanisms, including increased adhesion via the expression of specific proteins (InlA, FnBPA, and LAP), targeted antimicrobial activity through engineered sensing systems (Lactococcus lactis detecting Vibrio cholerae CAI-1), and enhanced immunomodulation through cytokine production. Results have demonstrated the potential of genetically modified probiotics in preventing and treating gastrointestinal infections through mechanisms that include competitive exclusion, bacteriocin production, intestinal barrier reinforcement, and immune modulation. However, challenges remain in ensuring genetic stability and preventing horizontal gene transfer. Future research should focus on optimizing probiotic strains for targeted applications while addressing biosafety concerns. By understanding the complex interplay between probiotics, pathogens, and host immunity, innovative strategies can be developed to harness the full therapeutic potential of probiotic interventions in maintaining gut health.},
}
@article {pmid40530689,
year = {2025},
author = {Budzak, J and Siegel, TN},
title = {Next generation genetic screens in kinetoplastids.},
journal = {Nucleic acids research},
volume = {53},
number = {11},
pages = {},
doi = {10.1093/nar/gkaf515},
pmid = {40530689},
issn = {1362-4962},
support = {ALTF 764-2022//EMBO Postdoctoral Fellowship/ ; 101105761 - VSG-SWITCH//Marie Sk&#x142;odowska-Curie Actions Postdoctoral Fellowship/ ; SI 1610/2-2//German Research Foundation/ ; SI 1610/4-1//German Research Foundation/ ; 101044320//ERC-CoG/ ; },
mesh = {*Kinetoplastida/genetics ; *Genetic Testing/methods ; Single-Cell Analysis/methods ; Genome, Protozoan ; CRISPR-Cas Systems ; Genomics/methods ; High-Throughput Nucleotide Sequencing ; },
abstract = {The genomes of all organisms encode diverse functional elements, including thousands of genes and essential noncoding regions for gene regulation and genome organization. Systematic perturbation of these elements is crucial to understanding their roles and how their disruption impacts cellular function. Genetic perturbation approaches, which disrupt gene expression or function, provide valuable insights by linking genetic changes to observable phenotypes. However, perturbing individual genomic elements one at a time is impractical. Genetic screens overcome this limitation by enabling the simultaneous perturbation of numerous genomic elements within a single experiment. Traditionally, these screens relied on simple, high-throughput readouts such as cell fitness, differentiation, or one-dimensional fluorescence. However, recent advancements have introduced powerful technologies that combine genetic screens with image-based and single-cell sequencing readouts, allowing researchers to study how perturbations affect complex cellular phenotypes on a genome-wide scale. These innovations, alongside the development of CRISPR-Cas technologies, have significantly enhanced the precision, efficiency, and scalability of genetic screening approaches. In this review, we discuss the genetic screens performed in kinetoplastid parasites to date, emphasizing their application to both coding and noncoding regions of the genome. Furthermore, we explore how integrating image-based and single-cell sequencing technologies with genetic screens holds the potential to deliver unprecedented insights into cellular function and regulatory mechanisms.},
}
@article {pmid40529587,
year = {2025},
author = {Lee, CK and Lee, HJ and Jeong, SH and Lee, SJ},
title = {Precision targeting of genetic variations in mixed bacterial cultures using CRISPR-Cas12a-programmed λ phages.},
journal = {Frontiers in microbiology},
volume = {16},
number = {},
pages = {1575339},
pmid = {40529587},
issn = {1664-302X},
abstract = {The CRISPR-Cas system, an adaptive immune mechanism in prokaryotes against bacteriophages, has been developed into a versatile tool for recognizing and cleaving target nucleic acid sequences. In this study, we developed a model system by integrating CRISPR-Cas12a into the genome of temperate bacteriophage λ, enabling precise regulation of lysogeny and lysis in Escherichia coli. We confirmed that λ phage, armed with Cas12a nuclease and CRISPR RNA (crRNA) targeting specific sequences, could inhibit the lysogenic cycle of E. coli cells. We demonstrated that the CRISPR-Cas12a-loaded temperate λ phage mimicked a lytic phage by selectively killing cells carrying the target genomic sequence. Furthermore, by employing truncated crRNA to enhance target recognition specificity, we found that the synthetic phage could distinguish single nucleotide variations in the genomic target DNA, enabling precise targeting and selective elimination of target cells in homogeneous bacterial cultures. To further validate its specificity, we tested this system in mixed bacterial cultures, wherein Cas12a nuclease and truncated crRNA-loaded bacteriophages selectively eliminated only those cells carrying the target sequences perfectly matching the crRNA. These results highlight the potential of this approach for advancing precision microbiome modulation.},
}
@article {pmid40528015,
year = {2025},
author = {Ottaviani, L and Lefeuvre, R and Montes, E and Widiez, T and Giorni, P and Mithöfer, A and Marocco, A and Lanubile, A},
title = {A loss-of-function of ZmWRKY125 induced by CRISPR/Cas9 improves resistance against Fusarium verticillioides in maize kernels.},
journal = {Plant cell reports},
volume = {44},
number = {7},
pages = {144},
pmid = {40528015},
issn = {1432-203X},
mesh = {*Zea mays/microbiology/genetics/immunology/metabolism ; *Fusarium/physiology/pathogenicity ; *Plant Diseases/microbiology/genetics/immunology ; *Disease Resistance/genetics ; *Plant Proteins/genetics/metabolism ; *CRISPR-Cas Systems/genetics ; Gene Expression Regulation, Plant ; Cyclopentanes/metabolism ; Oxylipins/metabolism ; Abscisic Acid/metabolism ; Plant Growth Regulators/metabolism ; Loss of Function Mutation ; Transcription Factors/genetics/metabolism ; },
abstract = {ZmWRKY125 negatively regulates maize resistance to Fusarium verticillioides infection through modulating phytohormone, ROS scavenging and secondary metabolite gene expression as well as jasmonic and abscisic acid biosynthetic pathway activity. Fusarium verticillioides causes heavy damage to maize growth and yield and is responsible for mycotoxin contamination. Despite its widespread occurrence, few resistant genes have been identified and functionally validated for their role in the defense mechanisms against this fungus in maize. WRKY transcription factors are known to be crucial in regulating the expression of defense-responsive genes towards pathogen attack. In this context, in our previous genome-wide association study one SNP in the gene ZmWRKY125 was found significantly associated with the responses to F. verticillioides infection in maize seedlings. Here, loss-of-function mutant lines of ZmWRKY125 were obtained by the clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (Cas9) system. The zmwrky125 edited lines were phenotypically evaluated showing a decrease by about 5 and 4 times of Fusarium ear rot (FER) severity and fumonisin contamination, respectively, compared to the wild-type genotype. The transient expression of ZmWRKY125 in maize protoplasts confirmed a nuclear localization as expected for a transcription factor. RNA-seq analysis comparison using two zmwrky125 edited lines and the wild-type genotype highlighted an enhanced modulation of the jasmonic acid (JA) and abscisic acid (ABA) hormones, redox state, cell wall modification, and secondary metabolism-associated genes after fungal infection. Moreover, the increased expression of JA- and ABA-related genes correlated with a wider accumulation of these two phytohormones in the mutant background in contrast to wild-type. This data provided new information for understanding the function of ZmWRKY125, despite further field evaluations will be required for validation of the resistance against FER.},
}
@article {pmid40527733,
year = {2025},
author = {Obuchi, W and Zargani-Piccardi, A and Leandro, K and Rufino-Ramos, D and Di Lanni, E and Frederick, DM and Maalouf, K and Nieland, L and Xiao, T and Repiton, P and Vaine, CA and Kleinstiver, BP and Bragg, DC and Lee, H and Miller, MA and Breakefield, XO and Breyne, K},
title = {Engineering of CD63 Enables Selective Extracellular Vesicle Cargo Loading and Enhanced Payload Delivery.},
journal = {Journal of extracellular vesicles},
volume = {14},
number = {6},
pages = {e70094},
pmid = {40527733},
issn = {2001-3078},
support = {//Ionis Pharmaceuticals/ ; NCI-CA232103/BC/NCI NIH HHS/United States ; //Friedreich's Ataxia Research Alliance Australia/ ; //Daiichi Sankyo Company/ ; NIH DP2-CA259675//National Science Foundation/ ; NIH DP2-CA281401//National Science Foundation/ ; NIH K22-CA2802019-01//National Science Foundation/ ; NIH R01-GM138790//National Science Foundation/ ; //Kayden-Lambert MGH Research Scholar Award/ ; },
mesh = {*Extracellular Vesicles/metabolism ; *Tetraspanin 30/metabolism/genetics ; Animals ; Humans ; Mice ; HEK293 Cells ; Protein Engineering/methods ; },
abstract = {Extracellular vesicles (EVs) are mediators of intercellular communication through the transfer of nucleic acids, lipids and proteins between cells. This property makes bioengineered EVs promising therapeutic vectors. However, it remains challenging to isolate EVs with a therapeutic payload due to the heterogeneous nature of cargo loading into EVs. In this study, enrichment of EVs with a desired cargo was possible through engineering of the hallmark CD63 transmembrane protein. E-NoMi refers to engineered CD63 with mCherry on the inside of the EV membrane and a tag (3xFLAG) exposed on the outside of the EV membrane. To facilitate EV loading during biogenesis, cargo proteins, such as EGFP, Cre recombinase and the CRISPR-Cas nuclease (SaCas9), were fused to a nanobody (Nb) protein with a high affinity for mCherry. FLAG-tag-based immunocapture from cell conditioned media allowed selection of cargo-loaded E-NoMi-EVs, and tobacco etch virus (TEV) protease cleavage sites were used to remove the 3xFLAG-tag from the surface of E-NoMi-EVs after capture. For functional payload delivery to recipient cells, the vesicular stomatitis virus G (VSV-G) fusogenic protein was incorporated into E-NoMi-EVs to form fusogenic EV-based vectors (EVVs) and proved to be 10-fold more effective at cargo delivery than EVs generated by size-exclusion chromatography. Functional delivery of cargo with E-NoMi-EVVs was validated in two mouse brain models in vivo.},
}
@article {pmid40527417,
year = {2025},
author = {Yaikhan, T and Singkhamanan, K and Suwannasin, S and Dechathai, T and Yingkajorn, M and Chusri, S and Surachat, K},
title = {Genomic insights into Metapseudomonas otitidis PA-NS83: The first clinical isolate from Thailand and its comparative genomic analysis.},
journal = {Infection, genetics and evolution : journal of molecular epidemiology and evolutionary genetics in infectious diseases},
volume = {},
number = {},
pages = {105786},
doi = {10.1016/j.meegid.2025.105786},
pmid = {40527417},
issn = {1567-7257},
abstract = {Metapseudomonas otitidis was first isolated from human middle ear fluid and has since been detected in both environmental and clinical samples, emerging as an opportunistic pathogen linked to chronic otitis media and other infections. This study reports the first clinical isolate of M. otitidis from Thailand, PA-NS83, and presents a comprehensive genomic characterization. Whole-genome sequencing and comparative analysis with 37 publicly available M. otitidis genomes revealed a diverse antimicrobial resistance (AMR) profile, with PA-NS83 carrying AMR genes commonly found in environmental isolates. Virulence gene analysis identified key determinants associated with biofilm formation, motility, secretion systems, and iron acquisition, highlighting its potential pathogenicity. Pan-genome analysis demonstrated substantial genomic diversity, with PA-NS83 clustering closely with M. otitidis CSMC7, an environmental isolate from polystyrene waste. However, PA-NS83 harbored 419 unique genes, including virulence-associated genes and a CRISPR-Cas system, suggesting adaptation to clinical settings. These findings underscore the genetic plasticity of M. otitidis and its potential role in human infections. Continued genomic surveillance and functional studies are essential to further assess its clinical significance and antimicrobial resistance mechanisms.},
}
@article {pmid40527291,
year = {2025},
author = {Wang, J and Zhang, W and Li, W and Xie, Q and Zang, Z and Liu, C},
title = {Enhancement of CRISPR-Cas12a system through universal circular RNA design.},
journal = {Cell reports methods},
volume = {5},
number = {6},
pages = {101076},
doi = {10.1016/j.crmeth.2025.101076},
pmid = {40527291},
issn = {2667-2375},
mesh = {*RNA, Circular/genetics/metabolism ; *CRISPR-Cas Systems/genetics ; Humans ; Gene Editing/methods ; *Endodeoxyribonucleases/metabolism/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; DNA Repair ; Bacterial Proteins ; CRISPR-Associated Proteins ; },
abstract = {Precise control of Cas12a activity is crucial to address incompatibility in isothermal amplification-CRISPR-Cas12a one-pot nucleic acid detection. We developed a light-triggerable circular RNA system for dynamic LbCas12a regulation. By employing circular CRISPR guide RNA (crRNA) or a split circular universal direct repeat region with a replaceable spacer, we resolved the incompatibility between isothermal amplification and CRISPR detection. This system demonstrated robust performance in detecting trace nucleic acids in clinical samples. Furthermore, DNA modifications on circular crRNA enabled CRISPR-Cas12a regulation via base excision repair (BER) enzymes, offering potential for BER enzyme detection and modulation of LbCas12a cleavage activity by BER enzymes. This programmable strategy holds promise for selective gene editing in cells with elevated BER enzyme expression, such as uracil DNA glycosylase (UDG) in colon cancer cells. The circular RNA-assisted approach represents a resource-efficient method with significant potential for medical diagnostics and future clinical gene therapy applications.},
}
@article {pmid40526416,
year = {2025},
author = {Ortega-Sanz, I and Rubio, A and Pérez-Pulido, AJ},
title = {Bacteriophages in Pseudomonas aeruginosa evade the CRISPR-Cas I-F system by depletion of PAM sequences.},
journal = {Microbial genomics},
volume = {11},
number = {6},
pages = {},
pmid = {40526416},
issn = {2057-5858},
mesh = {*Pseudomonas aeruginosa/virology/genetics ; *CRISPR-Cas Systems/genetics ; *Bacteriophages/genetics ; Genome, Viral ; Base Composition ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Humans ; },
abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) proteins systems provide bacteria with an adaptive immune system against exogenous sequences, such as plasmids and bacteriophages (viruses of prokaryotes). To avoid autoimmunity, the recognition of a very short sequence called the protospacer adjacent motif (PAM) is essential for efficient immune response triggering. This raises the question of whether viruses targeted by certain CRISPR-Cas systems have a higher or lower frequency of their PAM sequences than non-targeted viruses. This was tested here in the opportunistic human pathogen Pseudomonas aeruginosa that presents five different types of CRISPR-Cas systems. The most frequent of them is the subtype I-F (present in 36% of the strains), which has the PAM 5'-CC sequence. When the viral genomes targeted by this system were analysed, their PAM frequency was found to be lower than that of non-targeted viruses. Remarkably, targeted viruses have comparatively lower G+C content. All this could be the result of selection pressure on the genome of these viruses to escape from the CRISPR-Cas I-F system.},
}
@article {pmid40526281,
year = {2025},
author = {Bulle, M and Rahman, MM and Islam, MR and Abbagani, S},
title = {Strategies to develop climate-resilient chili peppers: transcription factor optimization through genome editing.},
journal = {Planta},
volume = {262},
number = {2},
pages = {30},
pmid = {40526281},
issn = {1432-2048},
mesh = {*Capsicum/genetics/physiology ; *Gene Editing/methods ; *Transcription Factors/genetics/metabolism ; Stress, Physiological ; Plant Proteins/genetics/metabolism ; CRISPR-Cas Systems ; Climate Change ; Genome, Plant ; },
abstract = {Chili peppers (Capsicum spp.), a globally significant crop revered for their nutritional, economic, and cultural importance, are increasingly imperiled by the converging burdens of climate-induced abiotic stresses, including drought, heat, and salinity, and relentless biotic assaults from pathogens and insect herbivores. These overlapping stressors not only destabilize yield but also compromise the metabolic intricacy responsible for the accumulation of health-promoting secondary metabolites. Although Capsicum exhibits remarkable genetic and phytochemical diversity, the integrated transcriptional, metabolic, and epigenetic frameworks that underpin its stress resilience remain poorly delineated. This review synthesizes recent advances in decoding core transcription factor families, such as CaNAC, CaWRKY, and CaMYB, that serve as pivotal regulators of osmotic adjustment, reactive oxygen species detoxification, hormonal crosstalk, and secondary metabolite biosynthesis under stress conditions. We further highlight how multi-omics-guided gene discovery, when paired with CRISPR/Cas-mediated genome editing, enables precise reprogramming of key regulatory loci to enhance adaptive responses. Emerging innovations, including base editing, prime editing, and novel nucleases like Cas12a and Cas13d, are expanding the functional genome-editing landscape, while the integration of morphogenic regulators and genotype-independent transformation platforms is beginning to circumvent long-standing obstacles in Capsicum genetic engineering. Lastly, we propose a transformative framework that converges transcription factor modulation, multi-omics strategies, precision phenotyping, and next-generation genome editing to accelerate the development of climate-resilient Capsicum cultivars with optimized metabolic traits. This strategic convergence of molecular insight and biotechnological innovation offers a robust foundation for building next-generation chili pepper varieties capable of withstanding intensifying environmental and pathogenic pressures, ultimately safeguarding yield, nutritional quality, and agricultural sustainability in the face of global climate change.},
}
@article {pmid40525624,
year = {2025},
author = {Zhang, Y and Lin, Y and Li, R and Jiang, D and Cai, R and Su, G and Yu, Y and Qu, G},
title = {Split G-Quadruplex-Integrated CRISPR-Cas Biosensor for One-Pot, Signal-On Visual Detection of Pathogen Nucleic Acids.},
journal = {Analytical chemistry},
volume = {},
number = {},
pages = {},
doi = {10.1021/acs.analchem.5c02037},
pmid = {40525624},
issn = {1520-6882},
abstract = {Pathogen nucleic acid analysis has emerged as an indispensable component of contemporary healthcare systems, serving dual roles in personalized clinical management and population-level disease surveillance. Herein, we present a novel G-quadruplex-integrated CRISPR-Cas biosensing platform that performs in a signal-on mechanism for colorimetric detection of pathogen nucleic acids in one-pot. By harnessing the unique properties of split G4 structures, we develop a universal visual probe that generates a distinct green colorimetric signal upon target recognition, which effectively couples with both Cas12 and Cas13 systems. Using the monkeypox virus (MPXV) B7R gene and a conserved respiratory syncytial virus (RSV) sequence as model targets for Cas12a and Cas13, respectively, we establish a detection workflow combining recombinase polymerase amplification (RPA) with CRISPR-mediated cleavage, visualized through enzymatic mediated color conversion. The tube-in-tube cartridge architecture adopted in this work enables seamless integration of RPA and CRISPR-based detection within a single closed-tube system, effectively eliminating cross-contamination risks. We successfully validate the platform for detection of MPXV in environmental samples and RSV in clinical specimens, achieving a detection limit of 1 copy per test and perfect concordance with PCR methods (40/40 agreement). The colorimetric biosensing platform developed herein demonstrates rapid (<60 min) and facial performance, establishing a novel molecular diagnostic paradigm that achieves laboratory-comparable accuracy for real-time surveillance and point-of-care applications.},
}
@article {pmid40524239,
year = {2025},
author = {Villora, SA and Zhao, Y and Silva, PC and Hahn, AA and Olanin, V and Groll, D and Maurer, S and Roetzer, V and Szymanski, W and Procida-Kowalski, T and Philipp, N and Koch, A and Bartkuhn, M and Graumann, J and Volckmann, R and Koster, J and Rossbach, O and Salzig, D and Dammann, R and Sigges, C and Halbritter, J and Haerteis, S and Richter, AM},
title = {Epigenetic silencing and CRISPR-mediated reactivation of tight junction protein claudin10b (CLDN10B) in renal cancer.},
journal = {Clinical epigenetics},
volume = {17},
number = {1},
pages = {102},
pmid = {40524239},
issn = {1868-7083},
support = {2023_1_01//FCMH Forschungscampus Mittelhessen/ ; 2023_1_01//FCMH Forschungscampus Mittelhessen/ ; 2023_1_01//FCMH Forschungscampus Mittelhessen/ ; 2024_1_02//FCMH Forschungscampus Mittelhessen,/ ; 2024_1_02//FCMH Forschungscampus Mittelhessen,/ ; 2024_1_02//FCMH Forschungscampus Mittelhessen,/ ; 031B1232B//BMBF/ ; 031B1232B//BMBF/ ; 031B1232B//BMBF/ ; },
mesh = {Humans ; *Claudins/genetics ; DNA Methylation ; *Kidney Neoplasms/genetics ; *Carcinoma, Renal Cell/genetics ; Epigenesis, Genetic ; Cell Line, Tumor ; Gene Expression Regulation, Neoplastic ; Gene Silencing ; Promoter Regions, Genetic ; CRISPR-Cas Systems ; Male ; Female ; Tight Junctions ; Mixed Function Oxygenases ; Proto-Oncogene Proteins ; },
abstract = {BACKGROUND: The kidney's tubular system relies on cell polarity and tight junctions to maintain structure and function and disruptions contribute to diseases like cancer. Loss of tight junction proteins such as Claudins can actively contribute to tumorigenesis.<br><br>RESULTS: We aimed to identify biomarkers for renal carcinoma, after kidney transplantation and conventional kidney tumors. We identified the epigenetic silencing of the Claudin 10 gene isoform B (CLDN10B) through DNA hypermethylation in renal cancers, including clear cell (ccRCC), papillary (pRCC) and post-transplantation renal carcinoma (PT-ccRCC). In contrast, CLDN10A was hypomethylated in ccRCC and pRCC. Differential methylation of the isoforms discriminates RCC from other malignancies. The epigenetic alteration of CLDN10B significantly correlated with reduced patient survival and advanced tumor staging. CLDN10B overexpression or induction significantly inhibited migration, cell cycle progression, and cellular growth. Using a CRISPR-based epigenetic editing tool&#xa0;reactivated CLDN10B to its endogenous level using VP160 and TET1 by promoter demethylation and significantly demonstrated its tumor-suppressive effects in 2D and 3D cell models.<br><br>CONCLUSION: Our findings suggest that CLDN10B acts as a tumor suppressor, and its epigenetic regulation may represent a therapeutic target for RCC. Ultimately, understanding CLDN10B's regulation and function could provide new insights into renal cancer treatment.},
}
@article {pmid40524010,
year = {2025},
author = {Levengood, H and Smith, L and Gillis, S and Zhou, Y and Zhang, C},
title = {Plantago species are emerging model organisms for functional genomics and stress biology.},
journal = {Plant cell reports},
volume = {44},
number = {7},
pages = {142},
pmid = {40524010},
issn = {1432-203X},
support = {1923557//National Science Foundation/ ; },
mesh = {*Genomics/methods ; *Plantago/genetics/physiology ; *Stress, Physiological/genetics ; Genome, Plant/genetics ; CRISPR-Cas Systems ; },
abstract = {Species in the Plantago genus are emerging model organisms to multiple research disciplines. The genus Plantago has long been recognized for its significance in various research fields, yet it remains underutilized as a model organism in scientific studies. Several Plantago species possess unique traits, including easily accessible vascular tissues, medicinal properties, gynodieocity, and remarkable adaptability to diverse environmental conditions. These characteristics position Plantago as a promising model for research in areas such as plant vascular biology, stress physiology, reproductive biology, ecology, and medicinal biochemistry. Recent advancements, including the development of genetic transformation systems, the availability of sequenced genomes, and the application of CRISPR-Cas9 technology, have significantly enhanced the capability of using Plantago as a model system. This review discusses the research potential of Plantago species, highlighting key historical discoveries and recent breakthroughs that demonstrate their value across multiple scientific disciplines.},
}
@article {pmid40523955,
year = {2025},
author = {Rong, C and Zhang, R and Xie, J and Li, J and Yan, T and Liu, Z and Liu, Y and Xu, R and Shi, X and Zhao, X and Song, J and Meng, Y and Chang, Z and Ding, Y and Ding, C},
title = {Type-A and -C response regulator genes positively impact rice plant height and panicle architecture.},
journal = {Plant cell reports},
volume = {44},
number = {7},
pages = {141},
pmid = {40523955},
issn = {1432-203X},
support = {No BK20231470//Basic Research Program of Jiangsu Province/ ; },
mesh = {*Oryza/genetics/growth &amp; development/metabolism/anatomy &amp; histology ; Gene Expression Regulation, Plant ; *Plant Proteins/genetics/metabolism ; Cytokinins/metabolism ; Signal Transduction/genetics ; Mutation ; Plant Growth Regulators/metabolism ; CRISPR-Cas Systems ; Genes, Plant ; },
abstract = {Thirteen type -A and two type -C RR genes exhibited distinct expression patterns and unique functions. Notably, RR2 and RR4 had the most significant positive effects impact on panicle development. Cytokinin signal transduction occurs through a "two-component system". Type-A and -C response regulators (RRs) are groups of proteins of similar structures constituting significant components of cytokinin signal transduction. In rice, 13 (Type-A) and 2 (Type-C) RRs have been identified to date; however, their functions remain partially known. In this study, we examined the expression patterns of Type-A and Type-C RRs in rice using RNA-Seq and confirmed their functions by constructing mutants of the 15 genes with CRISPR/Cas9. Almost all Type-A RRs played positive roles in the development of secondary branches and secondary spikelets, particularly RR2 and RR4. Notably, rr1 rr2 and rr8 rr12 rr13 higher-order mutants displayed small panicle sizes and decreased plant height. Additionally, both Type-C RRs played positive roles in regulating heading date. RNA-seq revealed several genes with significantly altered expression in the rr2 and rr4 mutants, with almost half of the differentially expressed genes (DEGs) overlapping between the two mutants. Many of the DEGs were associated with the cytokinin and abscisic acid pathways. Our findings provide new insights into the functions of Type-A and -C RRs in rice growth and may serve as a foundation for future studies focusing on cytokinin signaling.},
}
@article {pmid40522908,
year = {2025},
author = {Schloßhauer, JL and Shamloo, S and Schamrin, K and Imig, J},
title = {Dual CRISPR-Interference Strategy for Targeting Synthetic Lethal Interactions Between Non-Coding RNAs in Cancer Cells.},
journal = {Journal of visualized experiments : JoVE},
volume = {},
number = {219},
pages = {},
doi = {10.3791/67752},
pmid = {40522908},
issn = {1940-087X},
mesh = {Humans ; *RNA, Long Noncoding/genetics ; Cell Line, Tumor ; *CRISPR-Cas Systems ; Streptococcus pyogenes/genetics/enzymology ; *Melanoma/genetics/pathology ; Staphylococcus aureus/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {Long non-coding RNAs (lncRNAs) represent a vast and functionally diverse class of RNA molecules, with over 100,000 predicted in the human genome. Although lncRNAs are less conserved across species compared to protein-coding genes, they play critical roles in gene regulation, chromatin interactions, and cancer progression. Their involvement in cancer makes them promising therapeutic targets. CRISPR interference (CRISPRi), utilizing catalytically inactive Cas9 fused with a transcriptional repressor such as KRAB-MeCP2, offers a precise method for targeting nuclear lncRNAs and assessing their functions. This study introduces a dual CRISPRi system using orthogonal CRISPRi technologies from Staphylococcus aureus and Streptococcus pyogenes based on dCas9-KRAB-MeCP2, optimized for combinatorial targeting of lncRNAs in human melanoma cells. The protocol facilitates combinatorial gene knockdown or synthetic lethal screening of lncRNA pairs, providing a novel tool for cancer research. By exploring synthetic lethality between lncRNAs, this approach can help identify lncRNA interactions critical for cancer cell survival, offering new therapeutic strategies. The dual system's functionality is demonstrated, highlighting its potential in identifying critical cancer-specific lncRNA interactions.},
}
@article {pmid40522884,
year = {2025},
author = {Christenson, AE and Divekar, NS and Hernández Zamora, MG and Nguyen, CD and Nuñez, JK},
title = {CRISPR Epigenome Editing in Human Cells using Plasmid DNA Transfection and mRNA Nucleofection Delivery.},
journal = {Journal of visualized experiments : JoVE},
volume = {},
number = {219},
pages = {},
doi = {10.3791/68089},
pmid = {40522884},
issn = {1940-087X},
mesh = {Humans ; *Plasmids/genetics/administration &amp; dosage ; *RNA, Messenger/genetics/administration &amp; dosage ; *Transfection/methods ; *DNA/genetics/administration &amp; dosage ; *Gene Editing/methods ; *CRISPR-Cas Systems ; *Epigenome/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Epigenome Editing ; },
abstract = {Epigenetics refers to chemical modifications of histone proteins and DNA that can regulate the expression of genes. The human epigenome is altered dynamically during cell differentiation and aging, and many diseases are associated with aberrant epigenome patterning. Recent advances in CRISPR have led to the development of programmable tools to edit epigenetic modifications at targeted genomic loci, enabling precise rewriting of epigenetic modifications in human cells. CRISPR-based epigenome editors rely on catalytically dead Cas9 coupled with epigenetic modifiers that ultimately result in programmed repression or activation of targeted genes in mammalian genomes. Unlike traditional genome editing methods, epigenome editing does not require DNA breaks or changes in the human genome sequence and thus serves as a safer alternative to control gene expression. In this protocol, we highlight two different methods to perform dCas9-mediated epigenome editing in human cell lines using plasmid DNA transfections and nucleofection of mRNAs encoding CRISPR epigenome editors. We demonstrate programmable epigenome editing to transiently repress genes using CRISPR interference (CRISPRi) and for silencing genes durably for many weeks using CRISPRoff, a fusion of dCas9 to the KRAB domain and de novo DNA methyltransferase complex. We also provide guidance on quantitative methods to measure successful epigenome editing of target genes and key considerations on which epigenome editing tool to use, depending on experimental criteria.},
}
@article {pmid40522066,
year = {2025},
author = {Zhang, J and Zhu, A and Wei, S and Shi, Y and Chen, C and Huang, T and Tang, T and Zhao, J and Cai, Y and Han, C and Zhao, J and Wang, Y},
title = {Identification of Pan-Coronavirus Neutralizing Aptamers Through CRISmers Targeting the Conserved Fusion Peptide.},
journal = {Advanced healthcare materials},
volume = {},
number = {},
pages = {e2501869},
doi = {10.1002/adhm.202501869},
pmid = {40522066},
issn = {2192-2659},
support = {2023YFA0915000//National Key Research and Development Program of China/ ; 2022YFC2303700//National Key Research and Development Program of China/ ; 82273967//National Natural Science Foundation of China/ ; 82025001//National Natural Science Foundation of China/ ; 2021QN020576//Department of Science and Technology of Guangdong Province/ ; 2021B1515130005//Guangdong Basic and Applied Research Projects/ ; JSGG20200807171801013//Shenzhen Science and Technology Innovation Committee/ ; //Shenzhen Institute of Synthetic Biology Scientific Research Program/ ; 2023M742397//China Postdoctoral Science Foundation/ ; GZC20231724//Postdoctoral Fellowship Program of CPSF/ ; },
abstract = {A series of coronavirus outbreaks emphasize the necessity to develop pan-coronavirus therapeutics. Previously, the CRISmers system is developed, a Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated proteins (CRISPR/Cas)-mediated cellular screening platform for the identification of RNA aptamers. In this study, CRISmers are applied to target the fusion peptide (FP) of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), a conserved region across coronavirus genera, with E.coli serving as the screening host. From this approach, a lead aptamer, #FP-10 is identified, which demonstrates potent pan-coronavirus neutralization activity against multiple SARS-CoV-2 variants and alphacoronaviruses (HCoV-229E and HCoV-NL63), demonstrating its potential for development into a broad-spectrum therapeutic candidate.},
}
@article {pmid40521185,
year = {2025},
author = {Wu, D and Shu, H and Zhang, M and Wei, X and Ji, J and Shen, H and Zhang, H and Xie, L and Zhou, L and Yang, L and Jiang, J and Chen, C and Tian, S and Zhang, X and Long, X and He, X and Wang, H},
title = {Mannosylated neutrophil vesicles targeting macrophages alleviate liver inflammation by delivering CRISPR/Cas9 RNPs.},
journal = {Theranostics},
volume = {15},
number = {13},
pages = {6221-6235},
pmid = {40521185},
issn = {1838-7640},
mesh = {Animals ; *CRISPR-Cas Systems/genetics ; Mice ; *Macrophages/metabolism ; *Neutrophils/metabolism ; NLR Family, Pyrin Domain-Containing 3 Protein/genetics/metabolism ; *Ribonucleoproteins/genetics/administration &amp; dosage/metabolism ; Gene Editing/methods ; Liver/pathology ; Disease Models, Animal ; Mice, Inbred C57BL ; Mannose/metabolism/chemistry ; Inflammation/therapy ; Humans ; CRISPR-Associated Protein 9 ; Male ; Hepatitis/therapy ; Inflammasomes/metabolism ; },
abstract = {Background: Inflammation is a key driver of various liver diseases. NLRP3 inflammasome in hepatic macrophages is a key mediator of inflammation and has emerged as a promising target. Genome editing presents a powerful approach to modulate inflammation by directly disrupting genes such as NLRP3 directly. However, efficient and cell-specific delivery of CRISPR/Cas9 ribonucleoproteins (RNPs) remains challenging. Methods: We developed a novel delivery system by encapsulating CRISPR/Cas9 RNPs within mannosylated neutrophil membranes vesicles (Cas9/gNLRP3@M-N) to enhance targeting hepatic macrophages. Results: Cas9/gNLRP3@M-N selectively accumulated in hepatic macrophages, effectively disrupted the NLRP3 gene, attenuated inflammation in acute fulminant hepatitis, and improved disease outcomes in chronic steatohepatitis model. Conclusions: Cas9/gNLRP3@M-N represents a promising targeted gene-editing approach for the treatment of inflammatory liver diseases.},
}
@article {pmid40521034,
year = {2025},
author = {Al-Ouqaili, MTS and Ahmad, A and Jwair, NA and Al-Marzooq, F},
title = {Harnessing bacterial immunity: CRISPR-Cas system as a versatile tool in combating pathogens and revolutionizing medicine.},
journal = {Frontiers in cellular and infection microbiology},
volume = {15},
number = {},
pages = {1588446},
pmid = {40521034},
issn = {2235-2988},
mesh = {*CRISPR-Cas Systems ; *Gene Editing/methods ; Humans ; *Bacteria/genetics/immunology ; *Bacterial Infections/therapy/immunology ; Animals ; Genetic Therapy/methods ; Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) technology has emerged as an adaptable instrument for several uses. The CRISPR-Cas system employs Cas proteins and programmable RNA molecules to guide the recognition and cleavage of specific DNA regions, permitting accurate genome editing. It is derived from the bacterial immune system and allows for accurate and efficient modification of DNA sequences. This technique provides unparalleled gene editing, control, and precise alteration opportunities. This review aims to offer a comprehensive update of the core concepts of the CRISPR-Cas system and recent progress, while also providing an overview of the significant applications in diverse fields such as microbiology and medicine. The CRISPR-Cas9 gene editing technique has facilitated substantial advancements in comprehending gene function, simulating diseases, and creating innovative therapeutics. CRISPR-based therapeutics present a hopeful prospect for addressing intricate ailments, including genetic disorders, malignancies, and infectious diseases, as they serve as viable substitutes for conventional pharmaceuticals. In microbiology, this method serves as a diagnostic and therapeutic tool that proves highly efficient in eliminating bacteria that have developed resistance to various antibiotics. Despite its significant potential, CRISPR encounters ethical, safety, and regulatory obstacles that necessitate meticulous deliberation. Concerns regarding off-target effects, poor delivery to target tissues, and unwanted side effects emphasize the necessity to thoroughly examine the technology. It is necessary to balance the advantages and difficulties CRISPR presents. Consequently, more rigorous preclinical and clinical experiments are essential before using it in humans.},
}
@article {pmid40520364,
year = {2025},
author = {Nitulescu, AM and Du, W and Glaser, V and Kath, J and Aird, EJ and Cullot, G and Greensmith, R and Mikkelsen, NS and Stein, M and Bak, RO and Kaminski, M and Corn, JE and Wagner, DL},
title = {Single-stranded HDR templates with truncated Cas12a-binding sequences improve knock-in efficiencies in primary human T cells.},
journal = {Molecular therapy. Nucleic acids},
volume = {36},
number = {2},
pages = {102568},
pmid = {40520364},
issn = {2162-2531},
abstract = {CRISPR-Cas12a gene editing offers an alternative to Cas9-based methods, providing better targeting of AT-rich regions, simplified guide RNA manufacturing, and high specificity. However, the efficacy of donor-based editing is subject to various factors, with template format playing a crucial role. Currently, the predominant non-viral template format for homology-directed repair (HDR) after nuclease-induced DNA breaks is double-stranded DNA, which is toxic when transfected at high doses. Others have demonstrated that using single-stranded DNA (ssDNA) with flanking double-stranded Cas-target-sequences (CTS) as a template for Cas9-mediated gene editing can mitigate this toxicity and increase knock-in efficiency. Here, we investigate CTS design for AsCas12a Ultra by exploring PAM orientation and binding requirements. Additionally, we rule out ssDNase activity of AsCas12a under cell-physiological Mg[2+] conditions. Finally, we showcase the advantage of ssDNA donors with CTS (ssCTS) at high doses for delivering clinically relevant transgenes of varying sizes into three TCR-CD3 complex genes (TRAC, CD3ζ, CD3ε), achieving up to 90% knock-in rates for a 0.8kb-insert at the CD3ε locus. Long-read sequencing confirmed higher HDR rates and revealed that CTS reduced partial integration events compared to unmodified ssDNA. Overall, AsCas12a and ssCTS represent a platform for highly efficient knock-in in primary human T cells with minimal toxicity.},
}
@article {pmid40518326,
year = {2025},
author = {Zhao, Y and Tan, J and Fang, L and Jiang, L},
title = {Leveraging gene editing to combat methane emissions in ruminant agriculture.},
journal = {Trends in biotechnology},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.tibtech.2025.05.020},
pmid = {40518326},
issn = {1879-3096},
abstract = {Methane emissions from ruminants represent a major contributor to global greenhouse gases (GHGs), posing challenges for sustainable agriculture and climate change mitigation. Recent advances in clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated proteins (Cas)-based gene editing offer transformative approaches to address this issue by targeting both forage crops and rumen methanogens. Enhancing lipid content and secondary metabolites in forage crops can suppress methanogenesis and improve feed efficiency, while precise genome editing in methanogenic Archaea can disrupt pathways critical to methane production. However, challenges remain regarding delivery methods, gene targeting specificity, ecological impacts, and regulatory acceptance. In this review, we explore current progress, identify key knowledge gaps, and highlight the need for interdisciplinary strategies that integrate biotechnology, synthetic biology, and regulatory frameworks to develop effective and scalable methane mitigation solutions.},
}
@article {pmid40517395,
year = {2025},
author = {Marín-Sanz, M and Sánchez-León, S and Guzmán-López, MH and Starker, CG and Voytas, DF and Barro, F},
title = {Cas9- and Cas12a-mediated excision and replacement of the celiac disease-related α-gliadin immunogenic complex in hexaploid wheat.},
journal = {Plant biotechnology journal},
volume = {},
number = {},
pages = {},
doi = {10.1111/pbi.70200},
pmid = {40517395},
issn = {1467-7652},
support = {//European Union "NextGenerationEU"/PRTR/ ; QUAL21_023 IAS//Junta de Andaluc&#xed;a/ ; //Conexi&#xf3;n TRIGO/WheatNet/ ; PID2022-142139OB-I00//Ministerio de Ciencia, Innovaci&#xf3;n y Universidades/ ; TED2021-129733B-I00//Ministerio de Ciencia, Innovaci&#xf3;n y Universidades/ ; },
abstract = {Celiac disease (CD) is a chronic enteropathy affecting approximately 1% of the global population. Wheat α-gliadins are a major contributor to the autoimmune response, as they contain one of the most immunogenic peptides, the 33-mer, along with numerous variants. In this study, we used CRISPR/Cas technology to mutate genes encoding α-gliadins. This approach employed paired sgRNAs to precisely excise immunogenic regions while preserving non-immunogenic sequences within the α-gliadins. Furthermore, we replaced the excised region with an α-gliadin-based double-stranded oligodeoxynucleotide (dsODN) designed with nucleotide changes to reduce immunoreactivity and increase peptidase cleavage sites. Two different CRISPR systems, Cas9 and Cas12a, were applied to generate wheat protoplasts and plants with non-immunogenic regions. Cas9 demonstrated superior performance in terms of editing frequency, excision and replacement of immunogenic fragments. However, the Cas12a nuclease (Cpf1) showed promising editing efficiency, offering the potential for future wheat editing applications. Using the Cas9 system, we achieved a 74.2% excision rate of the 33-mer in wheat plants. Subsequent analyses showed a significant reduction in the reactivity to the G12 monoclonal antibody, capable of identifying the 33-mer peptide and a decrease in the prolamin levels compared to the wild-type. Additionally, we developed a high-throughput sequencing-based software specifically designed to identify mutations in multi-copy gene families. This innovative tool enabled fast, parallel screening of the samples in this study and facilitated the identification of the specific editing patterns produced by the designed constructs.},
}
@article {pmid40516885,
year = {2025},
author = {Cunha da Silva, G and Rossi, CC},
title = {Defense systems and mobile elements in Staphylococcus haemolyticus: a genomic view of resistance dissemination.},
journal = {Microbial pathogenesis},
volume = {206},
number = {},
pages = {107808},
doi = {10.1016/j.micpath.2025.107808},
pmid = {40516885},
issn = {1096-1208},
abstract = {Staphylococcus haemolyticus is a multidrug-resistant opportunistic pathogen and a major reservoir of antimicrobial resistance (AMR) genes within the Staphylococcaceae family. Its high genomic plasticity, frequent association with mobile genetic elements (MGEs), and prevalence in clinical settings underscore its relevance as both a threat and a conduit for resistance dissemination. In this study, we performed a comprehensive pan-genomic analysis of the S. haemolyticus defensome - including restriction-modification (RM), abortive infection (Abi), and CRISPR-Cas systems - across 692 high-quality genomes. Our results reveal a highly diverse and modular repertoire of immune systems, often organized in physical clusters and frequently associated with MGEs. We identified evidence of antagonistic interactions, with both defense and anti-defense elements encoded on plasmids and prophages. CRISPR spacer analysis showed a predominant targeting of phages, and genomes encoding CRISPR-Cas systems exhibited a lower abundance of MGEs and AMR genes, suggesting a trade-off between defense and gene acquisition. RNA-seq data from one reference strain indicate that only a fraction of the defensome is actively transcribed under standard conditions, hinting at environment-responsive regulation. Together, these findings provide new insights into the genomic strategies sustaining the persistence and adaptability of S. haemolyticus in clinical environments. The interplay between its immune systems and mobilome likely contributes not only to its evolutionary trajectory, but also to its role in the horizontal transfer of resistance determinants among pathogenic staphylococci. A deeper understanding of this immune-mobilome interface may help inform future strategies to limit the spread of resistance.},
}
@article {pmid40516718,
year = {2025},
author = {Bu, G and Ma, X and Jin, H and Wang, L},
title = {CRISPR-Cas and RNA sequencing reveal nutrient enhancement pathways in quinoa for plant-based athlete recovery diets and future food security.},
journal = {International journal of biological macromolecules},
volume = {318},
number = {Pt 4},
pages = {144785},
doi = {10.1016/j.ijbiomac.2025.144785},
pmid = {40516718},
issn = {1879-0003},
abstract = {Quinoa (Chenopodium quinoa Willd.) is a nutrient-dense, climate-resilient pseudocereal with growing relevance to food and nutritional security. However, its potential remains underutilized due to suboptimal levels of key micronutrients. In this study, to apply CRISPR-Cas9 for the simultaneous enhancement of multiple nutrients in quinoa, marking a significant advancement in crop biofortification, we employed multiplexed CRISPR-Cas9 genome editing to target five genes involved in lysine transport, phytic acid biosynthesis, and vitamin C and E biosynthetic pathways. Both knockout and homology-directed knock-in strategies were applied to induce heritable mutations in genes such as CqAAP1, CqIPK1, CqGGP, and CqHPT. Homology-directed knock-in refers to a precise gene-editing method that uses a template to insert specific genetic changes at targeted sites in the genome. Edited lines exhibited significant improvements in seed nutrient concentrations, including lysine (+35 %), zinc (+43 %), vitamin C (+114 %), and vitamin E (+45 %), without yield or growth penalties. Whole-transcriptome profiling via RNA sequencing (RNA-Seq) identified 1284 differentially expressed genes (FDR < 0.05), predominantly associated with amino acid metabolism, redox regulation, and vitamin biosynthesis. Gene Ontology (GO) and KEGG enrichment analyses confirmed the transcriptional activation of nutrient assimilation and antioxidant pathways. Integration of qRT-PCR with RNA-Seq confirmed the reliability of expression data. Structural validations of edits were performed using PCR, gel electrophoresis, and Sanger sequencing. Phenotypic assessments, including seed weight, morphology, and exploratory herbivory assays, confirmed agronomic stability. This study presents a technically validated framework for simultaneous nutritional trait stacking in quinoa using nucleic acid engineering tools. Our findings offer critical insights into the transcriptional plasticity of quinoa and establish a functional genomics platform for developing multi-nutrient biofortified crops to address hidden hunger and advance sustainable food systems.},
}
@article {pmid40515942,
year = {2025},
author = {Saha, T and Saha, RP and Singh, MK and Priya, K and Singh, S and Rajeev, M and Bhattacharya, D and Nag, M and Lahiri, D},
title = {An overview on in-vivo generation of CAR-T cells using CRISPR-loaded functionalized nanocarriers for treating B-cell lineage acute lymphoblastic leukemia.},
journal = {Molecular biology reports},
volume = {52},
number = {1},
pages = {596},
pmid = {40515942},
issn = {1573-4978},
mesh = {Humans ; Gene Editing/methods ; CRISPR-Cas Systems/genetics ; *Immunotherapy, Adoptive/methods ; Animals ; *Receptors, Chimeric Antigen/genetics/metabolism/immunology ; T-Lymphocytes/immunology/metabolism ; Mice ; Nanoparticles/chemistry ; *Precursor Cell Lymphoblastic Leukemia-Lymphoma/therapy/immunology/genetics ; *Precursor B-Cell Lymphoblastic Leukemia-Lymphoma/therapy/immunology ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; B-Lymphocytes ; },
abstract = {Chimeric antigen receptor T (CAR-T) cell therapy has become a milestone in the management of B cell lineage acute lymphoblastic leukemia. Yet, the traditional method-dependent on ex vivo manipulation, amplification, and reinfusion of autologous T cells-is high-cost, low-scalability, and severely immune-related toxicity. Here, we report a new nano-immunoengineering platform that allows in vivo production of chimeric antigen receptor T cells through the use of functionalized nanoparticles carrying clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein 9 (Cas9) gene editing elements. These nanoparticles are engineered to specifically target blood circulating T lymphocytes and deliver CRISPR/Cas9 complexes that have the ability to integrate chimeric antigen receptor constructs into the TRAC locus and knock out immune checkpoint genes like programmed cell death protein 1 (PD-1) simultaneously. Targeted delivery, endosomal escape, and efficient genome editing with minimal off-target effects are ensured through gold-based and DNA nanostructure-based carriers. Preclinical models show effective in vivo programming of functional chimeric antigen receptor T cells with vigorous antitumor efficacy, improved persistence, and decreased cytokine release syndrome. This method is a revolutionary breakthrough in cancer immunotherapy that provides a scalable, economical, and clinically flexible replacement for conventional chimeric antigen receptor T cell production.},
}
@article {pmid40515919,
year = {2025},
author = {Ye, Y and Zhang, M and Nie, H and Duan, Y and Ye, Z and Zheng, C},
title = {Screening for Host Proteins with Pro- and Antiviral Activity via High-Throughput CRISPR.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2940},
number = {},
pages = {273-281},
pmid = {40515919},
issn = {1940-6029},
mesh = {Humans ; *CRISPR-Cas Systems ; Antiviral Agents/pharmacology ; *High-Throughput Screening Assays/methods ; *Host-Pathogen Interactions/genetics ; Animals ; Cell Line ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Virus Diseases/virology/genetics ; Viruses/genetics ; Virus Replication ; },
abstract = {The whole-genome library for CRISPR screening serves as an important biotechnological tool aimed at probing gene function in mammalian cells, providing a foundation for the systematic discovery of essential genes corresponding to biological effects. Research indicates that whole-genome library cell lines can be used to identify host factors and potential drug targets associated with viral infections effectively at high throughput, providing crucial evidence for the development of novel antiviral drugs. Here, we primarily discuss the methods for constructing genome-scale CRISPR screens in cell lines and their applications in virology research. By cloning and expressing the whole genomic DNA of specific organisms, stable cell lines, which can be utilized for functional validation, drug screening, and gene function studies, can be established. Through gene knockout or overexpression techniques, in-depth analyses of the key roles that genes play in the viral life cycle could be conducted, revealing how viruses exploit the biological mechanisms of host cells for replication and evasion of immune responses. These findings not only enhance our understanding of the interactions between viruses and host cells but also yield new targets for the development of antiviral drugs and vaccines.},
}
@article {pmid40515918,
year = {2025},
author = {Wang, J and Zheng, C and Xiao, W},
title = {Zebrafish as a Model for Studying Virus-Host Interactions.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2940},
number = {},
pages = {261-272},
pmid = {40515918},
issn = {1940-6029},
mesh = {Animals ; *Zebrafish/virology/genetics ; *Host-Pathogen Interactions/genetics ; *Rhabdoviridae/physiology/genetics ; Disease Models, Animal ; CRISPR-Cas Systems ; *Rhabdoviridae Infections/virology/genetics ; *Fish Diseases/virology/genetics ; },
abstract = {During the long evolutionary process, viruses and hosts have engaged in a game of immune escape and viral clearance, resulting in an exquisite model of virus-host interaction. An appropriate animal model can help unravel the mysteries of this process. Owing to their susceptibility to multiple viruses, small size, high fecundity, low cost, ease of visualization, and efficient gene editing, zebrafish (Danio rerio) have recently emerged as a powerful vertebrate model system for studying virus-host interactions. In this chapter, we describe in detail the experimental protocols for analyzing virus-host interactions in a zebrafish model in which spring viremia of carp virus (SVCV) is used as an example. These protocols include the generation of knockout zebrafish via CRISPR/Cas9 technology, methods for viral infection of zebrafish larvae or adults, analysis of in vivo viral resistance of host genes, analysis of host and viral gene expression in zebrafish larvae via qRT-PCR and Western blotting, and detection of host protein binding to viral proteins via immunoprecipitation. These experimental protocols provide an effective reference for studying virus-host interactions in a zebrafish model.},
}
@article {pmid40515917,
year = {2025},
author = {Zhou, Q and Wu, T and Zheng, C and Zou, X},
title = {The Application of Base-Editing Technology to Investigate Virus-Host Interactions and Antiviral Therapeutic Strategies.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2940},
number = {},
pages = {251-260},
pmid = {40515917},
issn = {1940-6029},
mesh = {*Gene Editing/methods ; Humans ; CRISPR-Cas Systems ; *Host-Pathogen Interactions/genetics ; *Antiviral Agents/therapeutic use/pharmacology ; *Virus Diseases/virology/genetics/drug therapy/therapy ; *Viruses/genetics/drug effects ; Animals ; },
abstract = {Understanding viral pathogenesis and developing effective countermeasures hinge on deciphering the complex relationships between viruses and their hosts. While conventional approaches have shown limitations in unraveling these intricate interactions, researchers are now turning to cutting-edge genetic tools, particularly base-editing technology. This section explores how base editing can be innovatively applied to investigate virus-host dynamics and to create novel antiviral therapeutic approaches.},
}
@article {pmid40515905,
year = {2025},
author = {Huang, W and Zhao, X and Zheng, C and Shen, Y},
title = {CRISPR-Mediated Viral Gene Knock-In for Studying Viral-Host Interactions.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2940},
number = {},
pages = {109-119},
pmid = {40515905},
issn = {1940-6029},
mesh = {*CRISPR-Cas Systems ; *Herpesvirus 1, Human/genetics/physiology ; Red Fluorescent Protein ; Humans ; Luminescent Proteins/genetics/metabolism ; Virus Replication/genetics ; *Host-Pathogen Interactions/genetics ; *Gene Knock-In Techniques/methods ; Animals ; Capsid Proteins/genetics/metabolism ; },
abstract = {CRISPR-mediated gene knock-in is a powerful tool for studying virus-host interactions. In this protocol, we describe the methodology for the knock-in of a red fluorescent protein, mCherry, fused to the viral protein VP26, a component of the herpesvirus capsid. By integrating the mCherry gene into the HSV-1 genome, we enable detailed studies of viral protein dynamics, viral replication, and virus-host interactions in living cells. In particular, this approach allows real-time monitoring of the subcellular localization of the viral capsid. The protocol provides a step-by-step guide for designing the gene knock-in vectors, transfection, selection, and validation of the mCherry fusion to the VP26 protein in the HSV-1 genome.},
}
@article {pmid40514654,
year = {2025},
author = {Li, M and Zhou, S and Zhang, S and Xie, X and Nie, J and Wang, Q and Ma, L and Cheng, Y and Luo, J},
title = {Transdermal delivery of CRISPR/Cas9-mediated melanoma gene therapy via polyamines-modified thermosensitive hydrogels.},
journal = {Journal of nanobiotechnology},
volume = {23},
number = {1},
pages = {441},
pmid = {40514654},
issn = {1477-3155},
support = {2024BCA001//Project of Technological Innovation Plan in Hubei Province/ ; 2024BCA001//Project of Technological Innovation Plan in Hubei Province/ ; 2024040701010046//Natural Science Foundation of Wuhan City/ ; 2025AFB729//Hubei Provincial Natural Science Foundation of China/ ; },
mesh = {*Hydrogels/chemistry ; Animals ; *CRISPR-Cas Systems/genetics ; Doxorubicin/pharmacology/administration &amp; dosage/chemistry ; *Melanoma/therapy/genetics/drug therapy ; Humans ; Cell Line, Tumor ; *Genetic Therapy/methods ; *Polyamines/chemistry ; Mice ; Y-Box-Binding Protein 1/genetics/metabolism ; Gene Editing ; Administration, Cutaneous ; Drug Delivery Systems ; Mice, Nude ; },
abstract = {The main obstacles to the clinical application of the CRISPR/Cas9 system are off-target effects and low delivery efficiency. There is an urgent need to develop new delivery strategies and technologies. Three types of in situ injectable hydrogels with different electrical properties were created to find the most secure and efficient sustained-release drug delivery system. After in vitro and in vivo comparisons, we found that the positively charged hydrogels had higher cellular uptake, stronger gene editing efficiency, greater cytotoxicity, longer tumor accumulation, and better anti-tumor efficacy than negatively charged and neutral hydrogels. We designed single guide RNA targeting the Y-box binding protein 1 (YB-1) gene and then used it to create a ribonucleoprotein complex with Cas9 protein. Doxorubicin was co-encapsulated into this positively charged hydrogel to create a co-delivery system. By knocking down YB-1, the expression of YB-1 was reduced, inhibiting the growth and migration of melanoma cells. The strategy of combining YB-1 gene editing and intratumoral injection enhanced the therapeutic effect of doxorubicin while reducing side effects.},
}
@article {pmid40514251,
year = {2025},
author = {Qurtam, AA},
title = {Epigenetic reprogramming in breast cancer: The role of CRISPR-Cas 9.},
journal = {Bulletin du cancer},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.bulcan.2025.03.007},
pmid = {40514251},
issn = {1769-6917},
abstract = {Epigenetic alterations are known to be a significant factor in the development and advancement of breast cancer (BC), which continues to be a substantial cause of illness and death in women globally. The emergence of CRISPR-Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats and CRISPR-associated protein 9) technology has fundamentally transformed our capacity to edit the genome with unparalleled accuracy, providing novel opportunities for therapeutic intervention. This review examines the utilization of CRISPR-Cas9 to alter epigenetic landscapes to combat BC. We examine the fundamental processes of CRISPR-Cas9 and its derivatives, including dCas9, in their ability to specifically target DNA methylation and histone alterations. The highlighted review showcases the potential of CRISPR-Cas9 in reactivating silenced tumor suppressor genes and inhibiting oncogenes. In addition, we analyze the incorporation of CRISPR-based epigenetic editing into current medicines, offering valuable knowledge on the use of combination therapies to improve treatment effectiveness and overcome resistance. This review intends to highlight the revolutionary potential of CRISPR-Cas9 in generating targeted, personalized therapeutics for BC by explaining the present advancements and future applications. The incorporation of this state-of-the-art technology with conventional and developing therapies holds the potential to establish more efficient and long-lasting remedies in the battle against BC, ultimately enhancing patient results and rates of survival.},
}
@article {pmid40512855,
year = {2025},
author = {Boardman, DA and Mangat, S and Gillies, JK and Leon, L and Fung, VCW and Haque, M and Mojibian, M and Halvorson, T and Huang, Q and Tuomela, K and Wardell, CM and Brown, A and Lam, AJ and Levings, MK},
title = {Armored human CAR Treg cells with PD1 promoter-driven IL-10 have enhanced suppressive function.},
journal = {Science advances},
volume = {11},
number = {24},
pages = {eadx7845},
pmid = {40512855},
issn = {2375-2548},
mesh = {Humans ; *Interleukin-10/genetics/metabolism ; *T-Lymphocytes, Regulatory/immunology/metabolism ; *Programmed Cell Death 1 Receptor/genetics ; *Promoter Regions, Genetic ; Animals ; *Receptors, Chimeric Antigen/genetics/immunology/metabolism ; Mice ; Dendritic Cells/immunology ; CRISPR-Cas Systems ; Immunotherapy, Adoptive/methods ; },
abstract = {Regulatory T cell (Treg cell) therapy has been transformed through the use of chimeric antigen receptors (CARs). We previously found that human Treg cells minimally produce IL-10 and have a limited capacity to control innate immunity compared to type 1 regulatory T cells (Tr1 cells). To create "hybrid" CAR Treg cells with Tr1 cell-like properties, we examined whether the PDCD1 locus could be exploited to endow Treg cells with CAR-regulated IL-10 expression. CRISPR-mediated PD1 deletion increased CAR Treg cell activation, and knock-in of IL10 under control of the PD1 promoter resulted in CAR-induced IL-10 secretion. IL10 knock-in improved CAR Treg cell function, as determined by increased suppression of dendritic cells and alloantigen- and islet autoantigen-specific T cells. In vivo, IL10 knock-in CAR Treg cells were stable, safe, and suppressed dendritic cells and xenogeneic graft-versus-host disease. CRISPR-mediated engineering to simultaneously remove an inhibitory signal and enhance a suppressive mechanism is a previously unexplored approach to improve CAR Treg cell potency.},
}
@article {pmid40510793,
year = {2025},
author = {Zhang, Y and Lv, L and Xu, S and Chen, Y},
title = {Innovative nucleic acid detection of Clostridioides difficile utilizing the PAM-unconventional, one-step LAMP/CRISPR-Cas12b detection platforms.},
journal = {Frontiers in cellular and infection microbiology},
volume = {15},
number = {},
pages = {1594271},
pmid = {40510793},
issn = {2235-2988},
mesh = {*Clostridioides difficile/genetics/isolation &amp; purification ; *CRISPR-Cas Systems ; *Molecular Diagnostic Techniques/methods ; *Clostridium Infections/diagnosis/microbiology ; *Nucleic Acid Amplification Techniques/methods ; Sensitivity and Specificity ; Humans ; Bacterial Proteins/genetics ; Feces/microbiology ; Bacterial Toxins/genetics ; },
abstract = {INTRODUCTION: Clostridioides difficile (C. difficile), a human pathogen that causes diarrhea and colon lesions, has garnered widespread attention. Rapid and accurate detection of bacterial virulence factors is essential for the diagnosis of C. difficile infection (CDI). To date, numerous laboratory tests have been developed; however, none fully meet the combined requirements of speed, cost-effectiveness, portability, sensitivity, and specificity. Molecular diagnostic technologies based on CRISPR-Cas systems have provided a promising solution to this challenge. Nonetheless, the limited compatibility between pre-amplification and CRISPR cleavage, coupled with the inherent selectivity of CRISPR systems for protospacer adjacent motif (PAM) sequences near the target site, poses additional constraints on the broader adoption of this approach.<br><br>METHODS: Here, we developed PAM-unconventional, One-step LAMP/CRISPR-Cas12b (POLC) detection platforms for the toxin-encoding genes tcdA and tcdB of C. difficile.<br><br>RESULTS: The POLC platforms operated at 60 &#xb0;C, enabling result interpretation either through fluorescence intensity measurements or direct visualization under UV light. The limits of detection (LoDs) ranged from 3 to 14 copies/&#x3bc;L using a fluorescence reader and from 6 to 18 copies/&#x3bc;L via direct observation. Compared to qPCR, which typically requires over an hour, the POLC platforms reduced the detection time to approximately 40 minutes. Each reaction cost approximately USD 6.5, offering a substantial cost saving compared to qPCR-based commercial kits (over USD 10 per test). In clinical validation with 55 fecal samples, the tcdA POLC assay achieved 86.4% sensitivity and 84.8% specificity, while the tcdB POLC assay demonstrated 96.6% sensitivity and 100% specificity, using qPCR as the reference standard.<br><br>DISCUSSION: Our research presents innovative CRISPR-based one-step nucleic acid detection platforms that eliminate canonical PAM sequence requirements. These platforms exhibit high sensitivity and specificity while achieving rapid detection under simple conditions, making them promising candidates for clinical diagnostics and point-of-care testing (POCT).},
}
@article {pmid40507794,
year = {2025},
author = {Ho, CH and Tsai, CY and Chang, CC and Hu, CJ and Huang, CY and Lu, YC and Lin, PH and Lin, CH and Lin, HI and OuYang, CH and Hsu, CJ and Liu, TC and Chen, YT and Chan, YH and Cheng, YF and Wu, CC},
title = {Low Efficiency of Homology-Independent Targeted Integration for CRISPR/Cas9 Correction in the Vicinity of the SLC26A4 c.919-2A&gt;G Variant.},
journal = {International journal of molecular sciences},
volume = {26},
number = {11},
pages = {},
pmid = {40507794},
issn = {1422-0067},
support = {106-T04//National Taiwan University Hospital/ ; 107-2622-B-002-008-CC2//National Science and Technology Council of Taiwan/ ; },
mesh = {*Sulfate Transporters/genetics ; Humans ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; *Hearing Loss, Sensorineural/genetics ; Goiter, Nodular/genetics ; *Membrane Transport Proteins/genetics ; },
abstract = {Recessive variants of SLC26A4 are a common cause of hereditary hearing impairment and are responsible for non-syndromic enlarged vestibular aqueducts and Pendred syndrome. Patients with bi-allelic SLC26A4 variants often suffer from fluctuating hearing loss and recurrent vertigo, ultimately leading to severe to profound hearing impairment. However, there are currently no satisfactory prevention or treatment options for this condition. The CRISPR/Cas9 genome-editing technique is a well-known tool for correcting point mutations or manipulating genes and shows potential therapeutic applications for hereditary disorders. In this study, we used the homology-independent targeted integration (HITI) strategy to correct the SLC26A4 c.919-2A>G variant, the most common SLC26A4 variant in the Han Chinese population. Next-generation sequencing was performed to evaluate the editing efficiency of the HITI strategy. The results showed that only 0.15% of the reads successfully exhibited HITI integration, indicating that the c.919-2 region may not be a suitable region for HITI selection. This suggests that other site selection or insertion strategies may be needed to improve the efficiency of correcting the SLC26A4 c.919-2A>G variant. This experience may serve as a valuable reference for other researchers considering CRISPR target design in this region.},
}
@article {pmid40506744,
year = {2025},
author = {Kristof, A and Karunakaran, K and Allen, C and Mizote, P and Briggs, S and Jian, Z and Nash, P and Blazeck, J},
title = {Engineering novel CRISPRi repressors for highly efficient mammalian gene regulation.},
journal = {Genome biology},
volume = {26},
number = {1},
pages = {164},
pmid = {40506744},
issn = {1474-760X},
support = {DP2 CA280622/CA/NCI NIH HHS/United States ; NSF GRFP//National Science Foundation/ ; 1DP2CA280622-01//NIH Office of the Director/ ; },
mesh = {Humans ; *CRISPR-Cas Systems ; *Gene Expression Regulation ; *Repressor Proteins/genetics/metabolism ; RNA, Guide, CRISPR-Cas Systems/genetics ; HEK293 Cells ; Animals ; Gene Knockdown Techniques ; Cell Line ; Clustered Regularly Interspaced Short Palindromic Repeats ; CRISPR-Associated Protein 9 ; },
abstract = {BACKGROUND: CRISPR interference (CRISPRi), the repurposing of the RNA-guided endonuclease dCas9 as a programmable transcriptional repressor, allows highly specific repression (knockdown) of gene expression. CRISPRi platforms can often have incomplete knockdown, performance variability across cell lines and gene targets, and inconsistencies dependent on the guide RNA sequence employed.<br><br>RESULTS: Here, we explore the combination of novel repressor domains with strong Kr&#xfc;ppel-associated box (KRAB) repressors, screening >&#x2009;100 bipartite and tripartite fusion proteins for their ability to reduce gene expression as CRISPRi effectors. We show that these novel repressor fusions have reduced dependence on guide RNA sequences, better slow cell growth when used to knock down expression of essential genes, and function in either fusion or scaffold modalities. Furthermore, we isolate and characterize a particularly effective CRISPRi platform, dCas9-ZIM3(KRAB)-MeCP2(t), which shows improved gene repression of endogenous targets both at the transcript and protein level across several cell lines and when deployed in genome-wide screens.<br><br>CONCLUSIONS: We posit that these novel repressor fusions can enhance the reproducibility and utility of CRISPRi in mammalian cells.},
}
@article {pmid40506725,
year = {2025},
author = {Wang, T and Jiang, W and Huang, Z and Yuan, Z and Chen, Z and Lin, J},
title = {Multiplex detection of respiratory RNA viruses without amplification based on CRISPR-Cas13a immunochromatographic test strips.},
journal = {Virology journal},
volume = {22},
number = {1},
pages = {192},
pmid = {40506725},
issn = {1743-422X},
support = {2024T021//Fuzhou University Testing Fund of Precious Apparatus/ ; 2022-S-wr4//Fuzhou Health Science and Technology Plan Soft Science Research Project/ ; 2022-S-rc5//Young and Middle-aged Talent Research Project of Fuzhou City/ ; 2020J05279//Fujian Provincial Natural Science Foundation of China/ ; 2020Y9140//Joint Funds for the Innovation of Sience and Technology, Fujian Province/ ; 2021Y9107//Joint Funds for the Innovation of Science and Technology, Fujian Province/ ; 2021S263//Fuzhou Science and Technology Project/ ; 2021ZQNZD010//Major Research Projects for Young and Middle-aged Researchers of Fujian Provincial Health Commission/ ; 2022J01521//Natural Science Foundation of Fujian Province/ ; 2022Y4003//Fujian Provincial Science and Technology Plan Project/ ; 2022ZD01001//Fujian Provincial Major Health Research Project/ ; 2021GGA053//Fujian Provincial Health Technology Project/ ; 2023YFC3304304//National Key Research &amp; Development Plan/ ; },
mesh = {Humans ; *SARS-CoV-2/isolation &amp; purification/genetics/immunology ; Sensitivity and Specificity ; *Chromatography, Affinity/methods ; *COVID-19/diagnosis/virology ; *RNA Viruses/isolation &amp; purification/genetics ; CRISPR-Cas Systems ; *Respiratory Tract Infections/diagnosis/virology ; Reagent Strips ; Influenza, Human/diagnosis/virology ; },
abstract = {Acute respiratory infections, caused by RNA viruses like respiratory syncytial virus, influenza, rhinovirus, and coronavirus, are major global health threats. Real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR) is the gold standard for detecting these viruses but is time-consuming, complex, and requires specialized equipment. There is a need for rapid, convenient, and multi-target detection methods to improve disease prevention and control. This study developed a multi-target immunochromatographic detection method using LbuCas13a protein and "band elimination" test strips for detecting SARS-CoV-2 and influenza virus. The method's performance was evaluated by testing known 5 positive and 4 negative samples for SARS-CoV-2 and comparing results with fluorescent PCR and colloidal gold methods. Detection sensitivity was quantified using digital PCR and qPCR. The immunochromatographic test strips showed 100% concordance with fluorescent PCR and colloidal gold methods in initial clinical SARS-CoV-2 detection. Subsequently, we used dual-target immunochromatographic test strips to detect 9 SARS-CoV-2 positive samples and 9 H3N2 positive samples. However, false negatives were observed in dual-target detection of SARS-CoV-2 and H3N2 samples, likely due to low sample concentration or sample degradation. The method had a minimum detection limit of 381.75 copies/µL, as determined by digital PCR and qPCR. The developed multi-target immunochromatographic detection method offers a rapid, low-cost, and simple approach for detecting both SARS-CoV-2 and influenza viruses. With high sensitivity, specificity, and reliability, this method holds promise as a practical tool for RNA virus diagnosis and improving public health response to respiratory infections.},
}
@article {pmid40504161,
year = {2025},
author = {Tien, JC and Zhai, Y and Wu, R and Zhang, Y and Chang, Y and Cheng, Y and Todd, AJ and Wheeler, CE and Li, S and Mannan, R and Cheng, C and Magnuson, B and Cruz, G and Cao, Y and Mahapatra, S and Stolfi, C and Cao, X and Su, F and Wang, R and Yang, J and Zhou, L and Qiao, Y and Xiao, L and Cieslik, M and Wang, X and Wang, Z and Chou, J and Fearon, ER and Ding, K and Cho, KR and Chinnaiyan, AM},
title = {Defining CDK12 as a tumor suppressor and therapeutic target in mouse models of tubo-ovarian high-grade serous carcinoma.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {122},
number = {24},
pages = {e2426909122},
doi = {10.1073/pnas.2426909122},
pmid = {40504161},
issn = {1091-6490},
support = {P50-CA186786//HHS | NIH | National Cancer Institute (NCI)/ ; R35-CA231996//HHS | NIH | National Cancer Institute (NCI)/ ; U2C-CA271854//HHS | NIH | National Cancer Institute (NCI)/ ; 22037003//MOST | National Natural Science Foundation of China (NSFC)/ ; W81XWH-21-1-0458//U.S. Department of Defense (DOD)/ ; },
mesh = {Animals ; Female ; Mice ; *Ovarian Neoplasms/genetics/pathology/drug therapy/metabolism ; *Cyclin-Dependent Kinases/genetics/metabolism ; *Cystadenocarcinoma, Serous/genetics/pathology/drug therapy/metabolism ; Disease Models, Animal ; Humans ; CRISPR-Cas Systems ; Tumor Suppressor Protein p53/genetics/metabolism ; Mice, Knockout ; Cell Line, Tumor ; },
abstract = {Ovarian cancer is the sixth leading cause of cancer death among American women, with most fatalities attributable to tubo-ovarian high-grade serous carcinoma (HGSC). This malignancy usually develops resistance to conventional chemotherapy, underscoring the need for robust preclinical models to guide the development of novel therapies. Here, we introduce an HGSC mouse model generated via Ovgp1-driven Cre recombinase effecting CRISPR/Cas9-mediated deletion of Trp53, Rb1, and Nf1 tumor suppressors in mouse oviductal epithelium (m-sgPRN model). Cyclin-dependent kinase 12 (CDK12) inactivation-frequently observed in human HGSC-is associated with poorer outcomes, DNA damage accumulation (including tandem duplications), and increased tumor immunogenicity. In our system, coablation of Cdk12 (m-sgPRN;Cdk12KO) recapitulated hallmark features of HGSC, while accelerating tumor progression and reducing survival. In a conventional (Cre-lox-mediated) Trp53/Nf1/Rb1 triple knockout model with concurrent Cdk12 ablation (PRN;Cdk12KO mice), we observed T cell-rich immune infiltrates mirroring those seen clinically. We established both models as subcutaneous or intraperitoneal syngeneic allografts of CDK12-inactivated HGSC that exhibited sensitivity to immune checkpoint blockade. Furthermore, a CRISPR/Cas9 synthetic lethality screen in PRN;Cdk12KO-derived cell lines identified CDK13-an essential paralog of CDK12-as the most depleted candidate, confirming a previously reported synthetic lethal interaction. Pharmacologic CDK13/12 degradation (employing YJ1206) demonstrated enhanced efficacy in cell lines derived from both m-sgPRN;Cdk12KO and PRN;Cdk12KO models. Our results define CDK12 as a key tumor suppressor in tubo-ovarian HGSC and highlight CDK13 targeting as a promising therapeutic approach in CDK12-inactive disease. Additionally, we have established valuable in vivo resources to facilitate further investigation and drug development in this challenging malignancy.},
}
@article {pmid40503054,
year = {2025},
author = {Nawaz, A and Ariffin, NS and Wong, TW},
title = {Functionalized chitosan as nano-delivery platform for CRISPR-Cas9 in cancer treatment.},
journal = {Asian journal of pharmaceutical sciences},
volume = {20},
number = {3},
pages = {101041},
pmid = {40503054},
issn = {2221-285X},
abstract = {CRISPR-Cas system permanently deletes any harmful gene-of-interest to combat cancer growth. Chitosan (CS) is a potential cancer therapeutic that mediates via PI3K/Akt/mTOR, MAPK and NF-kβ signaling pathway modulation. CS and its covalent derivatives have been designed as nanocarrier of CRISPR-Cas9 alone (plasmid or ribonucleoprotein) or in combination with chemical drug for cancer treatment. The nanocarrier was functionalized with polyethylene glycol (PEG), targeting ligand, cell penetrating ligand and its inherent positive zeta potential to mitigate premature clearance and particulate aggregation, and promote cancer cell/nucleus targeting and permeabilization to enable CRISPR-Cas9 acting on the host DNA. Different physicochemical attributes are required for the CS-based nanocarrier to survive from the administration site, through the systemic circulation-extracellular matrix-mucus-mucosa axis, to the nucleus target. CRISPR-Cas9 delivery is met with heterogeneous uptake by the cancer cells. Choice of excipients such as targeting ligand and PEG may be inappropriate due to lacking overexpressed cancer receptor or availability of excessive metabolizing enzyme and immunoglobulin that defies the survival and action of these excipients rendering nanocarrier fails to reach the target site. Cancer omics analysis should be implied to select excipients which meet the pathophysiological needs, and chitosan nanocarrier with a "transformative physicochemical behavior" is essential to succeed CRISPR-Cas9 delivery.},
}
@article {pmid40501577,
year = {2025},
author = {Babajanyan, SG and Garushyants, SK and Wolf, YI and Koonin, EV},
title = {Evolution of antivirus defense in prokaryotes depending on the environmental virus prevalence and virome dynamics.},
journal = {bioRxiv : the preprint server for biology},
volume = {},
number = {},
pages = {},
doi = {10.1101/2025.05.27.656525},
pmid = {40501577},
issn = {2692-8205},
abstract = {UNLABELLED: Prokaryotes can acquire antivirus immunity via two fundamentally distinct types of processes: direct interaction with the virus as in CRISPR-Cas adaptive immunity systems and horizontal gene transfer (HGT) which is the main route of transmission of innate immunity systems. These routes of defense evolution are not mutually exclusive and can operate simultaneously, but empirical observations suggest that at least in some bacterial and archaeal species, one or the other route dominates the defense landscape. We hypothesized that the observed dichotomy stems from different life-history tradeoffs characteristic of these organisms. To test this hypothesis, we analyzed a mathematical model of a well-mixed prokaryote population under a stochastically changing viral prevalence. Optimization of the long-term population growth rate reveals two contrasting modes of defense evolution. In stable, predictable and fluctuating, unpredictable environments with a moderate viral prevalence, direct interaction with the virus and horizontal transfer of defense genes become the optimal routes of immunity acquisition, respectively. In the HGT-dominant mode, we observed a universal distribution of the fraction of microbes with different immune repertoires. Under very low virus prevalence, the cost of immunity exceeds the benefits such that the optimal state of a prokaryote is complete defense systems. By contrast, under very high virus prevalence, horizontal spread of defense systems dominates regardless of the stability of the virome. These findings might explain consistent but enigmatic patterns in the spread of antivirus defense systems among prokaryotes such as the ubiquity of adaptive immunity in hyperthermophiles contrasting their patchy distribution among mesophiles.<br><br>IMPORTANCE: The virus-host arms race is a major component of the evolutionary process in all organisms that drove the evolution of a broad variety of immune mechanisms. In the last few years, over 200 distinct antivirus defense systems have been discovered in prokaryotes. There are two major modes of immunity acquisition: innate immune systems spread through microbial populations via horizontal gene transfer (HGT) whereas adaptive-type immune systems acquire immunity via direct interaction with the virus. We developed a mathematical model to explore the short term evolution of prokaryotic immunity and show that in stable environments with predictable viral repertoires, adaptive-type immunity is the optimal defense strategy whereas in fluctuating environments with unpredictable virus composition, HGT dominates the immune landscape.},
}
@article {pmid40501453,
year = {2025},
author = {Kamoen, L and de Bruin, DA and Kralemann, LEM and Roos, K and Wildhagen, MMDA and van Schendel, R and Hooykaas, PJJ and de Pater, S and Tijsterman, M},
title = {Division of labor within polymerase theta in repair of CRISPR-induced DNA breaks in Arabidopsis thaliana.},
journal = {PNAS nexus},
volume = {4},
number = {6},
pages = {pgaf183},
pmid = {40501453},
issn = {2752-6542},
abstract = {To develop efficient strategies for precise mutagenesis in plants, it is crucial to characterize the mechanisms involved in the repair of CRISPR-induced double strand breaks (DSBs). Polymerase theta (Polθ)-mediated end joining (TMEJ) and classical nonhomologous end joining are key pathways that generate a wide array of mutations during DSB repair. To direct repair towards more predictable outcomes, we examined the impact of direct repeats flanking DSBs, which may trigger extended microhomology-mediated end joining (eMMEJ). Unexpectedly, we found that eMMEJ in Arabidopsis thaliana requires Polθ, in contrast to eMMEJ in animals. By reintroducing mutated versions of Polθ into Polθ-deficient plants we discovered that only the helicase activity of Polθ is needed for eMMEJ; we demonstrate that plants lacking Polθ's polymerase domain are incapable of TMEJ and are resistant to TMEJ-dependent T-DNA integration but still support extended microhomology-guided DSB repair at genomic sites with direct repeats. These findings reveal species-specific functionality of Polθ and point to functional divergence in TMEJ across species. Additionally, these insights provide new opportunities to direct targeted mutagenesis in plants toward single, predictable outcomes, paving the way for more efficient crop engineering. Classification: Biological, Health, and Medical Sciences.},
}
@article {pmid40500518,
year = {2025},
author = {Song, D and Xu, W and Zhuo, Y and Zhu, A and Long, F},
title = {Orthogonal CRISPR/Cas system facilitated dual-color fluorescence fiber-embedded optofluidic nano-biochip for parallel amplification-free on-site detection of bacterium and virus.},
journal = {Mikrochimica acta},
volume = {192},
number = {7},
pages = {417},
pmid = {40500518},
issn = {1436-5073},
support = {8242031//Natural Science Foundation of Beijing/ ; 2022YFF0609102//National Key R&amp;D Program of China/ ; },
mesh = {*CRISPR-Cas Systems ; *Escherichia coli O157/isolation &amp; purification/genetics ; *SARS-CoV-2/isolation &amp; purification/genetics ; Limit of Detection ; *Lab-On-A-Chip Devices ; COVID-19/diagnosis/virology ; Humans ; Optical Fibers ; Fluorescence ; Biosensing Techniques/methods ; },
abstract = {Bacterial and viral co-infections significantly exacerbate morbidity and mortality. Rapid, sensitive, and parallel detection of these pathogens remains a critical challenge. Here, an orthogonal CRISPR/Cas system facilitated dual-color fluorescence fiber-embedded optofluidic nano-biochip (CD-FOB) was fabricated. Leveraging the time-resolved effect, the CD-FOB achieved ultrasensitive parallel detection of Escherichia coli O157:H7 (E. coli O157:H7) and SARS-CoV-2 based on a multiple signal enhancement strategy, including the collateral cleavage activity of CRISPR/Cas, evanescent wave fluorescence enhancement, DNA-mediated signal amplification, and air-displacement fluorescence enhancement. Without the need for amplification, the CD-FOB system has a detection limit of 643 CFU/mL for E. coli O157:H7 and 3.48 copies/μL for SARS-CoV-2 within 50 min analysis time. To enable rapid on-site detection, a lyophilized CRISPR/Cas assay was prepared using stabilized freeze-dried reagents for detecting E. coli O157:H7 and SARS-CoV-2 in actual samples, achieving recoveries ranging from 70.5% to 200.5%. The unique combination of technical simplicity, multiplexing capability, and operational robustness positions CD-FOB as a versatile solution for combating current and future pathogen threats.},
}
@article {pmid40500390,
year = {2025},
author = {Chen, S and Chen, Q and You, X and Zhou, Z and Kong, N and Ambrosio, F and Cao, Y and Abdi, R and Tao, W},
title = {Using RNA therapeutics to promote healthy aging.},
journal = {Nature aging},
volume = {5},
number = {6},
pages = {968-983},
pmid = {40500390},
issn = {2662-8465},
mesh = {Humans ; *Healthy Aging/genetics ; Animals ; *Aging/genetics ; *RNA/therapeutic use ; RNA Interference ; Aptamers, Nucleotide/therapeutic use ; *Genetic Therapy/methods ; Neurodegenerative Diseases/therapy/genetics ; },
abstract = {Aging is characterized by a gradual decline of cellular and physiological functions over time and an increased risk of different diseases. RNA therapeutics constitute an emerging approach to target the molecular mechanisms of aging and age-related diseases via rational design and have several advantages over traditional drug therapies, including high specificity, low toxicity and the potential for rapid development and production. Here, we discuss the latest developments in RNA therapeutics designed to promote healthy aging, including RNA activation, messenger RNA therapy, RNA interference, antisense oligonucleotides, aptamers and CRISPR-Cas-mediated RNA editing. We also review the latest preclinical and clinical studies of RNA technology for treating age-related diseases, including neurodegenerative, cardiovascular and musculoskeletal diseases. Finally, we discuss the challenges of RNA technology aimed at supporting healthy aging. We anticipate that the fusion of RNA therapeutics and aging biology will have an important effect on the development of new medicines and maximization of their efficacy.},
}
@article {pmid40500074,
year = {2025},
author = {Lv, J and Yang, Y and Fabrick, JA and Wu, Y},
title = {Gene editing to enhance pesticide resistance in a beneficial predatory mite.},
journal = {Pesticide biochemistry and physiology},
volume = {212},
number = {},
pages = {106466},
doi = {10.1016/j.pestbp.2025.106466},
pmid = {40500074},
issn = {1095-9939},
mesh = {Animals ; *Gene Editing ; *Mites/genetics/drug effects ; CRISPR-Cas Systems ; Receptors, Nicotinic/genetics ; Drug Resistance/genetics ; Female ; Pest Control, Biological ; *Pesticides/pharmacology ; Acaricides/pharmacology ; Benzamides/pharmacology ; Predatory Behavior ; },
abstract = {Successful integrated pest management (IPM) often depends on a suite of control strategies that are compatible with one another. The predatory mite Neoseiulus californicus (Acari: Phytoseiidae) is a key biological control agent of spider mites (especially Tetranychus spp.) and other diminutive, yet important arthropod pests of agriculture. However, like many natural enemies, N. californicus is highly sensitive to chemical pesticides, limiting its overall effectiveness in the field. Here, we used CRISPR/Cas9 gene editing and Receptor-Mediated Ovary Transduction of Cargo (ReMOT Control) to create N. californicus harboring loss-of-function mutations in the nicotinic acetylcholine receptor α6 subunit (nAChRα6), the target of the pesticide, spinetoram. The resulting knockout strain (FZ-α6KO) exhibited a 23-fold increase in resistance to spinetoram compared to its parental strain. Inheritance of resistance to spinetoram in FZ-α6KO was autosomal, recessive, and tightly linked with the nAChRα6 gene. We demonstrate that by pairing gene editing with ReMOT Control, extremely small and fragile beneficial arthropods can be genetically manipulated. These results suggest that genetic modification to enhance pesticide resistance in beneficial predatory mites could improve the compatibility between the use of pesticides and biological control organisms.},
}
@article {pmid40500063,
year = {2025},
author = {Mocchetti, A and Nikoloudi, AA and Vontas, J and De Rouck, S and Van Leeuwen, T},
title = {CRISPR/Cas9 knock-out of nAChR α6 confers resistance to spinosyns in Frankliniella occidentalis and is associated with a higher fitness cost than target-site mutation G275E.},
journal = {Pesticide biochemistry and physiology},
volume = {212},
number = {},
pages = {106455},
doi = {10.1016/j.pestbp.2025.106455},
pmid = {40500063},
issn = {1095-9939},
mesh = {Animals ; *Receptors, Nicotinic/genetics ; *Insecticides/pharmacology ; *CRISPR-Cas Systems/genetics ; *Insecticide Resistance/genetics ; *Macrolides/pharmacology/toxicity ; *Thysanoptera/genetics/drug effects ; Drug Combinations ; Mutation ; Gene Knockout Techniques ; },
abstract = {Thrips are major agricultural pests globally and spinosyn insecticides like spinosad and spinetoram are commonly used for their control. However, numerous cases of resistance have emerged, often linked to mutations in the nicotinic acetylcholine receptor (nAChR) α6 subunit, the main molecular target of spinosyns. In this study, toxicological data for spinosad and spinetoram were obtained from a susceptible strain of Frankliniella occidentalis, as well as two field-collected resistant strains carrying the G275E resistance mutation. Notably, a new candidate resistance mutation never reported before, T202A, was identified in one of the field collected populations and its possible role in resistance is discussed. Further, CRISPR/Cas9-mediated knockout (KO) of α6 was performed in the susceptible strain to shed light on the phenotypic strength of this resistance mechanism previously observed in the field. The KO conferred complete insensitivity to spinosad and significant resistance to spinetoram, although higher doses of spinetoram remained lethal, suggesting potential interaction with a secondary target. Finally, in an experimental evolution approach, the α6 KO allele rapidly disappeared, indicating a substantial fitness cost. In contrast, G275E alleles persisted.},
}
@article {pmid40499559,
year = {2025},
author = {Vaz, M and Watson, KL and Moorehead, RA},
title = {Reduced JAG1 Expression Through miR-200 Overexpression or Crispr-Cas Mediated Knockout Impairs TNBC Growth and Metastasis.},
journal = {Molecular carcinogenesis},
volume = {},
number = {},
pages = {},
doi = {10.1002/mc.23937},
pmid = {40499559},
issn = {1098-2744},
support = {//This study was funded by a CIHR project grant PJT-162218 and a CIHR Priority grant PLL-192130/ ; },
abstract = {Studies from our lab demonstrated that increasing miR-200 expression in human triple negative breast cancer (TNBC) reduced tumor growth and metastasis In Vivo. In this study, we found that overexpression of miR-200s in TNBC cells significantly reduced the expression of JAG1. When JAG1 was knocked out in MDA-MB-231 cells proliferation and invasion were significantly reduced In Vitro. Moreover, loss of JAG1 inhibited mammary tumor growth and metastasis In Vivo. RNA sequencing revealed that loss of JAG1 altered the expression of genes associated with the ECM, angiogenesis, and EMT. These results imply that miR-200s may mediate some of their antitumor actions through reducing JAG1 expression and suggest that agents targeting JAG1 should be further evaluated as a therapeutic strategy for TNBC.},
}
@article {pmid40499557,
year = {2025},
author = {Kang, B and Lee, S and Ko, DH and Venkatesh, J and Kwon, JK and Kim, H and Kang, BC},
title = {Virus-induced systemic and heritable gene editing in pepper (Capsicum annuum L.).},
journal = {The Plant journal : for cell and molecular biology},
volume = {122},
number = {5},
pages = {e70257},
pmid = {40499557},
issn = {1365-313X},
support = {RS-2024-00322053//Rural Development Administration, Republic of Korea/ ; RS-2021-NR059217//the National Research Foundation of Korea/ ; },
mesh = {*Capsicum/genetics/virology ; *Gene Editing/methods ; Plants, Genetically Modified/genetics ; *Plant Viruses/genetics ; CRISPR-Cas Systems/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; Oxidoreductases/genetics ; },
abstract = {Genome editing using the CRISPR/Cas system enables rapid and efficient plant breeding by directly introducing desired traits into elite lines within a short time frame. However, challenges associated with conventional Agrobacterium tumefaciens-mediated transformation and regeneration have limited gene editing in pepper (Capsicum annuum L.). In this study, we applied and optimized a virus-induced gene editing (VIGE) system to overcome these limitations. We inoculated transgenic pepper seedlings already expressing Cas9 with vectors based on tobacco rattle virus 2 (TRV2) expressing single guide RNAs (sgRNAs) targeting Phytoene desaturase (PDS); shoots regenerated from inoculated cotyledons displayed photobleaching phenotypes. To promote sgRNA mobility and maintain its integrity, we modified the pTRV2-sgRNA vector by incorporating a self-cleaving hammerhead ribozyme (HH) sequence to produce an intact sgRNA fused to part of the mobile RNA of FLOWERING LOCUS T. Additionally, we tested alternative mobile elements, such as tRNA[Ile] and tRNA[Met]. Furthermore, we cultivated plants at the low temperature of 20°C following TRV inoculation to increase TRV persistence and spread. These optimizations, including vector modifications and cultivation conditions, resulted in a systemic editing efficiency of 36.3%, as evidenced by systemic leaves showing photobleaching phenotypes. We determined that 8.5% of progeny from plants inoculated with the pTRV-HH-CaPDS-sgRNA-FT construct were mutated at the CaPDS locus. In addition, we used our VIGE system to successfully edit FASCICULATE, producing mutants whose inflorescences showed a fasciculate phenotype. Direct inoculation with a TRV-based vector expressing a mobile sgRNA to bypass tissue culture, therefore, offers an effective tool for molecular studies and breeding in pepper.},
}
@article {pmid40499551,
year = {2025},
author = {Stadager, J and Bernardini, C and Hartmann, L and May, H and Wiepcke, J and Kuban, M and Najafova, Z and Johnsen, SA and Legewie, S and Traube, FR and Jude, J and Rathert, P},
title = {CRISPR GENome and epigenome engineering improves loss-of-function genetic-screening approaches.},
journal = {Cell reports methods},
volume = {5},
number = {6},
pages = {101078},
doi = {10.1016/j.crmeth.2025.101078},
pmid = {40499551},
issn = {2667-2375},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; Induced Pluripotent Stem Cells/metabolism/cytology ; *Epigenome/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; *Genetic Testing/methods ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Epithelial-Mesenchymal Transition/genetics ; *Loss of Function Mutation/genetics ; Gene Knockout Techniques ; Cell Differentiation/genetics ; Epigenome Editing ; },
abstract = {CRISPR-Cas9 technology has revolutionized genotype-to-phenotype assignments through large-scale loss-of-function (LOF) screens. However, limitations like editing inefficiencies and unperturbed genes cause significant noise in data collection. To address this, we introduce CRISPR gene and epigenome engineering (CRISPRgenee), which uses two specific single guide RNAs (sgRNAs) to simultaneously repress and cleave the target gene within the same cell, increasing LOF efficiencies and reproducibility. CRISPRgenee outperforms conventional CRISPR knockout (CRISPRko), CRISPR interference (CRISPRi), and CRISPRoff systems in suppressing challenging targets and regulators of cell proliferation. Additionally, it efficiently suppresses modulators of epithelial-to-mesenchymal transition (EMT) and impairs neuronal differentiation in a human induced pluripotent stem cell (iPSC) model. CRISPRgenee exhibits improved depletion efficiency, reduced sgRNA performance variance, and accelerated gene depletion compared to individual CRISPRi or CRISPRko screens, ensuring consistency in phenotypic effects and identifying more significant gene hits. By combining CRISPRko and CRISPRi, CRISPRgenee increases LOF rates without increasing genotoxic stress, facilitating library size reduction for advanced LOF screens.},
}
@article {pmid40499189,
year = {2025},
author = {Fernandes, PMB and Fernandes, AAR and Maurastoni, M and Rodrigues, SP},
title = {Lab Legends and Field Phantoms: The Tale of Virus-Resistant Plants.},
journal = {Annual review of virology},
volume = {},
number = {},
pages = {},
doi = {10.1146/annurev-virology-092623-101850},
pmid = {40499189},
issn = {2327-0578},
abstract = {Plant viruses present significant challenges to global agriculture, causing crop losses, threatening food security, and imposing economic burdens. Advances in biotechnology have revolutionized strategies to attack these threats, with genetically modified and genome-edited virus-resistant plants, developed using precision tools such as RNA interference and CRISPR/Cas technology, playing pivotal roles. Despite these breakthroughs, fragmented regulatory frameworks and divergent policies across regions including the European Union and the Global South hinder the global adoption of such innovations. Multifaceted approaches, including gene pyramiding, microbiome-based strategies, and pathogen-targeted defenses, show promise for enhancing plant resilience. This review explores the biological, regulatory, and ethical dimensions of deploying virus-resistant crops, emphasizing the need for harmonization of international regulation to maximize biotechnological benefits. By addressing these challenges, biotechnology can advance sustainable agriculture, secure food systems, and mitigate the effect of plant viral diseases.},
}
@article {pmid40499044,
year = {2025},
author = {Zhan, X and Jiang, Y and Li, Z and Hu, X and Lan, F and Ying, B and Wu, Y},
title = {Split DNA Tetrahedron-Mediated Spatiotemporal-Hierarchy CRISPR Cascade Integrated with Au@Pt Nanolabels and Artificial Intelligence for a Cervical Cancer MicroRNA Bioassay.},
journal = {ACS nano},
volume = {},
number = {},
pages = {},
doi = {10.1021/acsnano.5c01376},
pmid = {40499044},
issn = {1936-086X},
abstract = {The screening and monitoring of microRNAs as cancer molecular biomarkers is clinically significant, but traditional methods lack sufficient sensitivity, accuracy, and convenience. The CRISPR-colorimetric lateral flow assay (CLFA) integration offers a promising and efficient solution; however, cumbersome preamplification and poor quantification hinder clinical adoption. In this study, we developed a one-step isothermal CRISPR-Cas cascaded sensing system that is preamplification-free. At its core is a designed and selected split DNA tetrahedron activator, employing spatiotemporal-hierarchy mechanisms to precisely bidirectionally drive the kinetics of two Cas enzymes, accelerating the activation of Cas13a while delaying the initiation of Cas12a, to achieve optimal balance. This system enables ultrasensitive, single-step, single-tube, and rapid detection of a cervical cancer relative biomarker, microRNA-21, achieving a limit of detection of 38 aM with a broad linear range. The CRISPR system is further integrated with CLFA enhanced by ultrathin platinum-protected gold nanolabels (Au@Pt, also named Au@s-Pt), along with a smartphone equipped with dual convolutional neural network models (YOLO v5 and MobileNet v3), enabling more precise, rapid quantification of target miRNA. Using this integrated platform, miRNA-21 levels in cervical cancer and precancerous samples can be accurately quantified with approximately 30 min at low cost and without the need for large, sophisticated instruments, with results showing good concordance with quantitative real-time polymerase chain reaction. This platform provides an efficient, highly sensitive, user-friendly, and quantifiable point-of-care testing solution.},
}
@article {pmid40498336,
year = {2025},
author = {Patel, E and Das, P and Hazra, S and Sharma, M and Chhabra, G and Gill, BS and Sharma, S and Kaur, A and Singla, D and Sandhu, JS},
title = {Mutation in soybean Lox-2 PLAT/LH2 domain through CRISPR/Cas9 reduces seed lipoxygenase activity: responsible for undesirable flavour.},
journal = {Transgenic research},
volume = {34},
number = {1},
pages = {29},
pmid = {40498336},
issn = {1573-9368},
mesh = {*Glycine max/genetics/enzymology/growth &amp; development ; *Seeds/genetics/enzymology ; *CRISPR-Cas Systems/genetics ; *Lipoxygenase/genetics/metabolism ; Plants, Genetically Modified/genetics/growth &amp; development ; Mutation ; *Plant Proteins/genetics/metabolism ; },
abstract = {Soybean, a protein and oil rich legume is primarily used as livestock feed and to a lesser extent for human consumption due to undesirable flavour in the seeds caused by L-2 isozyme of lipoxygenase. Herein, soybean with reduced isozyme activity was developed through CRISPR/Cas9 targeted mutation in L-2 encoding Lox-2 gene. sgRNA designed from PLAT/LH2 domain in second exon of Lox-2 (Lox-2 E2) was validated by in vitro cleavage assay; inserted in CRISPR/Cas9 binary vector and used for genetic transformation of SL1074 cultivar hypocotyl segments. A total of 12 T0 putative plants were identified through PCR. Amongst these, four revealed mutation at the target sgRNA site by CEL1 assay and substitution of a base A with G six bp upstream of PAM converting lysine to glutamic acid at 119 position. T1 and T2 seeds derived from mutant T0-37 plant showed upto 25.49% reduction in isozyme activity as compared to SL1074. The base substitution was confirmed in T1 progeny; segregation analysis revealed homozygosity and heritability of mutation in T2 plants. The interaction between structural models of SL1074, mutant domains and negatively charged substrates revealed strong binding affinity of the substrates with positively charged lysine in SL1074 domain due to formation of two hydrogen bonds. On the contrary, weak binding of the substrates with negatively charged glutamic acid in mutant domain and absence of hydrogen bond explained reduction of isozyme activity in T2 seeds. The mutant soybean with reduced isozyme activity is an important source for introgressing the trait in plant breeding programs.},
}
@article {pmid40498069,
year = {2025},
author = {Chen, P and Li, X and Zhou, Q and Chen, J and Lu, L and Wang, P and Zhang, G and Sun, D and Huang, X and Liu, J and Wang, X},
title = {Configuration of adaptable template RNA architectures to unfold the editable space of a nuclease prime editor.},
journal = {Nucleic acids research},
volume = {53},
number = {11},
pages = {},
pmid = {40498069},
issn = {1362-4962},
support = {2021YFF1000700//National Key R&amp;D Program of China/ ; 32272848//National Natural Science Foundation of China/ ; CARS-39-03//China Agricultural Research System/ ; 2022GDTSLD-46//China Agricultural Research System/ ; },
mesh = {*Gene Editing/methods ; *RNA, Guide, CRISPR-Cas Systems/genetics/chemistry ; *RNA/chemistry/genetics ; Humans ; DNA Breaks, Double-Stranded ; CRISPR-Cas Systems ; },
abstract = {The nuclease prime editor (PEn) combines double-strand break (DSB) induction with reverse transcription for editing. Recently, high-activity PEn forms (e.g. uPEn) have been developed via the concomitant application of DNA repair regulator(s). While the standard uPEn introduces edits only downstream of the nuclease-induced DNA break, we seek innovative designs to enable upstream-directed editing by re-configuring guide/template RNAs to drive prime edits into the target strand (TS), instead of the conventional non-TS. We first devise a dual-RNA uPEn strategy by supplementing a cleavage-competent sgRNA with an accessory template RNA for modifying target strand (ActRNA:t). Characterization of the dual-RNA system allows us to next develop a bifunctional target strand-programming pegRNA (tsp-pegRNA). Both the dual- and single-RNA upstream-modifying uPEn forms (versions 3.1/3.2) successfully drive diverse types of accurate edits into a panel of locations refractory to the standard uPEn and the latest nickase PE. Moreover, we provide insights on the role of uPEn's helper module (i.e. i53) in driving TS prime edits. Additional co-administration of a DNA-dependent protein kinase inhibitor with uPEn3.2 leads to further optimization of editing purities. Together, these advances transform uPEn into a highly applicable tool with much-expanded editable space, and lay a strong foundation for future development of PEn/PE platforms.},
}
@article {pmid40498068,
year = {2025},
author = {Qiao, J and Zhang, J and Jiang, Q and Jin, S and He, R and Qiao, B and Liu, Y},
title = {Boosting CRISPR/Cas12a intrinsic RNA detection capability through pseudo hybrid DNA-RNA substrate design.},
journal = {Nucleic acids research},
volume = {53},
number = {11},
pages = {},
pmid = {40498068},
issn = {1362-4962},
support = {2022YFC2304304//National Key Research and Development Program of China/ ; 2023DJC136//Science and Technology Innovation Talent Plan of Hubei Province/ ; 2025AFB825//Natural Science Foundation of Hubei Province/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; Humans ; *CRISPR-Associated Proteins/metabolism/genetics ; MicroRNAs/genetics/analysis ; *DNA/chemistry/genetics ; *RNA/analysis/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; *Endodeoxyribonucleases/metabolism/genetics ; *Bacterial Proteins/metabolism/genetics ; Nucleic Acid Hybridization ; Limit of Detection ; },
abstract = {The CRISPR/Cas12a [clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR-associated protein 12a] system is known for its intrinsic RNA-guided trans-cleavage activity; however, its RNA detection sensitivity is limited, with conventional methods typically achieving detection limits in the nanomolar range. Here, we report the development of a "pseudo hybrid DNA-RNA" (PHD) assay that significantly enhances the RNA detection capability of Cas12a. The PHD assay achieves a striking detection limit of 7.7 pM using single CRISPR RNA (crRNA) and 33.8 fM using pooled crRNAs. Importantly, this assay exhibits ultra-high specificity, capable of distinguishing mutated RNA target sequences at the protospacer adjacent motif (PAM)-distal region. It can also detect ultrashort RNA sequences as short as 6-8 nt and long RNAs with complex secondary structures. Additionally, the PHD assay enables PAM-free attomolar-level DNA detection. We further demonstrate the practical utility of the PHD assay by successfully detecting miR-155 biomarkers and human pappilloma virus 16 DNA in clinical samples. We anticipate that the design principles established in this study can be extended to other CRISPR/Cas enzymes, thereby accelerating the development of powerful nucleic acid testing tools for various applications.},
}
@article {pmid40497031,
year = {2025},
author = {Galli, C},
title = {Current Techniques of Gene Editing in Pigs for Xenotransplantation.},
journal = {Transplant international : official journal of the European Society for Organ Transplantation},
volume = {38},
number = {},
pages = {13807},
pmid = {40497031},
issn = {1432-2277},
mesh = {Animals ; *Transplantation, Heterologous/methods ; *Gene Editing/methods ; Swine/genetics ; Humans ; CRISPR-Cas Systems ; Animals, Genetically Modified ; Nuclear Transfer Techniques ; Endogenous Retroviruses ; },
abstract = {Shortage of human organs for transplantation has created a demand for alternative solutions of which xenotransplantation is amongst the most promising one in the short term. However, the immune reaction following transplantation of a pig organ is greater than the one elicited during allotransplantation. Genetic engineering of the pig is required so that pig organs or tissues are made less immunogenic to humans by eliminating some antigens and by expressing human proteins that can reduce the damage by the host immune system. To generate founder animals with the desired mutations genetic engineering of somatic cells with multiplexed mutations combined with somatic cell nuclear transfer (SCNT) is the best solution with the technology available today. Safety concerns include potential zoonosis, primarily porcine endogenous retroviruses (PERVs). Ethical considerations might arise from the use animals involved in research. Genome editing techniques based CRISPR-Cas9, have greatly facilitated the modification of pig's genome to address coagulation and inflammation issues, to mention just a few, arising after the pig organ is transplanted into a human. However, further research is needed to ensure safety and efficacy of the genome edits introduced in the pig genome are compatible with the health and welfare of the pigs.},
}
@article {pmid40496602,
year = {2025},
author = {Jangra, S and Seal, DR and Ghosh, A},
title = {Biotechnological advancements for sustainable management of thrips.},
journal = {3 Biotech},
volume = {15},
number = {7},
pages = {204},
pmid = {40496602},
issn = {2190-572X},
abstract = {Thrips are minute, slender, polyphagous, thysanopteran insect pests that cause huge economic losses to crops by directly feeding on the phloem sap and transmitting several plant viruses. Thrips have emerged as a major threat to global agriculture and food security. Current management options mainly rely on chemical insecticides; however, thrips have evolved resistance to most commonly used insecticides, making management extremely difficult. The availability of host-plant resistance is limited in the case of thrips. Biotechnological approaches such as genetic engineering, RNA interference (RNAi), antisense oligonucleotides (ASOs), artificial microRNA (amiRNA), and genome editing have paved the way for the development of environmentally sustainable thrips management options. However, the adoption of these biotechnological approaches needs further refinement and validation. Transgenic plants with anti-herbivory proteins hold promise to be successful in managing thrips. Spray-induced gene silencing (SIGS) and gene editing would be novel alternatives to hazardous pesticides. This review discusses the progress made towards using modern biotechnological interventions in functional genomics, emphasizing their application in sustainable thrips management.},
}
@article {pmid40494869,
year = {2025},
author = {Basu, M and Xiao, JF and Kailasam Mani, SK and Qu, F and Lin, Y and Duex, J and Ye, H and Neang, V and Theodorescu, D},
title = {Genes driving three-dimensional growth of immortalized cells and cancer.},
journal = {Cell death &amp; disease},
volume = {16},
number = {1},
pages = {442},
pmid = {40494869},
issn = {2041-4889},
support = {R01 CA075115/CA/NCI NIH HHS/United States ; R29 CA075115/CA/NCI NIH HHS/United States ; CA075115//U.S. Department of Health &amp; Human Services | NIH | National Cancer Institute (NCI)/ ; },
mesh = {Humans ; Animals ; *Urinary Bladder Neoplasms/genetics/pathology ; Mice ; Cell Proliferation/genetics ; Mitogen-Activated Protein Kinase 1/genetics/metabolism/antagonists &amp; inhibitors ; Cell Line, Tumor ; Cell Transformation, Neoplastic/genetics ; CRISPR-Cas Systems ; },
abstract = {Unchecked growth in three-dimensions (3D) in culture is a key feature of immortalized cells on the path to malignant transformation and hence a potential target phenotype for prevention. Also, expression of genes driving this process, but not that of 2D growth, would likely be more specific to cancer development and their inhibition would be less toxic to normal cells, many of which can grow in 2D but rarely in 3D culture. To define such genes, we compared CRISPR depletion screens performed in HBLAK, a spontaneously immortalized, non-tumorigenic human urothelial cell line, grown in 2D to those in 3D. Using the CRISPR Bassik DTKP (drug target kinase phosphatase) deletion library targeting 2,333 genes, we identified 85 genes which were specifically lost in 3D cultures. Prioritizing hits to those associated with bladder cancer in patients provided us with a set of 11 genes. Only one gene, MAPK1 remained relevant if a human pan-cancer criteria was applied. Single gene in vitro validation confirmed that MAPK1 inhibition was specific to 3D growth. We also found that MAPK1 depletion led to significant growth reductions in human tumor xenografts in vivo. Inhibition of MAPK1 by Ulixertinib, an orally active MAPK1 inhibitor, led to human bladder cancer growth inhibition in both 3D in vitro and in vivo models. In summary, screening for genes specifically driving 3D growth in immortalized cells may provide targets for both prevention and early therapy in bladder and other cancers while potentially limiting therapeutic toxicity.},
}
@article {pmid40494841,
year = {2025},
author = {Liu, H and Zhang, M and Sun, L and Peng, Y and Sun, Y and Fan, Y and Li, H and Liu, D and Lu, H},
title = {Synergistic optimization enhancing the precision and efficiency of cytosine base editors in poplar.},
journal = {Communications biology},
volume = {8},
number = {1},
pages = {904},
pmid = {40494841},
issn = {2399-3642},
mesh = {*Populus/genetics/drug effects ; *Gene Editing/methods ; *Cytosine/metabolism ; *CRISPR-Cas Systems ; Genome, Plant ; Plants, Genetically Modified/genetics ; },
abstract = {CRISPR/Cas9 genome editing technology, particularly cytosine base editing (CBE) systems, emerges as a powerful tool for precise genomic modification in plants, offering transformative applications across agricultural and forestry research and breeding programs. However, current CBE systems in poplar exhibit low efficiency and imprecise base substitutions, and optimization of base editing systems specifically for poplar remains a significant challenge. To address these limitations, we engineer a high-efficiency poplar CBE system (hyPopCBE) by integrating the MS2-UGI system, fusing Rad51 DNA-binding domain, and modifying the nuclear localization signal. Through stepwise optimization, we develop hyPopCBE-V4, which exhibits a synergistic effect in woody plants. Compared to the original hyPopCBE-V1, hyPopCBE-V4 improves C to T editing efficiency while reducing byproducts and exhibiting a narrower editing window. The proportion of plants with clean C to T edits (without byproducts) increases from 20.93% to 40.48%, and the efficiency of clean homozygous C to T editing rises from 4.65% to 21.43%. Using hyPopCBE-V1 and its variants, we induce Pro197Leu mutation in the herbicide target gene PagALS. Poplar lines with edits in all four PagALS homologues exhibit high resistance to tribenuron and nicosulfuron. This study employs a multi-component synergistic optimization strategy that specifically enhances the efficiency and precision of CBE editing in poplar while improving synchronous editing of alleles. Through editing the herbicide resistance gene PagALS, we obtain the herbicide-resistant poplar germplasm. Our research provides a more precise and efficient CBE tool for genetic modification in poplar that can also be applied to other forestry species, demonstrating its potential for advancing forestry research and breeding programs.},
}
@article {pmid40493278,
year = {2025},
author = {Kaur, J and Viswanathan P, A and Bari, VK},
title = {CRISPR/Cas9-mediated editing of jasmonic acid pathways to enhance biotic &amp; abiotic stress tolerance: An overview &amp; prospects.},
journal = {Functional &amp; integrative genomics},
volume = {25},
number = {1},
pages = {125},
pmid = {40493278},
issn = {1438-7948},
mesh = {*Cyclopentanes/metabolism ; *Oxylipins/metabolism ; *Gene Editing/methods ; *CRISPR-Cas Systems ; *Stress, Physiological/genetics ; Crops, Agricultural/genetics ; Plant Growth Regulators/metabolism ; Signal Transduction ; },
abstract = {Food security is becoming increasingly important as the world's population grows, and the likelihood that climate change could impair agricultural supply complicates matters further. However, plants actively suppress growth as an adaptation strategy to enhance survival under stress conditions. Phytohormone jasmonates (JAs) regulate various physiological processes, including plant growth, development, and senescence. Plant biotic and abiotic stress responses cause dynamic shifts in the metabolism and signaling of JAs, suggesting that JAs response impacts plant development and resistance to various stresses. The JAs-associated responses depend on core components of JAs -signaling, including the transcriptional repressors protein JAZ and the transcription factor MYC2. While traditional breeding has greatly benefited the world, this approach has several disadvantages, such as the emergence of undesirable traits and species barriers. Genome editing technology has revolutionized plant biology research and has significant ramifications for agriculture and global food security, particularly in light of climate change and population growth. CRISPR/Cas9 and its derivative tools have been used for genome editing in numerous crops to improve or alter desired plant phenotypes. This review summarizes JA's role in plant stress and defense and how CRISPR/Cas9-editing technology modifies plant JA's responses, especially against biotic and abiotic stress.},
}
@article {pmid40493189,
year = {2025},
author = {Gorelik, A and Paulo, JA and Schroeter, CB and Lad, M and Shurr, A and Mastrokalou, C and Siddiqi, S and Suyari, O and Brognard, J and Walter, D and Matthews, J and Palmer, TM and Gygi, SP and Ahel, I},
title = {CRISPR screens and quantitative proteomics reveal remodeling of the aryl hydrocarbon receptor-driven proteome through PARP7 activity.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {122},
number = {24},
pages = {e2424985122},
doi = {10.1073/pnas.2424985122},
pmid = {40493189},
issn = {1091-6490},
support = {224095/Z/21/Z//Wellcome Trust (WT)/ ; 210634 223107 302632//Wellcome Trust (WT)/ ; BB/R007195/1 BB/W016613/1//UKRI | Biotechnology and Biological Sciences Research Council (BBSRC)/ ; C35050/A22284//Cancer Research UK (CRUK)/ ; GM67945//HHS | NIH (NIH)/ ; GM132129//HHS | NIH (NIH)/ ; },
mesh = {*Receptors, Aryl Hydrocarbon/metabolism/genetics ; Humans ; *Proteome/metabolism/genetics ; *Poly(ADP-ribose) Polymerases/metabolism/genetics ; Proteomics/methods ; Suppressor of Cytokine Signaling 3 Protein/metabolism/genetics ; Cell Line, Tumor ; Signal Transduction ; CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; Lung Neoplasms/metabolism/genetics ; Nucleoside Transport Proteins ; Basic Helix-Loop-Helix Transcription Factors ; },
abstract = {PARP7 is an enzyme that uses donor substrate NAD[+] to attach a single ADP-ribose moiety onto proteins related to immunity, transcription, and cell growth and motility. Despite the importance of PARP7 in these processes, PARP7 signaling networks remain underresearched. Here, we used genome-wide CRISPR screens and multiplex quantitative proteomics in distinct lung cancer cell lines treated with a PARP7 inhibitor to better understand PARP7 molecular functions. We find that manipulating the aryl hydrocarbon receptor (AHR) transcriptional activity mediates PARP7 inhibitor sensitivity and triggers robust changes to the AHR-controlled proteome (AHR-ome). One of the striking features of such AHR-ome remodeling was the downregulation of filamins A and B concurrent with the induction of the corresponding E3 ubiquitin ligase ASB2. We also show that suppressor of cytokine signaling 3 (SOCS3) crosstalks to AHR. Inhibition of PARP7 in SOCS3 knockout cells leads to reduced viability compared to wild-type cells treated with a PARP7 inhibitor. Our results reveal signaling interplay between PARP7, AHR, and SOCS3 and establish an invaluable resource to study the role of PARP7 in the regulation of AHR signaling and innate immunity through its ADP-ribosyl transferase activity.},
}
@article {pmid40491435,
year = {2025},
author = {Santarossa, BA and Mariani, &#xc9; and Corrêa, ADP and Costa, FC and Taylor, MC and Kelly, JM and Elias, MC and Calderano, SG},
title = {Stage-specific MCM protein expression in Trypanosoma cruzi: insights into metacyclogenesis and G1 arrested epimastigotes.},
journal = {Frontiers in cellular and infection microbiology},
volume = {15},
number = {},
pages = {1584812},
pmid = {40491435},
issn = {2235-2988},
mesh = {*Trypanosoma cruzi/growth &amp; development/genetics/metabolism ; *Protozoan Proteins/genetics/metabolism ; *Life Cycle Stages ; *Minichromosome Maintenance Proteins/genetics/metabolism ; CRISPR-Cas Systems ; *G1 Phase Cell Cycle Checkpoints ; },
abstract = {Trypanosoma cruzi is a protozoan parasite that is the etiological agent of Chagas disease, which is endemic to Latin America with reported cases in non-endemic regions such as Europe, Asia, and Oceania due to migration. During its lifecycle, T. cruzi alternates between replicative and non-replicative infective lifeforms. Metacyclogenesis is the most studied transition of the T. cruzi life cycle, where replicative epimastigotes differentiate into infective metacyclic trypomastigotes inside the gut of the triatomine vector. This early-branching organism expresses a divergent pre-replication complex (pre-RC) where the only conserved component is the MCM2-7 protein family. Given the role of pre-RC components in cell cycle regulation, we investigated whether MCM expression and location could be involved in proliferation control in epimastigotes and during metacyclogenesis. Using CRISPR/Cas9, we tagged MCM subunits and tracked their expression and subcellular localization. Our findings reveal that MCM subunits are consistently expressed and localized to the nucleus throughout the epimastigote cell cycle, including in G1/G0-arrested cells. However, MCM subunits are degraded during metacyclogenesis as cells enter the G0 state, marking the transition to replication arrest. Therefore, epimastigotes arrested in G1/G0 can either maintain MCM complex expression and resume the cell cycle when conditions become favorable, or they can undergo metacyclogenesis, exiting the cell cycle and entering a G0 state, where MCM subunits are degraded as part of the replication repression mechanism.},
}
@article {pmid40491004,
year = {2025},
author = {Capelli, L and Marzari, S and Spezzani, E and Bertucci, A},
title = {Synthetic CRISPR Networks Driven by Transcription Factors via Structure-Switching DNA Translators.},
journal = {Journal of the American Chemical Society},
volume = {147},
number = {24},
pages = {21184-21193},
doi = {10.1021/jacs.5c06913},
pmid = {40491004},
issn = {1520-5126},
mesh = {*CRISPR-Cas Systems ; *Transcription Factors/metabolism/chemistry ; *DNA/chemistry/metabolism/genetics ; Synthetic Biology ; CRISPR-Associated Proteins/metabolism/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats ; Gene Editing ; },
abstract = {CRISPR-Cas systems have advanced many domains in life sciences, enabling diverse applications in gene editing, diagnostics, and biosensing. Here, we introduce a platform that leverages transcription factors (TFs) to regulate CRISPR-Cas12a trans-cleavage activity via engineered DNA translators. These dynamic DNA structures respond to TF binding by switching conformations, modulating Cas12a activity. Using TATA-binding protein and Myc-Max as TF models, we optimized DNA translators for precise and tunable control with rapid response kinetics. We demonstrated the platform's specificity and versatility by integrating TF-induced regulation into synthetic biology networks, including the activation of a fluorogenic RNA aptamer (Mango III) and the creation of an artificial multimolecular communication pathway between Cas12a and Cas13a. This work establishes TFs as effective regulators of CRISPR-Cas systems, enabling novel protein-nucleic acid communication channels, showing potential for novel synthetic biology applications.},
}
@article {pmid40490981,
year = {2025},
author = {Zheng, Y and Du, F and Hang, Y and Ma, W and Yang, G and Wu, N and Sun, X},
title = {Recent Advances in Genome Base Editing Technology and Its Applications in Industrial Microorganism.},
journal = {Biotechnology journal},
volume = {20},
number = {6},
pages = {e70052},
doi = {10.1002/biot.70052},
pmid = {40490981},
issn = {1860-7314},
support = {2023YFA0914400//National Key Research and Development Program of China/ ; BK20230059//Natural Science Foundation of Jiangsu Province/ ; 32422068//National Natural Science Foundation of China/ ; 22378209//National Natural Science Foundation of China/ ; 22038007//National Natural Science Foundation of China/ ; },
mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; *Industrial Microbiology/methods ; Metabolic Engineering ; Synthetic Biology ; Saccharomyces cerevisiae/genetics ; },
abstract = {Base editing technology is a novel gene-editing approach derived from the CRISPR/Cas9 system, enabling precise and efficient base conversion. Due to operational simplicity, strong target specificity, high editing efficiency, and minimal editing byproducts, base editing has been widely applied in gene therapy, crop breeding, construction of model organisms, and microbial metabolic engineering. In this review, we systematically summarize the development history and recent advancements of several major base editors, including cytosine base editors (CBEs), adenine base editors (ABEs), CRISPR-free base editors, C-to-G base editors (CGBEs), glycosylase base editors (GBEs), and IscB-derived base editors. Furthermore, we comprehensively summarize optimization strategies for base editors and its application in constructing efficient industrial microorganism, such as Bacillus subtilis, Corynebacterium glutamicum, Saccharomyces cerevisiae, Yarrowia lipolytica, and Aspergillus niger. This review aims to facilitate the broader application of base editing technologies in synthetic biology and accelerate their translational potential.},
}
@article {pmid40490955,
year = {2025},
author = {Ruan, S and Wang, A and Zou, H and Lin, Y and Ye, L and Liang, S},
title = {Synthetic Genetic Circuits Enabled in Komagataella phaffii Through T7 RNAP/CRISPRa System.},
journal = {Biotechnology journal},
volume = {20},
number = {6},
pages = {e70036},
doi = {10.1002/biot.70036},
pmid = {40490955},
issn = {1860-7314},
support = {2021YFC2104000//National Key Research and Development Program/ ; 32272276//National Natural Science Foundation of China/ ; },
mesh = {*Gene Regulatory Networks/genetics ; *DNA-Directed RNA Polymerases/genetics/metabolism ; *Synthetic Biology/methods ; *Saccharomycetales/genetics/metabolism ; *CRISPR-Cas Systems/genetics ; *Viral Proteins/genetics/metabolism ; Promoter Regions, Genetic ; RNA, Guide, CRISPR-Cas Systems/genetics ; },
abstract = {The CRISPR activation (CRISPRa) transcriptional system has become a powerful synthetic biology tool for the regulation of endogenous gene expression, allowing for precise fine-tuning of target genes through the simple modification of sgRNA sequences. In this study, we demonstrate that sgRNAs can be effectively expressed using the T7 transcription system. The insertion of tRNA sequences between PT7 and sgRNAs significantly enhances the efficiency of transcriptional activation. Furthermore, the design of PT7-tRNA-sgRNA arrays facilitates the multiplexed activation of genes. sgRNA expression was regulated by the Tet-on induction system, split-T7 system, and RNA cleavage processing by HH-HDV, resulting in the creation of a Boolean logic gene circuit capable of performing both AND and OR logic operations. Finally, we developed a UPR self-responsive system by utilizing endogenous promoters that are responsive to UPR signals to control the expression of T7 RNAP. This system dynamically regulates the expression of the endogenous HAC1 transcription factor, thus enhancing the secretion of heterologous proteins. The findings from this study highlight the potential of utilizing the T7 transcription system for the construction of genetic circuits, providing a practical toolkit for gene regulation in the industrial Komagataella phaffii strain.},
}
@article {pmid40490752,
year = {2025},
author = {Masson, JD and Taglietti, V and Ruby, F and Ono, H and Mouri, N and Jorge, A and Guillaud, L and Tiret, L and Relaix, F},
title = {Extensive striated muscle damage in a rat model of Duchenne muscular dystrophy with Dmd exons 10-17 duplication.},
journal = {Skeletal muscle},
volume = {15},
number = {1},
pages = {16},
pmid = {40490752},
issn = {2044-5040},
support = {19507, 22946//AFM-T&#xe9;l&#xe9;thon/ ; EQU20200301021//Fondation pour la Recherche M&#xe9;dicale/ ; 101080690//European MAGIC Project consortium/ ; },
mesh = {Animals ; *Muscular Dystrophy, Duchenne/genetics/pathology ; Exons ; Disease Models, Animal ; Rats ; *Dystrophin/genetics ; Rats, Sprague-Dawley ; *Gene Duplication ; Male ; *Muscle, Striated/pathology ; CRISPR-Cas Systems ; Gene Editing ; },
abstract = {BACKGROUND: Duchenne muscular dystrophy (DMD) mainly affects young boys with out-of-frame mutations in the DMD gene, leading to dystrophin deficiency. This loss disrupts the assembly of the sarcolemmal dystrophin-associated glycoprotein complex, resulting in membrane fragility and damage during muscle contraction-relaxation cycles. Consequently, patients experience progressive muscle weakness, loss of ambulation and cardiorespiratory failure. Gene therapy represents one of the most promising therapeutic approaches, requiring rigorous preclinical validation of candidate strategies. While several preclinical models of dystrophin deficiency mimic point mutations or exon deletions, no existing rat model accurately replicates DMD gene duplications, which account for approximately 10% of DMD cases.<br><br>METHODS: Using CRISPR/Cas9 genome editing, we generated a&#x2009;~&#x2009;125 kbp duplication encompassing exons 10-17 of the Dmd gene in Sprague Dawley rats. To characterise disease progression in these rats, we assessed biochemical, histological and functional biomarkers at 6 and 10 months of age, comparing them to their healthy littermates.<br><br>RESULTS: We established the R-DMDdup10-17 line. The microstructure of limb, diaphragm and cardiac muscles of R-DMDdup10-17 (DMD) rats exhibited dystrophic changes at 6 and 10 months, including loss of myofibres and fibrosis. These alterations led to a significant body mass reduction, muscle weakness (including diaphragm deficiency) and cardiac electrical defects. Premature lethality was observed between 10 and 13 months.<br><br>CONCLUSION: Duplication of the Dmd genomic region encompassing exons 10 to 17 in rats results in dystrophin deficiency, severe striated muscle dystrophy, and premature death. The R-DMDdup10-17 line represents the first reported genetic model of a severe and early lethal duplication variant in the Dmd gene. It provides a critical tool for assessing targeted gene therapies aimed to correct such mutations.},
}
@article {pmid40489621,
year = {2025},
author = {Zheng, X and Wu, S and Qiu, J and Li, A and Li, L and Yan, G and Li, M and Meng, F and Zhang, K and Lin, S},
title = {Induction of a neurotoxin in diatoms by iron limitation via cysteine synthase.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {122},
number = {24},
pages = {e2424843122},
doi = {10.1073/pnas.2424843122},
pmid = {40489621},
issn = {1091-6490},
support = {U2106205//MOST | NSFC | National Natural Science Foundation of China-Shandong Joint Fund (-)/ ; tstp20231216//Special Foundation for Taishan Scholar of Shandong Province/ ; },
mesh = {*Iron/metabolism ; *Diatoms/metabolism/genetics ; *Neurotoxins/biosynthesis/metabolism ; *Amino Acids, Diamino/biosynthesis/metabolism ; *Cysteine Synthase/metabolism/genetics ; Humans ; Cyanobacteria Toxins ; CRISPR-Cas Systems ; },
abstract = {The β-N-methylamino-L-alanine (BMAA) is an emerging neurotoxin associated with human neurodegenerative diseases such as Alzheimer's disease. Here, we report the prevalence of BMAA synthesis in protein forms by marine diatoms and reconstruct its tentative biosynthesis pathway. Remarkably, the BMAA production is strongly induced by iron limitation. Transcriptomic analyses suggest that cysteine synthase (CysK) is involved in BMAA synthesis. This is verified as CRISPR/Cas9-based CysK knockout abolished BMAA production and addition of the recombinant CysK to the mutant restored BMAA synthesis. As diatoms are the most abundant primary producers in ocean, the prevalence of BMAA in diatoms has significant public health implications. The biosynthesis pathway provides biomarkers for further investigation of BMAA production in marine diatoms and insights for understanding the pathological mechanism for human neurodegenerative diseases.},
}
@article {pmid40489409,
year = {2025},
author = {Yuan, Y and Jiang, Z and Zeng, Y and Tang, J and Luo, J and Xie, C and Gong, Y},
title = {Genome-Wide CRISPR Screen for Unveiling Radiosensitive and Radioresistant Genes.},
journal = {Journal of visualized experiments : JoVE},
volume = {},
number = {219},
pages = {},
doi = {10.3791/67982},
pmid = {40489409},
issn = {1940-087X},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Radiation Tolerance/genetics ; Lung Neoplasms/genetics/radiotherapy ; Gene Editing/methods ; Cell Line, Tumor ; },
abstract = {The CRISPR-Cas9 system has been harnessed and repurposed into a powerful genome editing tool. By leveraging this technology, researchers can precisely cut, paste, and even rewrite DNA sequences within living cells. Nevertheless, the application of CRISPR screen technology goes far beyond mere experimentation. It serves as a pivotal tool in the fight against genetic diseases, systematically dissecting complex genetic landscapes, empowering researchers to unravel the molecular mechanisms underlying biological phenomena, and enabling scientists to identify and target the root causes of illnesses such as cancer, cystic fibrosis, and sickle cell anemia. Among all, cancer poses a formidable challenge for medicine, spurring eradication efforts. Radiotherapy, as a traditional treatment, yields results but has limitations. It eradicates cancer cells but also damages healthy tissues, causing adverse effects that reduce quality of life. Additionally, not all cancer cells respond to radiotherapy, and some may develop resistance, worsening the condition. To address this, a comprehensive whole-genome CRISPR screen technology is introduced, as it enables the efficient identification of radiosensitive and radioresistant genes, thereby advancing the field of cancer research and treatment. A genome-wide CRISPR screen was conducted in lung adenocarcinoma cells exposed to irradiation following the described protocol, through which both radioresistance- and radiosensitivity-associated genes were identified.},
}
@article {pmid40487915,
year = {2025},
author = {Wu, X and Liu, Y and He, Z and Zhou, X and Liesack, W and Peng, J},
title = {Coevolution and cross-infection patterns between viruses and their host methanogens in paddy soils.},
journal = {ISME communications},
volume = {5},
number = {1},
pages = {ycaf088},
pmid = {40487915},
issn = {2730-6151},
abstract = {Methanogens play a critical role in global methane (CH4) emissions from rice paddy ecosystems. Through the integration of metagenomic analysis and meta-analysis, we constructed a CRISPR spacer database comprising 14 475 spacers derived from 351 methanogenic genomes. This enabled the identification of viruses targeting key methanogenic families prevalent in rice paddies, including Methanosarcinaceae, Methanotrichaceae, Methanobacteriaceae, Methanocellaceae, and Methanomassiliicoccaceae. We identified 419 virus-host linkages involving 56 methanogenic host species and 189 viruses, spanning the families Straboviridae, Salasmaviridae, Kyanoviridae, Herelleviridae, and Demerecviridae, along with 126 unclassified viral entities. These findings highlight a virome composition that is markedly distinct from those observed in gut environments. Cross-infection patterns were supported by the presence of specific viruses predicted to infect multiple closely related methanogenic species. Evidence for potential virus-host coevolution was observed in 24 viruses encoding anti-CRISPR proteins, likely facilitating evasion of host CRISPR-mediated immunity. Collectively, this study reveals a complex and dynamic network of virus-host interactions shaping methanogen communities in rice paddy ecosystems.},
}
@article {pmid40483517,
year = {2025},
author = {Takahashi, G and Maeda, M and Shinozaki, K and Harada, G and Ito, S and Miyaoka, Y},
title = {High-throughput robotic isolation of human iPS cell clones reveals frequent homozygous induction of identical genetic manipulations by CRISPR-Cas9.},
journal = {Stem cell research &amp; therapy},
volume = {16},
number = {1},
pages = {295},
pmid = {40483517},
issn = {1757-6512},
support = {20K21409//Japan Society for the Promotion of Science/ ; 20H03442//Japan Society for the Promotion of Science/ ; 24K02028//Japan Society for the Promotion of Science/ ; 18K15054//Japan Society for the Promotion of Science/ ; 22K15386//Japan Society for the Promotion of Science/ ; 23jm0610092h0001//Japan Agency for Medical Research and Development (AMED)/ ; Takeda Science Foundation//Takeda Science Foundation/ ; Sumitomo Foundation//Sumitomo Foundation/ ; Ichiro Kanehara Foundation//Ichiro Kanehara Foundation/ ; },
mesh = {Humans ; *Induced Pluripotent Stem Cells/metabolism/cytology ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; Homozygote ; Clone Cells/cytology ; *Robotics ; },
abstract = {BACKGROUND: Genome editing in human iPS cells is a powerful approach in regenerative medicine. CRISPR-Cas9 is the most common genome editing tool, but it often induces byproduct insertions and deletions in addition to the desired edits. Therefore, genome editing of iPS cells produces diverse genotypes. Existing assays mostly analyze genome editing results in cell populations, but not in single cells. However, systematic profiling of genome editing outcomes in single iPS cells was lacking. Due to the high mortality of human iPS cells as isolated single cells, it has been difficult to analyze genome-edited iPS cell clones in a high-throughput manner.<br><br>METHODS: In this study, we developed a method for high-throughput iPS cell clone isolation based on the precise robotic picking of cell clumps derived from single cells grown in extracellular matrices. We first introduced point mutations into human iPS cell pools by CRISPR-Cas9. These genome-edited human iPS cells were dissociated and cultured as single cells in extracellular matrices to form cell clumps, which were then isolated using a cell-handling robot to establish genome-edited human iPS cell clones. Genome editing outcomes in these clones were analyzed by amplicon sequencing to determine the genotypes of individual iPS cell clones. We identified and distinguished the sequences of different insertions and deletions induced by CRISPR-Cas9 while determining their genotypes. We also cryopreserved the established iPS cell clones and recovered them after determining their genotypes.<br><br>RESULTS: We analyzed over 1,000 genome-edited iPS cell clones and found that homozygous editing was much more frequent than heterozygous editing. We also observed frequent homozygous induction of identical genetic manipulations, including insertions and deletions, such as 1-bp insertions and 8-bp deletions. Moreover, we successfully cryopreserved and then recovered genome-edited iPS cell clones, demonstrating that our cell-handling robot-based method is valuable in establishing genome-edited iPS cell clones.<br><br>CONCLUSIONS: This study revealed a previously unknown property of genome editing in human iPS cells that identical sequence manipulations tend to be induced in both copies of the target sequence in individual cells. Our new cloning method and findings will facilitate the application of genome editing to human iPS cells.},
}
@article {pmid40481308,
year = {2025},
author = {Du, W and Zhao, L and Diao, K and Zheng, Y and Yang, Q and Zhu, Z and Zhu, X and Tang, D},
title = {A versatile CRISPR/Cas9 system off-target prediction tool using language model.},
journal = {Communications biology},
volume = {8},
number = {1},
pages = {882},
pmid = {40481308},
issn = {2399-3642},
support = {82070199//National Natural Science Foundation of China (National Science Foundation of China)/ ; },
mesh = {*CRISPR-Cas Systems ; *Gene Editing/methods ; Humans ; RNA, Guide, CRISPR-Cas Systems/genetics ; Deep Learning ; Software ; Computational Biology/methods ; },
abstract = {Genome editing with the CRISPR/Cas9 system has revolutionized life and medical sciences, particularly in treating monogenic genetic diseases by enabling long-term therapeutic effects from a single intervention. However, the CRISPR/Cas9 system can tolerate mismatches and DNA/RNA bulges at target sites, leading to unintended off-target effects that pose challenges for gene-editing therapy development. Existing high-throughput detection and in silico prediction methods are often limited to specifically designed single guide RNAs (sgRNAs) and perform poorly on unseen sequences. To address these limitations, we introduce CCLMoff, a deep learning framework for off-target prediction that incorporates a pretrained RNA language model from RNAcentral. CCLMoff captures mutual sequence information between sgRNAs and target sites and is trained on a comprehensive, updated dataset. This approach enables accurate off-target identification and strong generalization across diverse NGS-based detection datasets. Model interpretation reveals the biological importance of the seed region, underscoring CCLMoff's analytical capabilities. The development of CCLMoff lays the foundation for a comprehensive, end-to-end sgRNA design platform, enhancing both the precision and efficiency of CRISPR/Cas9-based therapeutics. CCLMoff is a versatile tool and is publicly available at github.com/duwa2/CCLMoff .},
}
@article {pmid40480713,
year = {2025},
author = {Zhou, C and Zhang, Y and Yang, X and Zhao, Z and Xia, M and Wei, C and Wu, Q and Chang, Z and Ma, L and Yin, L},
title = {A magnetic CRISPR/Cas12a-SERS nanobiosensor for amplification-free and ultrasensitive detection of norovirus in water and food samples.},
journal = {Analytica chimica acta},
volume = {1363},
number = {},
pages = {344133},
doi = {10.1016/j.aca.2025.344133},
pmid = {40480713},
issn = {1873-4324},
mesh = {*Norovirus/isolation &amp; purification/genetics ; *Biosensing Techniques/methods ; *CRISPR-Cas Systems ; Spectrum Analysis, Raman/methods ; *Food Contamination/analysis ; Limit of Detection ; DNA, Single-Stranded/chemistry ; *Water Microbiology ; *Food Microbiology ; Magnetic Phenomena ; *CRISPR-Associated Proteins/metabolism ; },
abstract = {BACKGROUND: Norovirus (NoV) is the leading cause of foodborne disease outbreaks worldwide, typically spreading via contaminated food and water. Rapid, sensitive, and portable detection of NoV is crucial.<br><br>RESULTS: Here, we presented a magnetic CRISPR/Cas12a-SERS nanobiosensor capable of detecting NoV with high sensitivity, accuracy, speed, and portability. In this nanobiosensor, SERS nanoprobes linked to magnetic nanoprobes via linker single-stranded DNAs (ssDNAs). The presence of NoV nucleic acid triggered Cas12a's trans-cleavage activity, degrading the linker ssDNA. After magnetic separation, the dissociated SERS nanoprobes were efficiently separated from the magnetic nanoprobes. This enhanced the SERS signal in the supernatant, detectable using a portable Raman spectrometer. The detection limit for NoV is 100 copies/mL within 60&#xa0;min. The nanobiosensor was further assessed in real-world settings, demonstrating excellent sensitivity and selectivity for detecting trace NoV in complex food samples.<br><br>SIGNIFICANCE: This approach not only broadens CRISPR-based pathogen detection but also provides a reliable tool for monitoring foodborne viruses. Its potential extends beyond NoV, promising enhanced surveillance of various pathogens in food safety, environmental monitoring, and public health sectors.},
}
@article {pmid40480203,
year = {2025},
author = {McNamara, HM and Sozen, B},
title = {From genes to geometry: Controlling embryo models by programming genomic activation.},
journal = {Cell stem cell},
volume = {32},
number = {6},
pages = {857-858},
doi = {10.1016/j.stem.2025.04.013},
pmid = {40480203},
issn = {1875-9777},
mesh = {Animals ; Mice ; *Embryo, Mammalian/metabolism/cytology ; Mouse Embryonic Stem Cells/metabolism/cytology ; CRISPR-Cas Systems/genetics ; Embryonic Stem Cells/metabolism/cytology ; *Genome ; *Models, Biological ; },
abstract = {Embryo-like models derived from stem cells have emerged as powerful tools to study early development. In this issue, Lodewijk et al.[1] demonstrate that activating just two enhancers via CRISPR activation (CRISPRa) in mouse embryonic stem cells (ESCs) can drive self-organization into structured embryo-like models, offering a genome-driven approach in stem cell and developmental biology.},
}
@article {pmid40478881,
year = {2025},
author = {Ratajczyk, EJ and Bath, J and Šulc, P and Doye, JPK and Louis, AA and Turberfield, AJ},
title = {Controlling DNA-RNA strand displacement kinetics with base distribution.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {122},
number = {23},
pages = {e2416988122},
pmid = {40478881},
issn = {1091-6490},
support = {EP/W524311/1//UKRI | Engineering and Physical Sciences Research Council (EPSRC)/ ; CCF 2211794//National Science Foundation (NSF)/ ; },
mesh = {Kinetics ; *DNA/chemistry/genetics/metabolism ; *RNA/chemistry/genetics/metabolism ; CRISPR-Cas Systems/genetics ; Thermodynamics ; Gene Editing ; },
abstract = {DNA-RNA hybrid strand displacement underpins the function of many natural and engineered systems. Understanding and controlling factors affecting DNA-RNA strand displacement reactions is necessary to enable control of processes such as CRISPR-Cas9 gene editing. By combining multiscale modeling with strand displacement experiments, we show that the distribution of bases within the displacement domain has a very strong effect on reaction kinetics, a feature unique to DNA-RNA hybrid strand displacement. Merely by redistributing bases within a displacement domain of fixed base composition, we are able to design sequences whose reaction rates span more than four orders of magnitude. We extensively characterize this effect in reactions involving the invasion of dsDNA by an RNA strand, as well as the invasion of a hybrid duplex by a DNA strand. In all-DNA strand displacement reactions, we find a predictable but relatively weak sequence dependence, confirming that DNA-RNA strand displacement permits far more thermodynamic and kinetic control than its all-DNA counterpart. We show that oxNA, a recently introduced coarse-grained model of DNA-RNA hybrids, can reproduce trends in experimentally observed reaction rates. We also develop a simple kinetic model for predicting strand displacement rates. On the basis of these results, we argue that base distribution effects may play an important role in natural R-loop formation and in the function of the guide RNAs that direct CRISPR-Cas systems.},
}
@article {pmid40478482,
year = {2025},
author = {Syahrani, RA and Wanandi, SI and Nihayah, S and Arumsari, S and Watanabe, Y and Mizuno, S and Louisa, M and Wuyung, PE},
title = {Survivin knockout attenuates the progressiveness of BT549 triple negative-breast cancer cells: an in vitro study highlighting stemness and cellular stress response mechanisms.},
journal = {Molecular biology reports},
volume = {52},
number = {1},
pages = {560},
pmid = {40478482},
issn = {1573-4978},
mesh = {Humans ; *Survivin/genetics/metabolism ; *Triple Negative Breast Neoplasms/genetics/metabolism/pathology ; Cell Line, Tumor ; Cell Proliferation/genetics ; *Neoplastic Stem Cells/metabolism/pathology ; Apoptosis/genetics ; Female ; Gene Knockout Techniques ; Gene Expression Regulation, Neoplastic/genetics ; CRISPR-Cas Systems ; Stress, Physiological/genetics ; Cell Cycle/genetics ; },
abstract = {BACKGROUND: Survivin, an inhibitor of apoptosis proteins (IAPs), is more strongly expressed in triple negative-breast cancer (TNBC) than other subtypes of breast cancer and closely associated with aggressiveness characterized by rapid progression and poor prognosis. The main function of survivin is to regulate cell division and prevent apoptosis. However, other survivin mechanisms are complex and not fully understood. The aim of this study was to evaluate the effects of survivin knockout on TNBC progressiveness relating to proliferation, apoptosis, stemness, cellular stress response, and metastasis mechanisms.<br><br>METHODS AND RESULTS: The CRISPR/Cas9 (clustered regularly interspaced short palindrom repeat-associated Cas9) system was utilized to establish survivin knockout in the BT549 TNBC cell line. Both knockout and wild-type cells were used to study the role of survivin in various biological mechanisms. Apoptosis-, pluripotency-, and cellular stress-related proteins were examined via proteomic arrays. The cell cycle, apoptosis, and expression of breast cancer stem cell markers were analyzed via flow cytometry. Metastasis-related markers were evaluated via qRT&#x2012;PCR. Here, we report that survivin knockout inhibits proliferation and induces apoptosis in TNBC cells. Moreover, survivin knockout suppressed the expression of pluripotent markers and promoted a shift toward activation response to cellular stress, such as genotoxic, hypoxic, and oxidative stress. Additionally, survivin knockout suppressed metastasis.<br><br>CONCLUSION: The loss of survivin leads to attenuation of TNBC progression by altering various mechanisms, particularly by suppressing stemness and altering the cellular stress response. We propose that knocking out survivin could be a potential strategy for breast cancer therapy, especially TNBC.},
}
@article {pmid40474742,
year = {2025},
author = {Hung, YH and Wang, E and Agtarap, T and Klaas, G and Slotkin, RK},
title = {Engineering an RNA/protein-binding module for higher transgene protein production and improved long-term durability.},
journal = {The Plant journal : for cell and molecular biology},
volume = {122},
number = {5},
pages = {e70254},
doi = {10.1111/tpj.70254},
pmid = {40474742},
issn = {1365-313X},
support = {DBI-2050394//National Science Foundation/ ; MCB-1904326//National Science Foundation/ ; MCB-2230587//National Science Foundation/ ; },
mesh = {*Arabidopsis/genetics/metabolism ; *Transgenes/genetics ; Plants, Genetically Modified/genetics/metabolism ; *RNA-Binding Proteins/metabolism/genetics ; *Arabidopsis Proteins/genetics/metabolism ; Gene Editing ; RNA Interference ; Gene Expression Regulation, Plant ; RNA, Plant/genetics/metabolism ; CRISPR-Cas Systems ; DNA Methylation ; RNA, Messenger/metabolism/genetics ; },
abstract = {Improvement and research of plants depends on the long-term expression of transgenes. However, the durability of transgene expression is routinely hampered by silencing pathways that start as the post-transcriptional process of mRNA degradation by RNA interference (RNAi). To avoid transgene silencing, we aimed to inhibit the sorting of transgene mRNAs into RNAi. We manipulated a well-studied protein/RNA-binding module from Arabidopsis into a transgene transcript, where the transcript is now bound by an engineered RNA-binding protein that preferentially sorts the RNA into translation. We used the Cas9 transcript as a proof-of-principle and demonstrated higher Cas9 protein production and gene editing rates. In addition, transgenes with the engineered protein/RNA-binding module had improved long-term durability of transgene expression, as after several inbred generations these plants had higher Cas9 protein accumulation and lower levels of DNA methylation, a hallmark of transgene silencing. Our engineered system represents a successful manipulation of post-transcriptional RNA sorting for improved transgene performance, and could be applied to any transgene transcript.},
}
@article {pmid40473912,
year = {2025},
author = {Omura, SN and Alfonse, LE and Ornstein, A and Morinaga, H and Hirano, H and Itoh, Y and Munoz, G and Garrity, AJ and Hoffman, GR and DiTommaso, T and Yan, WX and Cheng, DR and Scott, DA and Maben, Z and Nureki, O},
title = {Structural basis for target DNA cleavage and guide RNA processing by CRISPR-Casλ2.},
journal = {Communications biology},
volume = {8},
number = {1},
pages = {876},
pmid = {40473912},
issn = {2399-3642},
support = {JP23fa627001//Japan Agency for Medical Research and Development (AMED)/ ; },
mesh = {*CRISPR-Cas Systems ; *RNA, Guide, CRISPR-Cas Systems/metabolism/genetics ; Cryoelectron Microscopy ; *DNA Cleavage ; *CRISPR-Associated Proteins/metabolism/genetics/chemistry ; Bacteriophages/genetics/enzymology ; Gene Editing ; },
abstract = {RNA-guided CRISPR-Cas nucleases are widely used as versatile genome-engineering tools. Among the diverse CRISPR-Cas effectors, CRISPR-Casλ-also referred to as Cas12n-is a recently identified miniature type V nuclease encoded in phage genomes. Given its demonstrated nuclease activity in both mammalian and plant cells, Casλ has emerged as a promising candidate for genome-editing applications. However, the precise molecular mechanisms of Casλ family enzymes remain poorly understood. In this study, we report the identification and detailed biochemical and structural characterizations of CRISPR-Casλ2. The cryo-electron microscopy structures of Casλ2 in five different functional states unveiled the dynamic domain rearrangements during its activation. Our biochemical analyses indicated that Casλ2 processes its precursor crRNA to a mature crRNA using the RuvC active site through a unique ruler mechanism, in which Casλ2 defines the spacer length of the mature crRNA. Furthermore, structural comparisons of Casλ2 with Casλ1 and CasΦ highlighted the diversity and conservation of phage-encoded type V CRISPR-Cas enzymes. Collectively, our findings augment the mechanistic understanding of diverse CRISPR-Cas nucleases and establish a framework for rational engineering of the CRISPR-Casλ-based genome-editing platform.},
}
@article {pmid40473600,
year = {2025},
author = {Zhou, J and Hepperla, A and Simon, JM and Kim, K and Hu, Q and Zhang, C and Dong, L and Hu, L and Zhang, C and Liao, C and Fang, A and Adachi, Y and Fu, H and Wang, T and Liang, Q and Zhao, F and Liu, H and Takeda, M and Fang, J and Zhong, H and Ly, P and Wang, L and Kapur, P and Xu, L and Jia, L and Malladi, S and Brugarolas, J and Simon, MC and Li, B and Zhang, Q},
title = {SWI/SNF ATPase silenced HLF potentiates lung metastasis in solid cancers.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {5226},
pmid = {40473600},
issn = {2041-1723},
support = {R35 CA220483/CA/NCI NIH HHS/United States ; R01 CA294133/CA/NCI NIH HHS/United States ; RR190058//Cancer Prevention and Research Institute of Texas (Cancer Prevention Research Institute of Texas)/ ; P30 CA142543/CA/NCI NIH HHS/United States ; R01CA211732//U.S. Department of Health &amp; Human Services | NIH | National Cancer Institute (NCI)/ ; R35 GM146979/GM/NIGMS NIH HHS/United States ; R01 CA211732/CA/NCI NIH HHS/United States ; P50 CA196516/CA/NCI NIH HHS/United States ; P50CA196516//U.S. Department of Health &amp; Human Services | NIH | National Cancer Institute (NCI)/ ; },
mesh = {Humans ; *Lung Neoplasms/secondary/genetics/metabolism ; *Transcription Factors/genetics/metabolism ; Animals ; *DNA Helicases/metabolism/genetics ; Cell Line, Tumor ; *Nuclear Proteins/metabolism/genetics ; Mice ; Cell Movement/genetics ; Gene Expression Regulation, Neoplastic ; *Carcinoma, Renal Cell/genetics/pathology/metabolism ; *Kidney Neoplasms/pathology/genetics/metabolism ; Female ; Gene Silencing ; CRISPR-Cas Systems ; },
abstract = {Metastasis is the main cause of cancer-related deaths, yet the underlying mechanisms remain elusive. Here, using clear cell renal cell carcinoma (ccRCC), a tumor type with frequent lung metastases, we conduct an in vivo genome-wide CRISPR-Cas9 screen and identify HLF as a potent suppressor of lung metastasis. HLF depletion enhances ccRCC cell migration and lung metastasis, whereas HLF overexpression abrogates these effects. In ccRCC patients, HLF expression is reduced at metastatic sites and associates with epigenetic silencing mediated by the SWI/SNF ATPase subunit BRG1. HLF levels negatively correlate with migration potential in collagen. Mechanistically, HLF regulates LPXN expression, modulating the integration of collagen's mechanical cues with the actin cytoskeleton through Paxillin, thereby suppressing cancer cell migration and lung metastasis. Overexpression of HLF or pharmacological inhibition of BRG1 reduces cell invasion across multiple cancer types. Our findings suggest that targeting the BRG1-HLF axis offers a promising therapeutic strategy for combating metastatic cancers.},
}
@article {pmid40472130,
year = {2025},
author = {Ghdayer Al Kaabi, NA and Kandhan, K and Hayat, F and Matar Al Blooshi, SA and Sheteiwy, MS and Alyafei, M},
title = {Shaping the future of date palm (Phoenix dactylifera) through new genetic improvement strategies.},
journal = {Functional plant biology : FPB},
volume = {52},
number = {},
pages = {},
doi = {10.1071/FP25021},
pmid = {40472130},
issn = {1445-4416},
mesh = {*Phoeniceae/genetics/metabolism ; *Plant Breeding/methods ; Gene Editing ; CRISPR-Cas Systems ; Genome-Wide Association Study ; Polymorphism, Single Nucleotide ; },
abstract = {Conventional breeding of date palm (Phoenix dactylifera) is inherently challenging due to its long generation time, dioecious nature, and high genetic heterogeneity. However, current developments in genomics and molecular biology offer promising avenues for accelerating breeding programs, particularly through high-throughput technologies including functional genomics. This article reviews genomic tools such as like CRISPR-Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats-CRISPR-associated protein 9) that may bring significant changes in date palm breeding. The CRISPR-Cas9 system enables scientists to accurately target genomic regions, which helps enhance breeding accuracy by adding advantageous traits and eliminating unfavorable genes through precision editing. Transcriptome and metabolome analyses have also explained the regulation of thousands of differentially expressed genes (DEGs) and metabolic pathways under environmental stress. These studies contribute to enhance the knowledge of stress tolerance mechanisms, which include the secondary metabolic process of flavonoids. Genomic studies illustrating single nucleotide polymorphism (SNP)-based diversity between cultivars from north African and the Arabian Gulf provide new genetic resources for selective breeding. The work relates genome-wide association studies (GWAS) and miRNA profiling to elucidate key regulatory networks involved in fruit development and stress resilience. The integration of such advanced technologies, especially the CRISPR-Cas9 system, is revolutionizing the landscape of date palm breeding, opening new avenues for accelerated development of superior cultivars that meet the needs of modern agriculture.},
}
@article {pmid40472129,
year = {2025},
author = {Sarfraz, Z and Zarlashat, Y and Ambreen, A and Mujahid, M and Iqbal, MS},
title = {Advanced gene editing techniques for enhancing disease resistance and climate resilience in crops.},
journal = {Functional plant biology : FPB},
volume = {52},
number = {},
pages = {},
doi = {10.1071/FP24357},
pmid = {40472129},
issn = {1445-4416},
mesh = {*Gene Editing/methods ; *Crops, Agricultural/genetics ; *Disease Resistance/genetics ; Climate Change ; CRISPR-Cas Systems ; Plants, Genetically Modified ; },
abstract = {Ensuring food security and solving the issues brought on by climate change require breeding and engineering of climate-resilient crops. Despite its contributions to reducing agricultural diseases, genetic engineering has several limitations, including high labor costs, lengthy processing times, and poor productivity. Genome editing has become a potential method to provide notable opportunities to explain complex biological processes, genetically solve the causes of diseases, and improve crops for disease resistance by effectively modifying multiple traits. Genome editing techniques including TALENs, ZFNs, and CRISPR/Cas9 increase agricultural productivity by developing climate-resistant crops and promoting climate-resilient agriculture. Among these approaches, CRISPR/Cas9 shows exceptional efficacy, minimal chance of off-target effects, and improved traits such as drought tolerance and disease resistance. This study explores advanced gene editing techniques for improving disease resistance in crops and developing climate-resilient varieties to reduce food insecurity and hunger. It demonstrates that these techniques have enhanced the nutritional content and resilience of many crops by fighting abiotic and biotic stresses. Future agricultural practices could alter the genes and improve disease-resistant crops by genome editing techniques.},
}
@article {pmid40471011,
year = {2025},
author = {Li, H and Shen, J and Zhao, X and Ji, J and Wang, Y and Yang, L and Zhuang, M and Liu, L and Zhang, Y and Lv, H},
title = {Rapid design of transgene-free cabbage with desired anthocyanin contents via HI-Edit.},
journal = {Journal of integrative plant biology},
volume = {},
number = {},
pages = {},
doi = {10.1111/jipb.13943},
pmid = {40471011},
issn = {1744-7909},
support = {CAAS-ASTIP-IVFCAAS//the Science and Technology Innovation Program of the Chinese Academy of Agricultural Sciences/ ; CARS-23//the China Agriculture Research System of MOF and MARA/ ; 2023YFD1201501//The National Key R&amp;D Program of China/ ; BE2023366//The Key Technology R&amp;D Program of Jiangsu Province/ ; },
abstract = {The HI-Edit system combines haploid induction and CRISPR/Cas-based genome editing to provide a promising way to design crops with desired traits in a rapid, precise and transgene-free manner. HI-Edit was applied to produce cabbages with desired anthocyanin contents.},
}
@article {pmid40470110,
year = {2025},
author = {Bao, H and Chen, Y and Zong, Y and Jin, K and Lan, H},
title = {Recent Advances in the Delivery of Bone Morphogenetic Proteins for Targeting Glioma: An Updated Review.},
journal = {International journal of nanomedicine},
volume = {20},
number = {},
pages = {7093-7112},
pmid = {40470110},
issn = {1178-2013},
mesh = {Humans ; *Glioma/drug therapy/therapy ; Animals ; *Brain Neoplasms/drug therapy ; *Bone Morphogenetic Proteins/administration &amp; dosage/therapeutic use/pharmacokinetics ; Genetic Therapy/methods ; *Drug Delivery Systems/methods ; Nanoparticles/chemistry ; Blood-Brain Barrier/metabolism ; Hydrogels/chemistry ; },
abstract = {Bone Morphogenetic Proteins might be the most prospective in glioma treatment because of the facts that they can differentiate glioma cells, inhibit tumor growth and manage glioma stem cells. Its clinical application is hindered by several challenges, including limited permeability across the blood-brain barrier, which impedes effective delivery to the central nervous system; high susceptibility to enzymatic degradation, which compromises stability and therapeutic efficacy; and nonselective binding, which reduces specificity and may result in unintended off-target effects. This review systematically covers the advanced BMP delivery systems such as nanoparticles, smart carriers, gene therapy, and exosome-based system. Hydrogels, scaffolds, and microspheres' local delivery methods are also discussed as prospective options. The in vitro studies reveal that BMPs are effective and using in vivo glioma models there is also evidence of the effectiveness of BMPs. In addition, new clinical trials reveal concern with safety, tolerability, and therapeutic effects of BMPs, especially their combination with chemotherapy and immunotherapy. BMP specificity and therapeutic performance are further optimized by Personalized medicine and CRISPR/Cas engineering. However, regulatory barriers and product commercialization are challenging issues. This review highlights the need for novel approaches and advanced technologies to address the challenges associated with BMP delivery, aiming to establish BMP-based therapies as an effective treatment strategy for glioma.},
}
@article {pmid40469526,
year = {2025},
author = {Isaacson, J and Bhanap, P and Putnam, N and Padilla, J and Fatima, N and Dotson, M and Hayoun, D and Ahmadi, M and Nonterah, G and Ji, Y},
title = {Enhancing CAR T-cell therapy manufacturing efficiency through semi-automated bioprocessing.},
journal = {Clinical &amp; translational immunology},
volume = {14},
number = {6},
pages = {e70025},
pmid = {40469526},
issn = {2050-0068},
abstract = {OBJECTIVES: Chimeric antigen receptor (CAR) T-cell therapies have revolutionised the treatment of blood-based malignancies. The use of manual CAR T-cell manufacturing methods is one of the challenges that contributes to these delays. As CAR T therapy emerges as a potential first- or second-line treatment option for these cancers, the demand for these therapies continues to rise. However, challenges persist in ensuring that the patients who need these therapies receive them in a timely manner. Automated CAR T-cell manufacturing methods that use software to control the equipment used in the process can help overcome the roadblocks associated with manual manufacturing, ultimately enabling a reduction in variability, increased efficiency, improved product quality and better data management. This paper aims to present an end-to-end semi-automated methodology for manufacturing CAR T cells using the Cell Therapy Systems (CTS™) Cellmation software - an off-the-shelf software solution - to control physically connected modular cell therapy instruments that eliminates the roadblocks associated with manual manufacturing.<br><br>METHODS: T&#xa0;cells from healthy donors were isolated and processed into CAR T&#xa0;cells using a semi-automated, connected, multi-instrument setup that leveraged electroporation and a CRISPR/Cas system for delivering the CD19-CAR construct to the T&#xa0;cells. Flow cytometry was used to assess cell type composition, cell viability and expression of T-cell activation markers throughout the process. We also measured exhaustion marker expression on T&#xa0;cells, T-cell receptor (TCR) knock-out, CAR knock-in and cytotoxic activity against NALM6 cells.<br><br>RESULTS: The results demonstrated the successful generation of functional CAR T&#xa0;cells using a semi-automated instrument workflow. The results were similar to the results from CAR T&#xa0;cells manufactured using non-automated processes; however, the successful connection and control of the instruments using automated software present an exciting opportunity for process developers and manufacturers who want to reduce manual touchpoints in their cell therapy manufacturing process.<br><br>CONCLUSION: The method that we describe in this paper could be beneficial to process development and manufacturing teams that might require flexibility in their CAR T&#xa0;cell manufacturing workflow and want to take advantage of modular systems that can be automated using the Cellmation software to reduce the problems associated with manual handling.},
}
@article {pmid40469284,
year = {2025},
author = {Lin, Z and Song, Z and Yu, H and Zhou, Y and Liu, D and Zhang, P and Wei, L and Dai, G and Liang, G and He, Z and Hu, X and Chen, Y and Zhao, P and Lu, H and Zheng, M},
title = {Ultra-sensitive in situ detection of intracellular Mycobacterium tuberculosis with CRISPR/Cas12a.},
journal = {Frontiers in immunology},
volume = {16},
number = {},
pages = {1597654},
pmid = {40469284},
issn = {1664-3224},
mesh = {*Mycobacterium tuberculosis/genetics/isolation &amp; purification ; Humans ; Animals ; *CRISPR-Cas Systems ; Mice ; *Macrophages/microbiology ; RAW 264.7 Cells ; *Tuberculosis/diagnosis/microbiology ; Bronchoalveolar Lavage Fluid/microbiology ; Sensitivity and Specificity ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {Mycobacterium tuberculosis (Mtb) invades and survives inside macrophages, evading detection and resisting antibiotic treatment, which results in severe clinical consequences such as fatal respiratory failure and systemic inflammation. Rapid and specific detection of intracellular Mtb is crucial for accurate diagnosis and optimizing treatment strategies. In this study, we developed a one-step CRISPR/Cas12a assay targeting the IS6110 gene for the specific and rapid detection of intracellular Mtb. Upon efficient delivery into RAW264.7 macrophages, the assay enabled direct visualization of Mtb IS6110 nucleic acid, generating detectable fluorescence signals. The diagnostic performance was further validated using bronchoalveolar lavage fluid (BALF) samples from clinical participants, achieving a sensitivity of 94%, which surpassed conventional methods such as culture (67%) and Xpert (78%), while maintaining a specificity of 100%. This CRISPR/Cas12a-based assay offers a highly sensitive, rapid, and innovative approach for intracellular Mtb detection, with significant potential to enhance tuberculosis diagnostic methodologies and improve clinical outcomes.},
}
@article {pmid40469012,
year = {2025},
author = {Zhu, F and Zhao, Q},
title = {Spherical Nucleic Acids as Modulators of CRISPR/Cas12a by a Steric Barrier Effect for Designing Versatile Biosensors.},
journal = {Analytical chemistry},
volume = {97},
number = {23},
pages = {12215-12222},
doi = {10.1021/acs.analchem.5c00937},
pmid = {40469012},
issn = {1520-6882},
mesh = {*Biosensing Techniques/methods ; *CRISPR-Cas Systems ; Gold/chemistry ; Metal Nanoparticles/chemistry ; *CRISPR-Associated Proteins/metabolism ; *Endodeoxyribonucleases/metabolism ; *Bacterial Proteins/metabolism ; Humans ; *Nucleic Acids/chemistry ; },
abstract = {Discovery of CRISPR/Cas12a has revolutionized broad fields, including gene editing, molecular diagnosis, and biosensing. Flexible regulation of Cas12a activity is important for diverse CRISPR/Cas12a applications, especially for biosensing, but it still faces limitations and challenges. We find gold nanoparticles (AuNPs) modified with a single-stranded DNA activator create a huge steric barrier that strongly locks activators in a one-to-many manner and inhibits Cas12a activity. This finding offers a new way to modulate the activity of Cas12a for applications, such as designing versatile biosensors. We report a spherical nucleic acid (SNA)-modulating CRISPR/Cas12a (SNA-Cas) platform using SNAs as signal translators for target sensing. A stimuli-responsive SNA was constructed by modifying AuNPs with a DNA activator containing a specific trigger element, and the target triggers specific reactions (e.g., thiol-exchange chemical reaction and RNA-cleaving by DNAzymes) to release activators into solution. A free activator initiates trans-cleavage activity of CRISPR/Cas12a, scissoring fluorescent DNA reporters to produce amplified signals. To show proof of concept, we demonstrate SNA-Cas to sensitively detect diverse non-nucleic acid targets, including biological thiol cysteine, heavy metal Pb[2+], and biomarkers of O[6]-methylguanine-DNA-methyltransferases (MGMT) and fat mass and obesity-related protein (FTO) demethylases. This work opens one door for SNA modulating CRISPR/Cas activity and shows great potential in designing versatile biosensors for detecting diverse targets.},
}
@article {pmid40468657,
year = {2025},
author = {Jun, JS and Kang, SJ and Hong, KW},
title = {Time-resolved analysis of Bacillus subtilis DB104 Spo0A-mutant transcriptome profile and enhancement of recombinant protein release.},
journal = {Journal of microbiology (Seoul, Korea)},
volume = {63},
number = {5},
pages = {e2411032},
doi = {10.71150/jm.2411032},
pmid = {40468657},
issn = {1976-3794},
support = {2021R1I1A2046284//National Research Foundation of Korea/ ; //Ministry of Education/ ; },
mesh = {*Bacillus subtilis/genetics/metabolism/growth &amp; development ; *Bacterial Proteins/genetics/metabolism ; Gene Expression Regulation, Bacterial ; *Transcriptome ; Spores, Bacterial/genetics/growth &amp; development ; *Recombinant Proteins/genetics/metabolism ; *Transcription Factors/genetics/metabolism ; Gene Expression Profiling ; Biofilms/growth &amp; development ; Mutation ; CRISPR-Cas Systems ; Quorum Sensing/genetics ; Gene Editing ; },
abstract = {Spo0A, the master regulator of sporulation initiation in Bacillus subtilis, controls over 500 genes directly or indirectly in early sporulation stages. Although the effects of Spo0A disruption on sporulation have been extensively studied, a comprehensive understanding of the genomic response throughout growth phases remain elusive. Here, we examined the transcriptomic changes in Spo0A mutant strain, R211E, and wild-type across a time-course RNA-seq to identify impacted biological processes and pathways. The R211E strain, which exhibits sporulation deficiency, was constructed using the clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR associated protein (Cas)9 system, highlighting the critical role of proper Cas9 dosing in gene editing. Functional analysis of 3,010 differentially expressed genes (DEGs) showed significant alterations in sporulation, quorum sensing, metabolism, and biofilm formation. The R211E disrupted the Spo0A-AbrB regulatory pathway, reducing biofilm formation and enhancing flagellar gene expression. Up-regulated metabolic pathways, including glycolysis, histidine, and purine biosynthesis, increased cell numbers during vegetative growth. Further, the mutant displayed elevated vegetative autolysin expression, resulting in reduced cell viability in the stationary phase. We also introduce the novel potential of R211E in a recombinant protein expression system that facilitated protein release into the supernatant, providing valuable insight for future research in metabolic engineering and efficient production systems in B. subtilis.},
}
@article {pmid40468633,
year = {2025},
author = {Cao, M and Brennan, A and Lee, CM and Park, SH and Bao, G},
title = {Deep Learning Based Models for CRISPR/Cas Off-Target Prediction.},
journal = {Small methods},
volume = {},
number = {},
pages = {e2500122},
doi = {10.1002/smtd.202500122},
pmid = {40468633},
issn = {2366-9608},
support = {RP210116//Cancer Prevention and Research Institute of Texas/ ; R01HG011459/HG/NHGRI NIH HHS/United States ; R01HL169761/HL/NHLBI NIH HHS/United States ; },
abstract = {CRISPR/Cas genome editing technologies enable effective and controlled genetic modifications; however, off-target effects remain a significant concern, particularly in clinical applications. Experimental and in silico methods are developed to predict potential off-target sites (OTS), including deep learning based methods, which can automatically and comprehensively learn sequence features, offer a promising tool for OTS prediction. Here, this work reviews the existing OTS prediction tools with an emphasis on deep learning methods, characterizes datasets used for deep learning training and testing, and evaluates six deep learning models -CRISPR-Net, CRISPR-IP, R-CRISPR, CRISPR-M, CrisprDNT, and Crispr-SGRU -using six public datasets and validates OTS data from the CRISPRoffT database. Performance of these models is assessed using standardized metrics, such as Precision, Recall, F1 score, MCC, AUROC and PRAUC. This work finds that incorporating validated OTS datasets into model training enhanced overall model performance, and improved robustness of prediction, particularly with highly imbalanced datasets. While no model consistently outperforms other models across all scenarios, CRISPR-Net, R-CRISPR, and Crispr-SGRU show strong overall performance. This analysis demonstrates the importance of integrating high-quality validated OTS data with advanced deep learning architectures to improve CRISPR/Cas off-target site predictions, ensuring safer genome editing applications.},
}
@article {pmid40466745,
year = {2025},
author = {Yang, P and Zeng, J and Li, L and Ma, R and Peng, J and Zhou, W and Fu, W and Wu, Y and Zhang, Y},
title = {From Cas proteins to cutting-edge biosensors: A new era in clinical pathogen diagnostics.},
journal = {The Journal of infection},
volume = {91},
number = {1},
pages = {106526},
doi = {10.1016/j.jinf.2025.106526},
pmid = {40466745},
issn = {1532-2742},
mesh = {*Biosensing Techniques/methods ; Humans ; *CRISPR-Cas Systems ; *Molecular Diagnostic Techniques/methods ; *Communicable Diseases/diagnosis ; },
abstract = {Infectious pathogens exert a profound impact on global health and socio-economic stability, positioning them as a critical focus of scientific inquiry. To safeguard public health, propel advancements in medical diagnostics, and ensure food safety, the development of efficient technologies for rapid, onsite detection of pathogens is imperative. In light of recent research breakthroughs, CRISPR/Cas-based technologies for pathogen biosafety and molecular diagnostics have emerged as particularly promising in the realm of infectious disease detection. This review succinctly introduces the working principles of CRISPR/Cas systems and thoroughly discusses the design and development of various CRISPR/Cas-based biosensors. Importantly, this paper explores the robust applications of CRISPR/Cas-assisted biosensing for emerging infectious diseases, highlighting its potential in pathogen diagnostics with features like cost-effectiveness, multiplex detection and POCT applications. Furthermore, challenges and future developments of CRISPR/Cas-based biosensors for rapid and accurate pathogen detection in specialized settings are also summarized, integrating CRISPR detection with portable POCT biosensors, nanomaterials and novel colorimetric materials. As it builds on a lot of foundational work and offers new insights and detailed reference to advance the development and application of CRISPR technologies in clinical pathogen diagnostics, opening new avenues in medical diagnostics and the prevention and control of infectious diseases.},
}
@article {pmid40466639,
year = {2025},
author = {Conti, A and Giannetti, K and Midena, F and Beretta, S and Gualandi, N and De Marco, R and Carsana, E and Varesi, A and Tavella, T and Alessandrini, L and Zarghamian, P and Weber, A and Ferrari, S and Brombin, C and Gilioli, D and Della Volpe, L and Xie, SZ and Merelli, I and Cathomen, T and Naldini, L and Di Micco, R},
title = {Senescence and inflammation are unintended adverse consequences of CRISPR-Cas9/AAV6-mediated gene editing in hematopoietic stem cells.},
journal = {Cell reports. Medicine},
volume = {6},
number = {6},
pages = {102157},
doi = {10.1016/j.xcrm.2025.102157},
pmid = {40466639},
issn = {2666-3791},
mesh = {*Gene Editing/methods ; *Hematopoietic Stem Cells/metabolism ; *CRISPR-Cas Systems/genetics ; *Inflammation/pathology/genetics ; *Cellular Senescence/genetics ; Animals ; Humans ; Tumor Suppressor Protein p53/metabolism ; NF-kappa B/metabolism ; Mice ; Interleukin 1 Receptor Antagonist Protein/pharmacology ; Interleukin-1/metabolism ; },
abstract = {Gene editing (GE) using homology-directed repair (HDR) in hematopoietic stem and progenitor cells (HSPCs) offers promise for long-range gene correction of inherited genetic disorders. However, cellular responses induced by CRISPR-Cas9/AAV6 engineering impair the long-term repopulating potential of HDR-edited HSPCs, adversely impacting the safety and efficacy of clinical translation. Our study uncovers a durable senescence-like response in genetically engineered HSPCs triggered by p53 and interleukin (IL)-1/nuclear factor κB (NF-κB) activation, which restricts graft size and clonal diversity in long-term transplantation assays. We show that transient p53 inhibition or blocking inflammatory pathways mitigates senescence-associated responses, improving the repopulating capacity of edited HSPCs. Importantly, we identify treatment with Anakinra, an IL-1 signaling antagonist, as a promising strategy to enhance polyclonal output in HDR-edited cells while minimizing genotoxicity risks associated with the editing procedure. Overall, our findings present strategies to overcome key hurdles in HDR-based HSPC gene therapies, providing a framework for enhancing their efficacy and safety in clinical applications.},
}
@article {pmid40466420,
year = {2025},
author = {Jiang, J and Kan, X},
title = {A smartphone-enabled colorimetric tumor-derived exosomes sensing based on multi-enzyme catalysis and dual-recognition triggered CRISPR/Cas12a trans-cleavage.},
journal = {Biosensors &amp; bioelectronics},
volume = {286},
number = {},
pages = {117644},
doi = {10.1016/j.bios.2025.117644},
pmid = {40466420},
issn = {1873-4235},
mesh = {Humans ; *Exosomes/chemistry ; *Biosensing Techniques/instrumentation/methods ; *Colorimetry/instrumentation/methods ; *Smartphone ; CRISPR-Cas Systems ; A549 Cells ; Aptamers, Nucleotide/chemistry ; Limit of Detection ; *Lung Neoplasms/diagnosis ; Tetraspanin 30/chemistry ; Biomarkers, Tumor ; Epithelial Cell Adhesion Molecule/chemistry ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {Detection of protein profiling on exosomes exhibits great promise for early non-invasive and accurate diagnosis of tumor in clinical diagnostics. However, it still faces multiple challenges, such as expensive instruments requirement and weak specificity by single biomarker. Herein, based on a dual-recognition strategy, a ternary hybrid of a trigger DNA (TDNA), EpCAM aptamer, and CD63 aptamer was used to capture A549 cells-derived exosomes to release TDNA, which initiated the trans-cleavage activity of CRISPR/Cas12a to nonspecifically cleave single-stranded DNA (ssNDA) and then resulted in the isolating of ssDNA linked nanozyme of Zr/Fe-CeO2@Ir@CaO2@HA (ZFCIrCH). ZFCIrCH not only achieved H2O2/O2 self-supply, but also possessed high peroxidase-like, oxidase-like, and superoxide dismutase-like activities, thereby generating a sensitive colorimetric signal for A549 cells-derived exosomes detection with a low limit of detection (LOD) of 31 particles/mL. Using a smartphone to analyze colorimetric images, exosome concentration also can be precisely quantified with a LOD of 29 particles/mL, which could also successfully distinguish healthy people from lung cancer patients. With the advantages of high sensitivity, good specificity, low cost, and convenient on-site detection of tumor-derived exosomes, the present colorimetric sensor has great promise in the accurate diagnosis of diseases.},
}
@article {pmid40465697,
year = {2025},
author = {Vo, QD},
title = {Gene editing therapy as a therapeutic approach for cardiovascular diseases in animal models: A scoping review.},
journal = {PloS one},
volume = {20},
number = {6},
pages = {e0325330},
pmid = {40465697},
issn = {1932-6203},
mesh = {Animals ; *Gene Editing/methods ; *Cardiovascular Diseases/therapy/genetics ; Disease Models, Animal ; *Genetic Therapy/methods ; CRISPR-Cas Systems ; Mice ; Humans ; Dependovirus/genetics ; },
abstract = {BACKGROUND: Cardiovascular diseases (CVDs) are the leading cause of mortality worldwide, with hereditary genetic factors contributing substantially to disease burden. Current treatments, including lifestyle modifications, pharmacotherapy, and surgical interventions, focus primarily on symptom management but fail to address underlying genetic causes, often resulting in disease progression or recurrence. Gene therapy has emerged as a transformative approach, offering a potential treatment. This review explores its efficacy and safety in animal models, identifying opportunities for future advancements.<br><br>METHODS: This review investigated studies on gene editing interventions in animal models of CVDs, retrieved from PubMed, ScienceDirect, and Web of Science up to December 2024.<br><br>RESULT: A total of 57 studies were included in this review. Mice (86%) were the predominant model, with CRISPR-Cas9 (53%) and AAV vectors (80%) as the most used tools. Key targets included PCSK9 (32%), LDLR (9%), and MYH6/7 (7%), achieving 25-85% editing efficiency in liver/heart tissues. Base editors (ABE/CBE) showed superior safety, with <1% off-targets versus CRISPR-Cas9's 2-5 off-targets per guide. Reported toxicity risks included liver injury (AAVs, 23%) and transient cytokine elevation (LNPs, 14%).<br><br>CONCLUSION: Gene editing therapy shows great potential for treating CVDs, with high efficiency, strong therapeutic outcomes, and favorable safety in animal models. Continued innovation and rigorous evaluation could transform cardiovascular treatment, benefiting patients with untreatable conditions.},
}
@article {pmid40465692,
year = {2025},
author = {Cai, Y and Zhao, P and Wu, F and Zhao, H and Shao, H and Marra, A and Patel, P and O'Connell, E and Fink, E and Miele, MM and Li, Z and De Stanchina, E and Cocco, E and Razavi, P and Toska, E and Fanning, SW and Xu, G and Sablina, AA and Scaltriti, M and Chandarlapaty, S},
title = {Inhibition of NR2F2 restores hormone therapy response to endocrine refractory breast cancers.},
journal = {Science translational medicine},
volume = {17},
number = {801},
pages = {eadk7786},
doi = {10.1126/scitranslmed.adk7786},
pmid = {40465692},
issn = {1946-6242},
mesh = {Humans ; *Breast Neoplasms/drug therapy/genetics/metabolism/pathology ; Female ; *COUP Transcription Factor II/metabolism/antagonists &amp; inhibitors/genetics ; Animals ; Cell Line, Tumor ; *Drug Resistance, Neoplasm/drug effects/genetics ; Neurofibromin 1/metabolism/genetics ; Gene Expression Regulation, Neoplastic/drug effects ; Mice ; Receptors, Estrogen/metabolism ; CRISPR-Cas Systems/genetics ; },
abstract = {Endocrine resistance is frequently encountered in estrogen receptor-positive (ER+) breast cancer, often because of somatic mutations such as neurofibromin 1 (NF1) loss. The mechanisms by which ER-directed proliferation is lost in such cases are unknown, limiting the potential use of additional endocrine treatments. Here, we performed CRISPR-Cas9 knockout (KO) screens and found that nuclear receptor subfamily 2 group F member 2 (NR2F2), an orphan nuclear receptor, was essential for NF1 loss-induced endocrine resistance. Induction of NR2F2 was observed in ER+ cell line models and patient samples and occurred via activation of the mitogen-activated protein kinase (MAPK) pathway upon NF1 loss or other MAPK pathway genetic alterations. Mechanistically, increased NR2F2 orchestrated a repressed ER transcriptional program by repartitioning the ER cistrome, altering the balance of its associated transcriptional coregulators, and modifying global chromatin accessibility. Accordingly, genetic depletion or pharmacologic inhibition of NR2F2 restored sensitivity to hormone therapies in multiple models, including ER+ cell lines, patient-derived xenografts, and patient-derived organoid-based xenografts harboring diverse endocrine-resistance mechanisms such as NF1, AT-rich interactive domain-containing protein 1A (ARID1A), phoshatase and tensin homolog (PTEN) loss, or Kirsten rat sarcoma virus (KRAS) overexpression. Together, these findings underscore NR2F2 as a critical modulator of the hormone response pathway and suggest its inhibition as a promising strategy to overcome endocrine resistance in breast cancer.},
}
@article {pmid40465008,
year = {2025},
author = {Jin, Y and Liang, X and Wang, X},
title = {Alternative splicing in stem cells and development: research progress and emerging technologies.},
journal = {Cell regeneration (London, England)},
volume = {14},
number = {1},
pages = {20},
pmid = {40465008},
issn = {2045-9769},
support = {YD9110002078//USTC Research Funds of the Double First-Class Initiative/ ; 32170557//National Natural Science Foundation of China/ ; 32400436//National Natural Science Foundation of China/ ; 2023IHM01034//Research Funds of Centre for Leading Medicine and Advanced Technologies of IHM/ ; 2024C879//Anhui Postdoctoral Scientific Research Program Foundation/ ; },
abstract = {Alternative splicing is a key regulatory mechanism that generates transcriptomic diversity by selectively splicing pre-RNA molecules in different ways, leading to the production of multiple RNA isoforms from a single gene. This process is crucial for the fine-tuning of gene expression and is tightly regulated during various biological processes. Recent studies have highlighted how alternative splicing contributes to stem cells self-renewal and differentiation, as well as how dysregulation of splicing factors can impact stem cells behavior and lead to developmental abnormalities or diseases. This review summarizes the current understanding of alternative splicing in stem cells and development, focusing on the molecular mechanisms that govern alternative splicing regulation, the role of splicing factors, and the impact of splicing isoforms on stem cell fate determination and developmental processes. We also discuss emerging technologies, such as CRISPR/Cas-based tools, single-cell long-read RNA sequencing, imaging technologies and 3D culture systems, which are advancing our ability to study alternative splicing in vitro and in vivo. Overall, this field is rapidly evolving, revealing new insights into how alternative splicing shapes the molecular landscape and functions of stem cells and developmental processes.},
}
@article {pmid40464988,
year = {2025},
author = {Waqas, MAB and Awan, MJA and Amin, I and Arif, M and Mukhtar, Z and Mansoor, S},
title = {Engineering high yield basmati rice by editing multiple negative regulators of yield.},
journal = {Molecular biology reports},
volume = {52},
number = {1},
pages = {545},
pmid = {40464988},
issn = {1573-4978},
mesh = {*Oryza/genetics/growth &amp; development ; *Gene Editing/methods ; CRISPR-Cas Systems/genetics ; Plant Breeding/methods ; Plants, Genetically Modified/genetics ; Plant Proteins/genetics ; Genes, Plant/genetics ; Quantitative Trait Loci/genetics ; Edible Grain/genetics ; Gene Expression Regulation, Plant ; },
abstract = {BACKGROUND: Yield improvement in Basmati rice is important to meet the growing demand of Basmati and pivotal to world food security. Yield in rice is a complex quantitative trait controlled by many genes and yield-determining factors. Although dominant genes introgression through breeding have been extensively used but it is a laborious and time-consuming process. Yield genes OsD27, OsGW2, OsTGW6 and OsGN1a have been shown to negatively regulate an increase in the number of tillers, grain width, grain weight, and number of grains per panicle respectively in different genetic backgrounds of rice separately. The aim of our study is to establish a multiplex system in Super Basmati to check if it acts similarly and what is their combinatorial effect on yield enhancement.<br><br>METHODS: Here we generated Super Basmati quadruple edited lines for all four genes OsD27, OsGW2, OsTGW6 and OsGN1a through CRISPR-Cas9 polycistronic tRNA-gRNA (PTG) endogenous processing efficient multiplex editing system. Quadruple edited lines were characterized for key yield parameters such as number of tillers, number of grains, grain weight, overall yield increase, lodging resistance and cooking quality related traits.<br><br>RESULTS: We found that all quadruple edited plants produced more tillers, a greater number of grains with high grain weight in field evaluations along with decreased plant height. Overall, the contribution of all four genes was additive resulting in boosting the yield in Super Basmati remarkably. Our data hence demonstrated a promising multiplex genome editing approach for rapid generation of superior alleles for all four yield contributing factor genes in elite Basmati variety with 30% co-editing efficiency.<br><br>CONCLUSIONS: We conclude that multiplex CRISPR-Cas9 genome editing of yield-negative regulators may boost the yield of elite Basmati rice in a short time without causing any drastic effect on other agronomic and quality traits such as aroma, chalkiness, and cooking qualities. Data suggests that the tRNA-gRNA multiplexing CRISPR-Cas9 system is efficient in the Basmati rice background for simultaneously editing multiple genes. Due to its capability of rapid generation of beneficial alleles, this multiplex system is well suited for pyramiding of multiple alleles in Basmati rice and may prove a promising approach to enhance yield in Basmati rice while it may provide useful germplasm resource for high-yielding future rice breeding programs.},
}
@article {pmid40464976,
year = {2025},
author = {Sharma, SS and Pandey, A and Kashyap, A and Goyal, L and Garg, P and Kushwaha, R and Sharma, J and Tripathi, S and Kumari, S and Thomas, G and Verma, M and Gupta, NC and Gupta, AK and Bhattacharya, R and Sharma, S and Rao, M},
title = {CRISPR/Cas9: efficient and emerging scope for Brassica crop improvement.},
journal = {Planta},
volume = {262},
number = {1},
pages = {14},
pmid = {40464976},
issn = {1432-2048},
support = {NASF/BGAM-9021/2022-23/NIPB//ICAR - National Agricultural Science Fund/ ; 2001-2019//Indian Council of Agricultural Research/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; *Brassica/genetics ; *Gene Editing/methods ; *Crops, Agricultural/genetics ; Genome, Plant ; Plant Breeding/methods ; Plants, Genetically Modified ; },
abstract = {CRISPR/Cas9 revolutionizes Brassica crop improvement by enhancing yield, quality, and stress resistance, providing a precise and versatile tool for genetic and agronomic advancements. The rapidly advancing CRISPR/Cas9 (Clustered regularly interspaced short palindromic repeats/CRISPR associated protein 9) technologies are being employed in both diploid and polyploid species of Brassica for gene functions and precise genetic improvements. CRISPR/Cas technology has sparked significant attention among the scientific community due to its affordability, precision, and effectiveness compared to other genome editing techniques. The recent discoveries highlight the diverse applications of the CRISPR/Cas9 genome editing tool in enhancing agriculturally important traits in Brassica species. This technology has been utilized to improve yield, quality, and resistance to both biotic and abiotic stresses globally. Here, we present an overview that encourages researchers to explore and improve the functionality and genetic progress of Brassica U-triangle species utilizing genome editing technologies. In addition, ethical considerations and concerns associated with CRISPR technologies are addressed, providing valuable insight into how CRISPR/Cas9 tools and have revolutionized crop improvement with special emphasis on Brassica for various agronomically and nutritionally important traits.},
}
@article {pmid40464068,
year = {2025},
author = {Liu, Y and Zhang, S and Hu, C},
title = {Cas7 meets Cas14: a strategic partnership in the type VII CRISPR-Cas.},
journal = {Protein &amp; cell},
volume = {16},
number = {2},
pages = {79-82},
pmid = {40464068},
issn = {1674-8018},
support = {23-0178-A0001//National University of Singapore Presidential Young Professorship (PYP)/ ; 23-1065-P0001//Singapore Ministry of Education Tier 1/ ; },
}
@article {pmid40461565,
year = {2025},
author = {Yang, C and Fang, Q and Li, M and Zhang, J and Li, R and Zhou, T and Wang, K and Deng, J and Wang, X and Huang, C and Feng, Y and Zhang, X and Shi, L and Bi, C and Zhang, X and Yu, J and Hao, J},
title = {Prime editor with rational design and AI-driven optimization for reverse editing window and enhanced fidelity.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {5144},
pmid = {40461565},
issn = {2041-1723},
support = {32471478//National Natural Science Foundation of China (National Science Foundation of China)/ ; 82203238//National Natural Science Foundation of China (National Science Foundation of China)/ ; },
mesh = {*Gene Editing/methods ; Humans ; CRISPR-Cas Systems/genetics ; CRISPR-Associated Protein 9/genetics/metabolism ; RNA, Guide, CRISPR-Cas Systems/genetics ; HEK293 Cells ; *Artificial Intelligence ; },
abstract = {Prime editing (PE) is a precise tool for introducing genetic mutations in eukaryotes. Extending the efficient editing scope and mitigating undesired byproducts are possible. We introduce reverse PE (rPE), a SpCas9-directed variant that enabled DNA editing at the 3' direction of HNH-mediated nick site. The rPE leveraging nCas9-D10A and rPE gRNA targeting the 5' direction of HNH-mediated nick site inscribes genetic alterations, achieving a reverse editing window and potentially high fidelity. HNH and reverse transcriptase engineered using protein language models in conjunction with La facilitate circular erPEmax and erPE7max, achieving editing efficiency up to 44.41% without nick gRNA or positive selection. Furthermore, our findings underscore the capability of rPE in inserting functionally enhanced variant (PIK3CD[E527G]) for cell therapy. By expanding the editing scope and enhancing genomic manipulability, rPE represents a meaningful advancement in prime editing, improving its utility for research and therapeutic applications.},
}
@article {pmid40461259,
year = {2025},
author = {Esser, SP and Turzynski, V and Plewka, J and Nuy, J and Moore, CJ and Banas, I and Soares, AR and Lee, J and Woyke, T and Probst, AJ},
title = {Differential Expression of Core Metabolic Functions in Candidatus Altiarchaeum Inhabiting Distinct Subsurface Ecosystems.},
journal = {Environmental microbiology reports},
volume = {17},
number = {3},
pages = {e70096},
pmid = {40461259},
issn = {1758-2229},
support = {DFG PR1603/2-1//Ministerium f&#xfc;r Kultur und Wissenschaft des Landes Nordrhein- Westfalen ("Nachwuchsgruppe Dr. Alexander Probst") and the German Research Foundation/ ; //Aker B.P.: GeneOil Project/ ; //U.S. Department of Energy Joint Genome Institute/ ; //DOE Office of Science User Facility/ ; //the Office of Science of the U.S. Department of Energy operated/ ; },
mesh = {*Ecosystem ; Germany ; Groundwater/microbiology ; Symbiosis ; Transcriptome ; Gene Expression Profiling ; Genome, Archaeal ; Multigene Family ; },
abstract = {Candidatus Altiarchaea are widespread across aquatic subsurface ecosystems and possess a highly conserved core genome, yet adaptations of this core genome to different biotic and abiotic factors based on gene expression remain unknown. Here, we investigated the metatranscriptome of two Ca. Altiarchaeum populations that thrive in two substantially different subsurface ecosystems. In Crystal Geyser, a high-CO2 groundwater system in the USA, Ca. Altiarchaeum crystalense co-occurs with the symbiont Ca. Huberiarchaeum crystalense, while in the Muehlbacher sulfidic spring in Germany, an artesian spring high in sulfide concentration, Ca. A. hamiconexum is heavily infected with viruses. We here mapped metatranscriptome reads against their genomes to analyse the in situ expression profile of their core genomes. Out of 537 shared gene clusters, 331 were functionally annotated and 130 differed significantly in expression between the two sites. Main differences were related to genes involved in cell defence like CRISPR-Cas, virus defence, replication, transcription and energy and carbon metabolism. Our results demonstrate that altiarchaeal populations in the subsurface are likely adapted to their environment while influenced by other biological entities that tamper with their core metabolism. We consequently posit that viruses and symbiotic interactions can be major energy sinks for organisms in the deep biosphere.},
}
@article {pmid40460174,
year = {2025},
author = {Tan, YS and Mo, BT and Li, GC and Guo, YR and Zhang, JZ and Wang, CZ},
title = {HarmGR13 mediates myo-inositol taste perception in Helicoverpa armigera larvae.},
journal = {PLoS genetics},
volume = {21},
number = {6},
pages = {e1011744},
pmid = {40460174},
issn = {1553-7404},
mesh = {Animals ; *Inositol/metabolism ; Larva/genetics/physiology ; *Taste Perception/genetics ; *Insect Proteins/genetics/metabolism/chemistry ; Sensilla/metabolism/physiology ; *Moths/genetics/physiology ; CRISPR-Cas Systems ; Phylogeny ; Ribose/metabolism ; *Receptors, Cell Surface/genetics/metabolism ; Taste/genetics ; Helicoverpa armigera ; },
abstract = {Myo-inositol, a sugar alcohol produced by most plants, serves as a nutrient and feeding stimulant for many phytophagous insects. Inositol-sensitive taste sensilla have been characterized in many Lepidoptera larvae, but their molecular bases remain unclear. In this study, we determined the gustatory receptors (GRs) for myo-inositol in larva of Helicoverpa armigera, a worldwide crop pest. First, electrophysiological analyses revealed that medial sensilla styloconica strongly responded to myo-inositol and ribose, with weaker responses to xylose, and one GRN inside sensillum may mediate the response to these three chemicals. Based on phylogenetic analysis of sugar GRs of Lepidoptera insects and previous results on Bombyx mori, we then selected two candidate GRs, HarmGR13 and HarmGR11. Using CRISPR-Cas9, we generated knockout mutants for the two GR genes. Knocking out HarmGR13 abolished the responses of the sensilla to myo-inositol, ribose, and xylose, while knocking out HarmGR11 showed no changes. Behavioral assays confirmed that larvae of HarmGR13 homozygous mutant lost the feeding preference to myo-inositol which the wild-type larvae had. Further functional analysis with Xenopus oocytes expressing system and two-electrode voltage-clamping demonstrated that myo-inositol and ribose specifically induced concentration-dependent currents in HarmGR13-expressing oocytes. Structural predictions and molecular docking of HarmGR13 revealed three amino acid residues might be involved in ligand binding. Mutation of these residues resulted in loss of oocyte responses to myo-inositol and ribose. We reveal that HarmGR13 is a receptor that mediates the activity of the cells sensitive to inositol and ribose in larvae, providing new molecular targets for the strategy of regulating the feeding behavior of pests by modifying taste.},
}
@article {pmid40460129,
year = {2025},
author = {Jiang, W and Wang, S and Ahlheit, D and Fumagalli, T and Yang, Z and Ramanathan, S and Jiang, X and Weber, T and Dahlin, J and Borodina, I},
title = {High-throughput metabolic engineering of Yarrowia lipolytica through gene expression tuning.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {122},
number = {23},
pages = {e2426686122},
pmid = {40460129},
issn = {1091-6490},
support = {NNF20CC0035580//Novo Nordisk Fonden (NNF)/ ; NNF20OC0060809//Novo Nordisk Fonden (NNF)/ ; NNF21OC0072559//Novo Nordisk Fonden (NNF)/ ; No. 101123257//EC | H2020 | PRIORITY 'Excellent science' | H2020 European Research Council (ERC)/ ; },
mesh = {*Yarrowia/genetics/metabolism ; *Metabolic Engineering/methods ; Transcription Factors/genetics/metabolism ; Promoter Regions, Genetic ; *Gene Expression Regulation, Fungal ; CRISPR-Cas Systems ; Plasmids/genetics ; Gene Library ; Fungal Proteins/genetics/metabolism ; },
abstract = {The challenge of accurately predicting which genetic alternations lead to the desired phenotype necessitates high-throughput metabolic engineering approaches where numerous hypotheses can be tested simultaneously. We describe the CRISPR-Cas9-based method TUNE[YALI] that enables high-throughput tuning of gene expression in the common industrial yeast Yarrowia lipolytica. The method is based on replacing the promoters of the target genes with native Y. lipolytica promoters of varying strengths or removing the promoters entirely. To demonstrate the method's capabilities, we created a plasmid library that targets 56 transcription factors (TFs) and changes the expression of each TF to seven different levels. We transformed this library into reference and betanin-producing strains of Y. lipolytica and screened the resulting clones for changes in morphology, thermotolerance, or improved betanin production. The genetic markup of the yeast clones with the desired phenotypic changes was determined by sequencing the inserted plasmids. We identified multiple TFs whose regulatory changes increased thermotolerance, two TFs that eliminated pseudohyphal growth, and several TFs that increased betanin production. Analogous libraries can be designed to target any chosen group of genes and even all the genes. The libraries can be shared and reused, accelerating applied strain development projects and fundamental functional genomics research (TUNE[YALI]-TF kit and TUNE[YALI]-TF library are available via AddGene under catalog numbers #1000000255 and #217744).},
}
@article {pmid40459995,
year = {2025},
author = {Wu, JL and Zheng, SS and Wang, L and Xiang, X and Li, F and Lv, ML and Wu, Q and Huang, ZX and Miao, HB},
title = {CRISPR-Cas System-Mediated Genetic Modification in Bacillus spp.: Current Status and Future.},
journal = {Journal of agricultural and food chemistry},
volume = {73},
number = {24},
pages = {14760-14775},
doi = {10.1021/acs.jafc.5c03140},
pmid = {40459995},
issn = {1520-5118},
mesh = {*CRISPR-Cas Systems ; *Bacillus/genetics/metabolism ; *Gene Editing/methods ; Bacterial Proteins/genetics/metabolism ; },
abstract = {Bacillus spp. are a group of Gram-positive bacteria that have shown significant potential for development in recent years. It is capable of utilizing low-cost substrates to produce various high-value-added compounds, making it widely applicable in fields such as feed, pharmaceuticals, and food. The clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated (Cas) system-mediated genetic modification is recognized as one of the most efficient technologies. The application of this technology for the genetic improvement of Bacillus spp. greatly enhanced the production performance of these strains. In this review, we summarize the various CRISPR-Cas systems that have been applied to Bacillus spp., with a particular focus on systematically outlining the strategies for implementing CRISPR-Cas-mediated genetic modification in these bacteria. Notably, homologous recombination is the most widely used strategy, while base editing is emerging as a novel and precise approach. Additionally, we discuss the importance of expression regulation strategies in establishing Bacillus spp. as a cell factory. Finally, we propose potential solutions to current technical challenges, providing insights for the development of high-performance genetically modified Bacillus spp. production strains.},
}
@article {pmid40459840,
year = {2025},
author = {Keerthana, R and Rakshana, P and Salunkhe, SR and Sakthi, AR and Kokiladevi, E and Saraswathi, T and Pushpam, R and Raveendran, M and Sudha, M},
title = {CRISPR-Cas9 mediated enhancement of abiotic stress resilience in tomato: a comprehensive review of target genes.},
journal = {Molecular biology reports},
volume = {52},
number = {1},
pages = {538},
pmid = {40459840},
issn = {1573-4978},
mesh = {*Solanum lycopersicum/genetics ; *CRISPR-Cas Systems/genetics ; Gene Editing/methods ; *Stress, Physiological/genetics ; Plant Breeding/methods ; Plants, Genetically Modified/genetics ; },
abstract = {Tomato (Solanum lycopersicum L.), a major vegetable crop grown worldwide, is consumed as both fresh and processed products. Concerns about the impact of abiotic stresses on tomato production are growing worldwide as climate change alters global weather patterns, adversely affecting crop yield and produce quality due to stresses like salt, heat, cold, and drought. Conventional breeding approaches such as hybridization, marker-assisted selection (MAS), and mutation breeding, have long been utilized to improve tomato resilience against abiotic stresses. These approaches are often hampered by extensive field trials, and require multiple generations limiting their efficiency in rapidly developing stress-tolerant cultivars. The efforts of traditional breeding systems are hindered by the narrow genetic base of tomatoes which poses a major bottleneck. Researchers have utilized CRISPR-Cas genome-editing technology to address this challenge to offer a precise and accelerated alternative for enhancing stress resilience in tomato. This versatile tool has gained attention for its simple, precise, and effective gene-editing capabilities. CRISPR-Cas based genome editing has successfully modified key genes related to stress-response pathways, enhancing abiotic stress resilience. Developing resistant cultivars help mitigate the impact of abiotic stress, thereby contributing to increased food production and food security. This review highlights recent progress in use of CRISPR-Cas9 gene editing to enhance tomato resilience to abiotic stresses.},
}
@article {pmid40459690,
year = {2025},
author = {Lakhani, H and Kumar, N and Jangra, A and Negi, S and Dholariya, T and Tiwari, S},
title = {Streamlined protoplast transfection system for in-vivo validation and transgene-free genome editing in Banana.},
journal = {Transgenic research},
volume = {34},
number = {1},
pages = {28},
pmid = {40459690},
issn = {1573-9368},
support = {BT/PR25789/GET/119/97/2017//Department of Biotechnology, Ministry of Science and Technology, India/ ; BIRAC/Tech Transfer/08/I2/QUT-BBF//Biotechnology Industry Research Assistance Council/ ; },
mesh = {*Musa/genetics/growth &amp; development ; *Protoplasts/metabolism ; *Gene Editing/methods ; *Transfection/methods ; *Plants, Genetically Modified/genetics/growth &amp; development ; CRISPR-Cas Systems/genetics ; Transgenes/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; Genome, Plant ; },
abstract = {The advancement in the CRISPR/Cas system has significantly streamlined genome editing in plants, rendering it simple, reliable, and efficient. However, the development of transgene-free crops is a challenging task for vegetatively propagated plants like banana. In the present study, we established banana protoplasts-based versatile and efficient platform for genome editing to overcome this limitation. Herein, a protocol has been optimized for protoplast isolation by considering leaf and embryogenic cell suspension (ECS) of banana cultivar Grand Naine. Freshly prepared ECS was identified as the best source for protoplast isolation. The protoplast viability and competency were checked by transfection with plasmid and RNP complex. Polyethylene glycol (PEG)-mediated protoplast transfection using pCAMBIA1302 and pJL50TRBO vectors showed GFP expression with 30 and 70% efficiency, respectively, eventually proving the protocol's efficacy. Further, gRNAs targeting banana β-carotene hydroxylase gene are validated by in-vitro cleavage test and subsequently used for RNP complex formation with varied ratios (1:1, 1:2, 1:5, and 1:10) of SpCas9 to gRNA1. Among these, a 1:2 molar ratio proved best to generate indel frequency with 7%. Sequencing analysis of the target amplicon revealed mutations upstream of the PAM region, specifically with gRNA1, among the three in-vitro validated gRNAs. This study evaluated the effectiveness of gRNAs in-vitro and in-vivo, yielding inconsistent results that highlight the need for comprehensive in-vivo validation of their functionality. Conclusively, the optimized protocol for banana transfection has the potential to be harnessed for the generation of transgene-free genetically improved banana.},
}
@article {pmid40457455,
year = {2025},
author = {Zhang, T and Meng, Z and Yu, H and Zhang, Z and Liu, G and Qiu, A and Zheng, W and Ding, P and Kai, T},
title = {An integrated multi-mode detection platform based on CRISPR/Cas 12a and aptamers for ultra-sensitive identification of sulfamethazine and genes associated with sulfonamide resistance.},
journal = {Journal of nanobiotechnology},
volume = {23},
number = {1},
pages = {408},
pmid = {40457455},
issn = {1477-3155},
support = {2025ZZTS0167//Graduate Research Innovation Program of Central South University/ ; 82373635//the National Natural Science Foundation of China/ ; 2022RC1206//the Science and Technology Innovation Program of Hunan Province/ ; 2023QYJC013//the Central South University Research Program of Advanced Interdisciplinary Studies/ ; },
mesh = {*Sulfamethazine/analysis/pharmacology ; *Aptamers, Nucleotide/chemistry ; *CRISPR-Cas Systems/genetics ; *Sulfonamides/pharmacology ; Biosensing Techniques/methods ; *Anti-Bacterial Agents/analysis/pharmacology ; Electrochemical Techniques ; *Drug Resistance, Bacterial/genetics ; Metal-Organic Frameworks/chemistry ; Benzidines/chemistry ; Limit of Detection ; },
abstract = {The production and buildup of sulfamethazine (SMZ) and resistance genes for sulfonamide antibiotics (sul1) pose a serious risk to environmental and public health safety. Creating advanced sensing systems that are both highly sensitive and selective for the prolonged observation of SMZ concentrations in the environment, along with the quantification of sul1 gene prevalence, aims to identify trends in resistance, posing a considerable challenge. Here, we devised a platform (SMZ-sul1 multi-mode detection platform) that allows for the fluorescence detection of SMZ in environmental samples. This is achieved through the competition for the aptamer between the complementary base and SMZ, along with the colorimetric, photothermal, and electrochemical tracking of sul1, using a magnetic separation unit (FP@cDNA). MOF-818@PtPd (MPP) nanozymes with high peroxide mimetic enzyme activity were linked to FP@cDNA through Zr-O-P bond and employed as a catalyst for the 3,3',5,5'-tetramethylbenzidine (TMB) oxidation, as well as for electrocatalytic hydrogen peroxide (H2O2) reduction. The ability of Cas12a to perform trans cleavage was activated by its precise identification of the sul1, leading to the non-selective cutting of single-stranded DNA (ssDNA). Thereafter, the MPP nanoparticles were released into the supernatant, where they catalyzed the oxidation of TMB. Alternatively, the functioning CRISPR/Cas12a system specifically targeted and cleaved ssDNA present on the electrode, resulting in altered loading of MPP nanozymes and a decrease in the current associated with the catalytic reduction of H2O2. The remarkable magnetic separation capabilities of FP@cDNA, combined with the superior target recognition features of CRISPR/Cas12a and aptamer, facilitated the creation of a highly sensitive detection system, achieving detection limits of 0.67 pM for SMZ and 7.6 fM for sul1, and exhibit great potential for monitoring and prediction in the field of public health.},
}
@article {pmid40456823,
year = {2025},
author = {Hong, D and Shu, M and Liu, J and Liu, L and Cheng, H and Zhu, M and Du, Y and Xu, B and Hu, D and Liu, Z and Zhao, Y and Dai, J and Lu, F and Huang, J},
title = {Divergent combinations of enhancers encode spatial gene expression.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {5091},
pmid = {40456823},
issn = {2041-1723},
support = {92474104//National Natural Science Foundation of China (National Science Foundation of China)/ ; 32370586//National Natural Science Foundation of China (National Science Foundation of China)/ ; },
mesh = {Animals ; *Enhancer Elements, Genetic/genetics ; Mice ; Humans ; Brain/metabolism/embryology ; Chromatin/metabolism/genetics ; Basic Helix-Loop-Helix Transcription Factors/genetics/metabolism ; Breast Neoplasms/genetics ; Spinal Cord/metabolism/embryology ; Gene Expression Regulation, Developmental ; Melanoma/genetics ; CRISPR-Cas Systems ; },
abstract = {Spatial transcriptomics and epigenomics have enabled mapping gene regulation in the tissue context. However, it remains poorly understood how spatial gene expression patterns are orchestrated by enhancers. Here we build eSpatial, a computational framework that deciphers spatially resolved enhancer regulation of gene expression by integrating spatial profiles of gene expression and chromatin accessibility. Applying eSpatial to diverse spatial datasets, including mouse embryo and brain, as well as human melanoma and breast cancer, we reveal a "spatial enhancer code", in which divergent combinations of enhancers regulate the same gene in spatially segregated domains. We validate the spatial enhancer code using public spatial datasets such as VISTA, Allen in situ hybridization (ISH), and H3K27ac MERFISH. Moreover, we conduct transgenic reporter assays and in vivo CRISPR/Cas9-mediated perturbation experiments to confirm the Atoh1 spatial enhancer code in determining Atoh1 spatial expression in mouse embryonic spinal cord and brain. Our study establishes the spatial enhancer code concept, revealing how combinations of enhancers dynamically shape gene expression across diverse biological contexts, providing insights into tissue-specific regulatory mechanisms and tumor heterogeneity.},
}
@article {pmid40456709,
year = {2025},
author = {Hanlon, MB and Shohet, JM and Wolfe, SA},
title = {Selective targeting of genome amplifications and repeat elements by CRISPR-Cas9 nickases to promote cancer cell death.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {5126},
pmid = {40456709},
issn = {2041-1723},
support = {R01 CA275945/CA/NCI NIH HHS/United States ; R01CA275945//U.S. Department of Health &amp; Human Services | NIH | National Cancer Institute (NCI)/ ; },
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Gene Amplification ; *Neuroblastoma/genetics/pathology ; Cell Line, Tumor ; N-Myc Proto-Oncogene Protein/genetics ; Cell Death/genetics ; *Deoxyribonuclease I/metabolism/genetics ; DNA Breaks, Double-Stranded ; Neoplasms/genetics ; },
abstract = {Focal gene amplification serves as an oncogenic driver during tumorigenesis and is a hallmark of many forms of cancer. Oncogene amplifications promote genomic instability, which is integral to cancer cell survival and evolution. However, focal gene amplification potentially affords an opportunity for therapeutic exploitation. As a proof-of-concept, we leverage CRISPR-Cas9 nickase to selectively promote cancer cell death in MYCN-amplified neuroblastoma in a gene amplification-dependent manner. Our analysis demonstrates that CRISPR-Cas9 nickase can generate a lethal number of highly toxic, replication-dependent double-strand breaks in cells harboring amplified loci. Furthermore, we demonstrate that Cas9 nickase-mediated toxicity can be modulated in combination with small molecule inhibitors targeting key regulators of the DNA-damage response or cell death pathways. Importantly, our findings in MYCN-amplified neuroblastoma translate to other cancer types with distinct oncogene amplifications.},
}
@article {pmid40456230,
year = {2025},
author = {Huda, NU and Hasan, KA and Saleem, F and Naz, S and Khan, S and Mirani, ZA and Noman Syed, M and Alswat, AS and Sohail, M},
title = {Coriander borne Salmonella superbug: genomic assessments of chromosomal and plasmid-associated resistance, virulence and MGEs of XDR Salmonella enterica Typhi NH1.},
journal = {Pathogens and global health},
volume = {},
number = {},
pages = {1-15},
doi = {10.1080/20477724.2025.2513769},
pmid = {40456230},
issn = {2047-7732},
abstract = {The drug-resistant strains of Salmonella enterica serovar Typhi (S. Typhi) are serious threats to health sectors worldwide. This study focuses on genomic characterization of environmentally derived an extensively drug resistant (XDR) strain of Salmonella Typhi, highlighting its potential to cause significant outbreak. The XDR strain (labeled as NH1) was isolated from fresh coriander, and characterized through whole-genome sequencing to investigate its lineage diversity, niche adaptation, sources and resistant mechanism. The NH1 strain exhibits phylogenomic association with clinical lineages S. Typhi NCTC8385 and ASM3025476. Its genome, assembled in 67 contigs, exhibited expected coverage and GC content, harboring a mega-plasmid, virulence factors, and intrinsic and extrinsic antimicrobial resistance genes. Plasmid annotation revealed IncQ1 and IncY plasmids responsible for the acquired resistance. SPIFinder identified SPI1-10 pathogenicity islands, and the CRISPR-Cas locus, associated with bacterial defense, bile salt resistance and biofilm formation, was present. NH1 strain also possesses numerous mobile elements (proteins and transposases), 12 prophages, and secretion systems (types I-IV, VI), aiding its survival in environment like vegetables. The genomic characterization of S. Typhi NH1 highlights the emergence of coriander-borne XDR strain, underscoring the need for targeted prevention to mitigate its public health impact.},
}
@article {pmid40455764,
year = {2025},
author = {De Pablo-Moreno, JA and González-Brusi, L and Miguel-Batuecas, A and Bermejo-Álvarez, P and Revuelta, L and Liras, A},
title = {Development of a novel and viable knock-in factor V deficiency murine model: Utility for an ultra-rare disease.},
journal = {PloS one},
volume = {20},
number = {6},
pages = {e0321864},
pmid = {40455764},
issn = {1932-6203},
mesh = {Animals ; Mice ; *Disease Models, Animal ; *Factor V Deficiency/genetics/pathology ; *Factor V/genetics/metabolism/chemistry ; *Gene Knock-In Techniques ; Humans ; Female ; Male ; CRISPR-Cas Systems ; Phenotype ; Homozygote ; Mutation, Missense ; *Rare Diseases/genetics ; Partial Thromboplastin Time ; Prothrombin Time ; },
abstract = {Factor V deficiency is a congenital coagulation disorder characterized by the absence or malfunction of factor V (FV). The purpose of this study was to develop a viable FV-deficient mouse model using CRISPR/Cas9 technology. A viable pathological model of the disease was not available to develop new therapies. A previous in silico study was performed to select a mutation causing a mild disease phenotype in humans (Thr1898Met missense). Such mutation was replicated in mice by CRISPR-mediated homology directed repair. Following crossing, homozygous individuals were subjected to coagulometry assays, including FV levels, prothrombin time (PT), and activated partial thromboplastin time (aPTT). The in silico study suggested that the mutation destabilizes FV structure of both mouse and human variants, putatively producing a mild phenotype of the disease in mice. Mendelian inheritance was observed in the offspring. No spontaneous signs of blood clotting disturbances, premature deaths or gestational dysfunctions were observed. FV levels in homozygous animals were 24.5% ± 5.1; 39.7 sec ± 2.8; PT was 61.8% ± 6.3; 23.4 sec ± 1.6 (INR = 1.47 ± 0.12); and aPTT was 46.9 sec ± 3.2. A viable FV-deficient mouse model was generated by introducing a missense mutation in FV. The model exhibits a mild phenotype of the disease, akin to that observed in humans.},
}
@article {pmid40455288,
year = {2025},
author = {Hu, J and Yu, W and Cui, J and Zhang, L and Yu, W},
title = {Recent advances in diagnostic technologies for postoperative central nervous system infections: a review.},
journal = {Neurological sciences : official journal of the Italian Neurological Society and of the Italian Society of Clinical Neurophysiology},
volume = {},
number = {},
pages = {},
pmid = {40455288},
issn = {1590-3478},
abstract = {Postoperative central nervous system infections (PCNSIs), including meningitis, cerebral abscesses, and implant-associated infections, represent critical complications following neurosurgical procedures. These infections pose significant risks to patient outcomes due to delayed diagnosis, escalating antimicrobial resistance, and limited therapeutic efficacy. Conventional diagnostic approaches, such as cerebrospinal fluid (CSF) analysis, microbial cultures, and neuroimaging, exhibit notable limitations in sensitivity, specificity, and rapidity. This review highlights transformative technologies reshaping PCNSI diagnostics, including molecular assays (e.g., quantitative PCR, digital droplet PCR), metagenomic next-generation sequencing (mNGS), CRISPR-based pathogen detection platforms, metabolomics, and advanced molecular imaging modalities. Furthermore, we address translational challenges in clinical adoption, including cost barriers, standardization gaps, and the need for interdisciplinary collaboration. Emerging artificial intelligence (AI)-driven strategies are proposed to optimize pathogen identification, predict antimicrobial resistance profiles, and tailor personalized therapeutic regimens.},
}
@article {pmid40455261,
year = {2025},
author = {Zhang, M and Zhou, L and Afridi, M and Guo, H and Cheng, H},
title = {An efficient hairy root system for validation of CRISPR/Cas system activities in cotton.},
journal = {Functional &amp; integrative genomics},
volume = {25},
number = {1},
pages = {116},
pmid = {40455261},
issn = {1438-7948},
support = {NK2022010303//the Ministry of Agriculture/ ; B23CJ0208//Hainan Seed Industry Laboratory/ ; YSPTZX202502//Hainan Province Academician Innovation Platform/ ; },
mesh = {*Gossypium/genetics/growth &amp; development/microbiology ; *Plant Roots/genetics/growth &amp; development ; *CRISPR-Cas Systems ; Agrobacterium/genetics ; *Gene Editing/methods ; Plants, Genetically Modified/genetics ; },
abstract = {The hairy root induction system has been widely applied in studying gene expression and function in plant species due to its rapidity and efficiency. The hairy root system is an efficient tool for evaluating the activities of CRISPR/Cas systems. Cotton hairy roots were primarily induced through cotton tissue culture under aseptic conditions and by injecting cotton stem tips under non-aseptic conditions. However, both methods are lab-intensive and time-consuming. In this study, an efficient cotton hairy root induction procedure was established via infecting cotton hypocotyls with Agrobacterium rhizogenes under non-sterile conditions. Cotton seedlings with expanded cotyledons were decapitated with a slanted cut, and the residual hypocotyl (maintained 1 cm apical portion) was inoculated with A. rhizogenes. Over 90% of the infected explants from all three tested varieties could produce hairy roots after 8 days of inoculation. The effictiveness of the method was tested by overexpressing two reporter genes (eGFP and GUS). The transformation efficiency of the GUS and eGFP were ranged from50-68.18% and 40.9-68.18%. In addition, the editing efficiency of target sites in different CRISPR/Cas systems were also tested in hairy root. This method provided technical support for screening suitable target sites for cotton gene editing.},
}
@article {pmid40454797,
year = {2025},
author = {Wu, J and Yan, Q and Qiu, H and Gao, EB},
title = {Integrating Enzyme-DNA Complex and CRISPR/Cas12a for Robust Norovirus Detection.},
journal = {Journal of medical virology},
volume = {97},
number = {6},
pages = {e70426},
doi = {10.1002/jmv.70426},
pmid = {40454797},
issn = {1096-9071},
support = {//The Ningbo Clinical Research Center for Children's Health and Diseases (no. 2019A21002), the Ningbo public welfare project (no. 2022S127), the Ningbo key discipline Pediatrics (no. 2022-B17), the Innovation Project of Distinguished Medical Team in Ningbo (no. 2022020405). Ningbo Medical and Health Brand Discipline (PPXK2024-06)./ ; },
mesh = {*Norovirus/isolation &amp; purification/genetics ; Humans ; *Caliciviridae Infections/diagnosis/virology ; *CRISPR-Cas Systems ; Sensitivity and Specificity ; *Gastroenteritis/virology/diagnosis ; *Nucleic Acid Amplification Techniques/methods ; RNA, Viral/genetics ; *Molecular Diagnostic Techniques/methods ; Clustered Regularly Interspaced Short Palindromic Repeats ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {Human norovirus (NoV) is a primary cause of acute gastroenteritis in children, making accurate and rapid detection essential for effective disease prevention and control. In this study, we developed a sensitive and efficient platform for pathogen nucleic acid detection by integrating asymmetric nucleic acid sequence-based amplification (asymmetric NASBA), enzyme-DNA molecular complex, and the clustered regularly interspaced short palindromic repeats (CRISPR) system, namely an A-enDMC platform. The target recognition capability of the enzyme-DNA complex operates independently from the signal amplification function of the CRISPR system. By decoupling the CRISPR reaction from the dependence on specific target sequences, the platform's universality and modularity are enhanced. The assay is fast (< 1.5 h), highly sensitive (< 5 copies/µL), and demonstrates no cross-reactivity with other common viruses. Compared to the widely used RT-qPCR method, the platform demonstrates high consistency in detection results, with the detection coincidence rate of 96.77% and a kappa value of 0.87. This platform provides a versatile technological tool for highly sensitive and specific RNA detection, demonstrating its extensive potential in real sample analysis.},
}
@article {pmid40453079,
year = {2025},
author = {Lund, S and Gong, C and Yu, X and Staudt, LM and Hodson, DJ and Scheich, S},
title = {Strategies for CRISPR-based knock-ins in primary human B cells and lymphoma cell lines.},
journal = {Frontiers in immunology},
volume = {16},
number = {},
pages = {1589729},
pmid = {40453079},
issn = {1664-3224},
mesh = {Humans ; *CRISPR-Cas Systems ; *Gene Knock-In Techniques/methods ; *Gene Editing/methods ; *B-Lymphocytes/metabolism/immunology ; Cell Line, Tumor ; *Lymphoma, Large B-Cell, Diffuse/genetics ; },
abstract = {Since its advent about ten years ago, the CRISPR-Cas9 system has been frequently used in biomedical applications. It has advanced various fields, and CRISPR-Cas9-based therapeutics have shown promising results in the treatment of specific hematological diseases. Furthermore, CRISPR gene editing technologies have revolutionized cancer research by enabling a broad range of genetic perturbations, including genetic knockouts and precise single nucleotide changes. This perspective focuses on the state-of-the-art methodology of CRISPR knock-ins to engineer immune cells. Since this technique relies on homology-directed repair (HDR) of double-strand breaks (DSBs) induced by the Cas9 enzyme, it can be used to introduce specific mutations into the target genome. Therefore, this methodology offers a valuable opportunity to functionally study specific mutations and to uncover their impacts not only on overall cell functions but also on the mechanisms behind cancer-related alterations in common signaling pathways. This article highlights CRISPR knock-in strategies, protocols, and applications in cancer and immune research, with a focus on diffuse large B cell lymphoma.},
}
@article {pmid40451886,
year = {2025},
author = {Okamoto, M and Sasamoto, K and Takahashi-Nakaguchi, A and Zhao, F and Yamaguchi, M and Chibana, H},
title = {CRISPR-Cas9 RNP-Mediated Deletion of ERG25 in Non-albicans Candida Species, Including Candida auris.},
journal = {Medical mycology journal},
volume = {66},
number = {2},
pages = {35-43},
doi = {10.3314/mmj.24-00023},
pmid = {40451886},
issn = {2186-165X},
mesh = {*CRISPR-Cas Systems ; Candida glabrata/genetics ; *Candida auris/genetics ; *Fungal Proteins/genetics ; *Gene Deletion ; *Candida/genetics ; Candidiasis/microbiology ; Antifungal Agents/pharmacology ; },
abstract = {The incidence of infections caused by non-albicans Candida (NAC) species, including Candida glabrata and Candida tropicalis, has recently increased. Furthermore, Candida auris, a multidrug-resistant yeast, poses a serious threat to global health. The development of novel antifungal agents with alternative mechanisms of action is necessary to combat these fungi. However, genetic analyses of the virulence factors in these NAC species are insufficient for this purpose. Recent advancements in the clustered regularly interspaced short palindromic repeat (CRISPR)-Cas9 system have facilitated enhanced the genetic analysis of NAC species. The RNP-based system, in which the Cas9-gRNA complex is assembled in vitro and introduced into cells, offers a simplified approach to genetic modification, eliminating the need for species-specific plasmids. Previous our research identified the ERG25 gene, which encodes C-4 sterol methyl oxidase, as a promising antifungal target in C. glabrata. This study demonstrated deletion of the ERG25 homolog in C. glabrata and C. auris using an RNP-based CRISPR-Cas9 system. The deletion of ERG25 in C. auris and C. glabrata indicated that Erg25 is crucial for the survival of these pathogenic yeasts within the host. Furthermore, we have successfully deleted the ERG25 alleles in C. tropicalis and Candida parapsilosis, demonstrating the effectiveness of using both the CRISPR-Cas9 and Cre-loxP systems in these species for the first time.},
}
@article {pmid40451196,
year = {2025},
author = {Smirnov, AV and Yunusova, AM},
title = {Novel CRISPR/Cas9-Based Approaches for Quantitative Study of DSB Repair Mechanics.},
journal = {Biochemistry. Biokhimiia},
volume = {90},
number = {4},
pages = {437-456},
doi = {10.1134/S0006297924601813},
pmid = {40451196},
issn = {1608-3040},
mesh = {*CRISPR-Cas Systems ; *DNA Breaks, Double-Stranded ; *DNA Repair ; Humans ; Animals ; },
abstract = {This review examines modern approaches to studying double-strand break (DSB) DNA repair in mammalian cells, employing the CRISPR/Cas9 system. Due to its flexibility and efficacy, the Cas9 nuclease is used in numerous genetic reporters. We discuss various fluorescence-based genetic reporters used to monitor the repair process. In addition, among the innovative Cas9-based methods, special attention is given to the techniques that examine both single and multiple DSBs, including approaches such as DSB-TRIP and ddXR. These methods open new possibilities for investigating structural rearrangements or analyzing random genomic sites. Additionally, the review considers how DSBs induced by Cas9 differ from those made by other nucleases and how these peculiarities could impact DNA repair mechanisms. Understanding these differences is crucial for planning experiments aimed at studying DSB repair.},
}
@article {pmid40451118,
year = {2025},
author = {Piñeiro-Silva, C and Gadea, J},
title = {Optimizing gene editing in pigs: The role of electroporation and lipofection.},
journal = {Animal reproduction science},
volume = {278},
number = {},
pages = {107874},
doi = {10.1016/j.anireprosci.2025.107874},
pmid = {40451118},
issn = {1873-2232},
mesh = {Animals ; *Electroporation/veterinary/methods ; Swine/genetics ; *Gene Editing/veterinary/methods ; *Animals, Genetically Modified ; },
abstract = {The production of genetically modified pigs is becoming increasingly important in both the agricultural and biomedical fields. Optimization of these processes is a key objective to improve the precision, scalability and viability of genetically modified animals for research and commercial applications. Among the available techniques, electroporation and lipofection have emerged as promising alternatives to traditional methods such as microinjection and somatic cell nuclear transfer (SCNT) due to their simplicity, cost-effectiveness, and potential for high-throughput applications. These methods allow the direct delivery of CRISPR/Cas components into zygotes and embryos, reducing the technical expertise required and bypassing some of the challenges associated with cloning. This review examines the application, efficacy, and outcomes of electroporation and lipofection as gene editing techniques in porcine gametes and embryos. We provide a comprehensive synthesis of recent advances, compare their efficacy, and discuss their potential in agricultural and biomedical research. The principles and mechanisms of both methods are reviewed, highlighting their advantages, such as cost-effectiveness and ease of implementation, over traditional approaches such as microinjection. In addition, we address their limitations, including variability in efficiency, and discuss recent protocol optimizations aimed at improving reproducibility and applicability. By analyzing these developments, this review provides valuable insights into the evolving role of electroporation and lipofection in porcine genetic modification strategies.},
}
@article {pmid40450609,
year = {2025},
author = {Pal, S and Krishna, R and Dedhia, L and Panwar, HS and Karkute, SG and Rai, N and Kumar, R and Pandey, S and Singh, AK},
title = {CRISPR mediated gene editing for economically important traits in horticultural crops: progress and prospects.},
journal = {Transgenic research},
volume = {34},
number = {1},
pages = {26},
pmid = {40450609},
issn = {1573-9368},
mesh = {*Gene Editing/methods ; *Crops, Agricultural/genetics/growth &amp; development ; *CRISPR-Cas Systems/genetics ; *Plants, Genetically Modified/genetics/growth &amp; development ; Plant Breeding ; Genome, Plant ; },
abstract = {Horticultural crops, with their cost-effectiveness, rich mineral and vitamin content, and high yield potential, have become indispensable worldwide for ensuring food and nutritional security. With the world's population on the rise and climate change becoming more prominent, it is crucial to focus on creating resilient, high-yielding crop varieties that can withstand the changing climate. Genetic improvement of different horticultural crops using conventional tools is both time-consuming and labourious. However, the breeding period can be cut short by adopting modern breeding techniques, including CRISPR/Cas-mediated genome editing. In the present review, we discuss the progress made so far through genome editing to improve several horticultural crops for various traits like stress resistance, morphology, nutritional attributes, quality, shelf life, male sterility, architecture and economic yield. We have also discussed the emerging CRISPR technologies like base editing, epigenome editing, CRE editing, transposon-based editing, prime editing etc., along with their pros and cons and the future prospects. The ethical considerations for commercialization and current regulatory frameworks for gene-edited products have also been discussed.},
}
@article {pmid40450056,
year = {2025},
author = {Ortiz-Rodríguez, LA and Cabanzo, R and Jaimes-Dueñez, J and Mendez-Sanchez, SC and Duque, JE},
title = {TropD-detector a CRISPR/LbCas12a-based system for rapid screening of Trypanosoma cruzi in Chagas vectors and reservoirs.},
journal = {Scientific reports},
volume = {15},
number = {1},
pages = {19107},
pmid = {40450056},
issn = {2045-2322},
mesh = {*Trypanosoma cruzi/genetics/isolation &amp; purification ; *Chagas Disease/parasitology/diagnosis ; Animals ; *CRISPR-Cas Systems ; *Disease Reservoirs/parasitology ; Rhodnius/parasitology ; Cytochromes b/genetics ; Endodeoxyribonucleases/genetics ; Histones/genetics ; CRISPR-Associated Proteins/genetics ; Bacterial Proteins ; },
abstract = {Chagas disease, also known as American Trypanosomiasis, is a zoonosis with global distribution caused by the parasite Trypanosoma cruzi, primarily transmitted through the feces of infected triatomines. The emergence of new cases highlights the importance of early pathogen detection in vectors and reservoirs to generate effective control strategies and establish preventive policies. The objective of this study was to design and validate a detection system of T. cruzi based on specific DNA cleavage, activation of Cas12a and trans-cleavage, targeting the genes Cytochrome B (Cytb), 18 S ribosomal subunit (SR18 s), and histone (H2 A). This system was validated for their uses in both vectors and reservoirs of the parasite. The initial step involved performing a bioinformatic analysis of the target genes, followed by the design of RNA guides specific to each cleavage site, along with primers for amplifying the target region through PCR and RPA. Subsequently, we sequenced the amplified DNA target and validated the detection system using T. cruzi DNA extracted from naturally infected Rhodnius pallescens in the metropolitan area of Bucaramanga, Colombia. After standardizing the method, we tested the CRISPR/Cas system with Silvio X10 laboratory strain of T. cruzi and scaled up to blood samples of naturally infected Didelphis marsupialis. As a result, we observed DNA cleavage using the CRISPR/Cas system with the Cytb guide, achieving a detection sensitivity of 118 parasite equivalents/mL in PCR and 116 parasite equivalents/mL with RPA amplification. Sequencing of the Cytb gene showed no mutations in the cleavage site. However, point mutations and indels were found in SR18S and H2 A, avoiding the formation of the CRISPR/LbCas12 complex. Furthermore, we introduce the design of a fluorescent detection prototype with CRISPR/LbCas12a called "Tropical Diseases Detector" (TropD-Detector). This device operates with an excitation wavelength of 480 nm emitted by an LED and a high-pass light filter with a cutoff wavelength of 500 nm. We detected positive samples using any photographic camera system. The TropD-Detector provides a visual, viable, and sensitive method for detecting T. cruzi in both vectors and reservoirs from endemic areas.},
}
@article {pmid40449999,
year = {2025},
author = {Schweitzer, AY and Adams, EW and Nguyen, MTA and Lek, M and Isaacs, FJ},
title = {Precision multiplexed base editing in human cells using Cas12a-derived base editors.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {5061},
pmid = {40449999},
issn = {2041-1723},
support = {R01 GM117230/GM/NIGMS NIH HHS/United States ; GM117230//U.S. Department of Health &amp; Human Services | NIH | National Institute of General Medical Sciences (NIGMS)/ ; CF22-1046//Carlsbergfondet (Carlsberg Foundation)/ ; },
mesh = {Humans ; *Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics/metabolism ; *CRISPR-Associated Proteins/genetics/metabolism ; *Endodeoxyribonucleases/metabolism/genetics ; Genome, Human ; HEK293 Cells ; Cell Line ; Bacterial Proteins ; },
abstract = {Base editors enable the direct conversion of target nucleotides without introducing DNA double strand breaks, making them a powerful tool for creating point mutations in a human genome. However, current Cas9-derived base editing technologies have limited ability to simultaneously edit multiple loci with base-pair level precision, hindering the generation of polygenic phenotypes. Here, we test the ability of six Cas12a-derived base editing systems to process multiple gRNAs from a single transcript. We identify base editor variants capable of multiplexed base editing and improve the design of the respective gRNA array expression cassette, enabling multiplexed editing of 15 target sites in multiple human cell lines, increasing state-of-the-art in multiplexing by three-fold in the field of mammalian genome engineering. To reduce bystander mutations, we also develop a Cas12a gRNA engineering approach that directs editing outcomes towards a single base-pair conversion. We combine these advances to demonstrate that both strategies can be combined to drive multiplex base editing with greater precision and reduced bystander mutation rates. Overcoming these key obstacles of mammalian genome engineering technologies will be critical for their use in studying single nucleotide variant-associated diseases and engineering synthetic mammalian genomes.},
}
@article {pmid40449852,
year = {2025},
author = {Kolesova, E and Pulone, S and Kostyushev, D and Tasciotti, E},
title = {CRISPR/Cas bioimaging: From whole body biodistribution to single-cell dynamics.},
journal = {Advanced drug delivery reviews},
volume = {224},
number = {},
pages = {115619},
doi = {10.1016/j.addr.2025.115619},
pmid = {40449852},
issn = {1872-8294},
abstract = {This review explores the transformative role of CRISPR/Cas systems in optical bioimaging, emphasizing how advancements in nanoparticle (NP) technologies are revolutionizing the visualization of gene-editing processes both in vitro and in vivo. Optical imaging techniques, such as near-infrared (NIR) and fluorescence imaging, have greatly benefited from the integration of nanoformulated contrast agents, improving resolution, sensitivity, and specificity. CRISPR/Cas systems, originally developed just for gene editing, are now being coupled with these imaging modalities to enable real-time monitoring and quantitative measurements of metabolites, vitamins, proteins, nucleic acids and other entities in specific areas of the body, as well as tracking of CRISPR/Cas delivery, editing efficiency, and potential off-target effects. The development of CRISPR/Cas-loaded NPs allows for enhanced imaging and precise monitoring across multiple scales with multiplexed and multicolor imaging in complex settings, including potential in vivo diagnostics. CRISPR/Cas therapeutics as well as diagnostics are hindered by the lack of efficient and targeted delivery tools. Biomimetic NPs have emerged as promising tools for improving biocompatibility, enhancing targeting capabilities, and overcoming biological barriers, facilitating more efficient delivery and bioimaging of CRISPR/Cas systems in vivo. As the design of these NPs and delivery mechanisms improves, alongside advancements in endolysosomal escape, CRISPR/Cas-based bioimaging will continue to advance, offering unprecedented possibilities in precision medicine and theranostic applications.},
}
@article {pmid40448764,
year = {2025},
author = {Jin, H and Sophocleous, A and Azfer, A and Ralston, SH},
title = {Analysis of Transcriptional Regulation in Bone Cells.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2885},
number = {},
pages = {247-269},
pmid = {40448764},
issn = {1940-6029},
mesh = {CRISPR-Cas Systems ; *Transcription, Genetic ; Promoter Regions, Genetic ; *Gene Expression Regulation ; Chromatin Immunoprecipitation/methods ; Humans ; Electrophoretic Mobility Shift Assay/methods ; Animals ; *Bone and Bones/cytology/metabolism ; *Osteoblasts/metabolism ; Mice ; Genes, Reporter ; },
abstract = {Transcription is a process by which the rate of RNA synthesis is regulated. Here we describe the techniques for carrying out promoter-reporter assays: electrophoretic mobility shift assays, chromosome conformation capture (3C) assays, chromatin immunoprecipitation assays, and CRISPR-Cas9 assay-five commonly used methods for studying and altering gene transcription.},
}
@article {pmid40448746,
year = {2025},
author = {Kamli, H and Khan, NU},
title = {Revolutionising cancer intervention: the repercussions of CAR-T cell therapy on modern oncology practices.},
journal = {Medical oncology (Northwood, London, England)},
volume = {42},
number = {7},
pages = {228},
pmid = {40448746},
issn = {1559-131X},
mesh = {Humans ; *Neoplasms/therapy/immunology ; *Immunotherapy, Adoptive/methods ; *Receptors, Chimeric Antigen/immunology ; Gene Editing/methods ; Tumor Microenvironment/immunology ; CRISPR-Cas Systems ; Medical Oncology/methods/trends ; },
abstract = {Chimeric Antigen Receptor T-cell (CAR-T) therapy represents a groundbreaking advance in oncology, leveraging patient-specific immune cells to target malignant tumours precisely. By equipping T cells with synthetic receptors, CAR-T therapy achieves remarkable antitumor effects and offers hope for durable cancer control. However, several limitations persist, including antigen scarcity, immunosuppressive tumour microenvironments, and T-cell exhaustion. CRISPR-Cas9 gene editing has enhanced CAR-T potency by knocking out immune checkpoints (PD-1, CTLA-4) and improving persistence, while RNA interference (RNAi) silences immune-evasion genes (e.g. SOCS1). Nanozyme-based delivery systems enable precise CRISPR-Cas9 delivery (> 70% editing efficiency) and tumour targeting, overcoming instability and off-target effects. Innovations like SUPRA CARs, armoured CAR-T cells (e.g. IL-12/IL-21-secreting TRUCKs), and dual checkpoint inhibition synergize to reprogram the tumour microenvironment, reducing relapse by 40% in trials. Despite progress, high costs, manufacturing hurdles, and ethical concerns (e.g. germline editing risks) remain critical barriers. Emerging solutions include universal off-the-shelf CAR-Ts, hybrid nano-CRISPR systems, and AI-driven design, paving the way for scalable, personalised immunotherapy. This review highlights breakthroughs in CRISPR, RNAi, and nanotechnology, underscoring CAR-T therapy's transformative potential while addressing translational challenges for broader clinical adoption.},
}
@article {pmid40447846,
year = {2025},
author = {Blaskovich, MAT and Cooper, MA},
title = {Antibiotics re-booted-time to kick back against drug resistance.},
journal = {npj antimicrobials and resistance},
volume = {3},
number = {1},
pages = {47},
pmid = {40447846},
issn = {2731-8745},
abstract = {After decades of neglect and a decline in antibiotic research and development, we are now finally witnessing the advent of new funding programs dedicated to new therapies. In addition to traditional new chemical entities that directly kill or arrest the growth of bacteria, alternative approaches are being identified and advanced towards proof-of-concept trials in the clinic. We briefly review the current pipeline of conventional new antibiotics and highlight in more depth promising alternatives, including potentiators of antibiotic action, bacteriophage, lysins and microbiome modulation. More innovative approaches, such as adaptive and innate immune modulators, CRISPR-Cas and diagnostic guided 'theranostics' are discussed and contrasted. Such exploratory therapies may require the development of alternative regulatory and clinical development pathways, but represent a potential circuit breaker from the current 'arms race' between bacteria and traditional antibiotics.},
}
@article {pmid40447637,
year = {2025},
author = {Schwaemmle, H and Soldati, H and Lykoskoufis, NMR and Docquier, M and Hainard, A and Braun, SMG},
title = {CRISPR screen decodes SWI/SNF chromatin remodeling complex assembly.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {5011},
pmid = {40447637},
issn = {2041-1723},
support = {PCEFP3_194305//Schweizerischer Nationalfonds zur F&#xf6;rderung der Wissenschaftlichen Forschung (Swiss National Science Foundation)/ ; },
mesh = {Animals ; Mice ; *Chromatin Assembly and Disassembly/genetics ; *Chromosomal Proteins, Non-Histone/metabolism/genetics ; *Transcription Factors/metabolism/genetics ; Chromatin/metabolism/genetics ; RNA-Binding Proteins/metabolism/genetics ; Mouse Embryonic Stem Cells/metabolism ; *CRISPR-Cas Systems ; Humans ; Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {The SWI/SNF (or BAF) complex is an essential chromatin remodeler, which is frequently mutated in cancer and neurodevelopmental disorders. These are often heterozygous loss-of-function mutations, indicating a dosage-sensitive role for SWI/SNF subunits. However, the molecular mechanisms regulating SWI/SNF subunit dosage to ensure complex assembly remain largely unexplored. We performed a CRISPR KO screen, using epigenome editing in mouse embryonic stem cells, and identified Mlf2 and Rbm15 as regulators of SWI/SNF complex activity. First, we show that MLF2, a poorly characterized chaperone protein, promotes SWI/SNF assembly and binding to chromatin. Rapid degradation of MLF2 reduces chromatin accessibility at sites that depend on high levels of SWI/SNF binding to maintain open chromatin. Next, we find that RBM15, part of the m[6]A writer complex, controls m[6]A modifications on specific SWI/SNF mRNAs to regulate subunit protein levels. Misregulation of m[6]A methylation causes overexpression of core SWI/SNF subunits leading to the assembly of incomplete complexes lacking the catalytic ATPase/ARP subunits. These data indicate that targeting modulators of SWI/SNF complex assembly may offer a potent therapeutic strategy for diseases associated with impaired chromatin remodeling.},
}
@article {pmid40447601,
year = {2025},
author = {Hu, H and Guo, S and Li, Y and Dong, K and Lu, Y and Ye, K and Li, L and Zhou, X and Cheng, L and Xiao, X},
title = {Spatially blocked split CRISPR-Cas12a system for ultra-sensitive and versatile small molecule activation and detection.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {5035},
pmid = {40447601},
issn = {2041-1723},
mesh = {*CRISPR-Cas Systems/genetics ; *Endodeoxyribonucleases/metabolism/genetics ; Humans ; *CRISPR-Associated Proteins/metabolism/genetics ; *Bacterial Proteins/metabolism/genetics ; },
abstract = {Detecting small molecules is pivotal across fields like clinical diagnostics, environmental monitoring, and food safety. The CRISPR-Cas12a system, known for its simplicity and sensitivity, offers a promising basis for small molecule detection. However, current CRISPR-based detection methods face challenges, including complex design requirements, high background noise, and limited adaptability to different targets. In our study, we introduce the SBS-Cas system, leveraging a split crRNA mode to induce spatial hindrance on the scaffold strand through molecular binding. This approach prevents the assembly with Cas12a, effectively masking its trans-cleavage activity. By introducing small molecules that competitively bind to the macromolecule, we eliminate this spatial hindrance, activating Cas12a. Our results demonstrate high sensitivity, versatility, and adaptability in small molecule detection across multiple reactions, with successful intracellular imaging and responsive fluctuations in complex environments underscoring the system's robustness. This innovative CRISPR-Cas12a-based approach establishes a low-background, highly sensitive platform for small molecule detection. SBS-Cas promises not only to enhance tools for clinical, environmental, and food safety applications but also to advance CRISPR research, providing insights and expanding possibilities in molecular detection science.},
}
@article {pmid40447589,
year = {2025},
author = {Liang, R and Wang, S and Cai, Y and Li, Z and Li, KM and Wei, J and Sun, C and Zhu, H and Chen, K and Gao, C},
title = {Circular RNA-mediated inverse prime editing in human cells.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {5057},
pmid = {40447589},
issn = {2041-1723},
mesh = {Humans ; *Gene Editing/methods ; *RNA, Circular/genetics/metabolism ; *CRISPR-Cas Systems/genetics ; HEK293 Cells ; CRISPR-Associated Protein 9/genetics/metabolism ; Genome, Human ; RNA, Guide, CRISPR-Cas Systems/genetics ; },
abstract = {Prime editors are restricted to performing precise edits downstream of cleavage sites, thereby limiting their editing scope. Therefore, we develop inverse prime editors (iPEs) that act upstream of the nickase cleavage site by replacing nCas9-H840A with nCas9-D10A, but the editing efficiencies are limited. To address this limitation, we develop circular RNA-mediated iPEs (ciPEs), achieving editing efficiencies ranging from 0.1% to 24.7%. Further optimization using Rep-X helicase increases editing efficiencies to a range of 2.7%-55.4%. The Rep-X-assisted ciPE system thus expands the scope of editing and improves efficiencies at genomic sites that are previously difficult to target. The Rep-X-assisted ciPE system will complement canonical PE system in enabling more extensive and efficient editing across a wider range of the human genome.},
}
@article {pmid40447514,
year = {2025},
author = {Yu, T and Zou, S and Long, Y and Ou, Y and Liu, S and Kang, T and Song, L and Sun, C and Liu, G},
title = {Glass fiber-interfaced CRISPR/Cas biosensing adaptable for diverse biomarker detection.},
journal = {Trends in biotechnology},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.tibtech.2025.05.001},
pmid = {40447514},
issn = {1879-3096},
abstract = {Developing a generic sensitive platform for detecting diverse biomarkers is essential for a comprehensive understanding of disease states, guiding precision medicine. Herein, we introduce a versatile platform based on glass fiber interfaced CRISPR/Cas with a universal reagent setting (g-CURS), which used a fixed pair of CRISPR RNA (crRNA) and a single-stranded DNA (ssDNA) activator to enable detection of multiple nucleic acids or proteins with ultrahigh sensitivity. The fixed ssDNA activator was labeled on multiple specific ligation products or detection antibodies conjugated on glass fiber to initiate CRISPR/Cas12a-assisted rapid and exponential cascade amplification through circular reporters (CRs), generating fluorescence signals readable by a portable detector. g-CURS was able to detect viral nucleic acids with attomolar sensitivity within 30 min and multiple low-abundance proteins in extracellular vesicles of Parkinson's disease (PD) serum with subpicomolar sensitivity within 80 min. g-CURS simplifies CRISPR/Cas biosensing using a standard reagent setting, holding promise for biomarker discovery free from bulky instruments.},
}
@article {pmid40447397,
year = {2025},
author = {Gao, Y and Li, Y and Gao, Q and Zhang, T and Zhang, Y and Liu, T and Han, K},
title = {Multi-functional dumbbell DNA probe design and its application in signal amplification cascade -based assay of human immunodeficiency virus.},
journal = {Analytica chimica acta},
volume = {1362},
number = {},
pages = {344141},
doi = {10.1016/j.aca.2025.344141},
pmid = {40447397},
issn = {1873-4324},
mesh = {*DNA Probes/chemistry/genetics ; Humans ; *Biosensing Techniques/methods ; *Nucleic Acid Amplification Techniques/methods ; CRISPR-Cas Systems ; *DNA, Viral/analysis/genetics ; Limit of Detection ; *HIV/isolation &amp; purification/genetics ; },
abstract = {Sensitive assay of Human immunodeficiency virus (HIV) is the premise of accurate prevention and con-trol of AIDS. Dumbbell DNA showed promising potential in biosensing, imaging applications. This study presents a highly sensitive biosensing platform integrating dumbbell-shaped DNA probes with rolling circle transcription (RCT)-regulated CRISPR-Cas12a to achieve cascade signal amplification. The arrangement of the functional regions in the same unclosed dumbbell probes (UDPs) might affect final performance in the biosensor. Three different types UDPs (A/B/C) were specifically designed and the performances of the UDPs were evaluated in the RCT-CRISPR based cascade platform. Among these, type A UDP shows the best performance with a detection limit of 44.8 aM targeting HIV-DNA as well as high sensitivity and specificity. Also, type A UDP had the best behavior in the clinical sample assay. The difference of UDPs' performance might attribute to the variation of opening form of the UDPs considering the arrangement of various functional regions. The platform's modular design supports customization for diverse nucleic acid targets, making it adaptable for early disease detection and precision diagnostics.},
}
@article {pmid40446796,
year = {2025},
author = {Yan, J and Luo, R and Rosen, BP and Liu, D and Wong, W and Leslie, CS and Huangfu, D},
title = {Discovery of NANOG enhancers and their essential roles in self-renewal and differentiation in human embryonic stem cells.},
journal = {Stem cell reports},
volume = {20},
number = {6},
pages = {102511},
pmid = {40446796},
issn = {2213-6711},
mesh = {Humans ; *Human Embryonic Stem Cells/cytology/metabolism ; *Cell Differentiation/genetics ; *Enhancer Elements, Genetic ; *Nanog Homeobox Protein/genetics/metabolism ; *Cell Self Renewal/genetics ; Animals ; Mice ; Cell Line ; CRISPR-Cas Systems ; },
abstract = {Human embryonic stem cells (hESCs) are notable for their ability to self-renew and to differentiate into all tissue types in the body. NANOG is a core regulator of hESC identity, and dynamic control of its expression is crucial to maintain the balance between self-renewal and differentiation. Transcriptional regulation depends on enhancers, but NANOG enhancers in hESCs are not well characterized. Here, we report two NANOG enhancers discovered from a CRISPR interference screen in hESCs. Deletion of a single copy of either enhancer significantly reduced NANOG expression, compromising self-renewal and increasing differentiation propensity. Interestingly, these two NANOG enhancers are involved in a tandem duplication event found in certain primates including humans but not in mice. However, the duplicated counterparts do not regulate NANOG expression. This work expands our knowledge of functional enhancers in hESCs and highlights the sensitivity of the hESC state to the dosage of core regulators and their enhancers.},
}
@article {pmid40446748,
year = {2025},
author = {Zuo, Z and Liang, R and Fan, S and Shan, H and Zhang, H and Wang, X and Fei, T},
title = {Genome-wide CRISPR screens identify key regulators of adipogenesis and glucose uptake in beige adipocytes.},
journal = {Biochemical and biophysical research communications},
volume = {774},
number = {},
pages = {152093},
doi = {10.1016/j.bbrc.2025.152093},
pmid = {40446748},
issn = {1090-2104},
mesh = {*Adipogenesis/genetics ; *Glucose/metabolism ; *Adipocytes, Beige/metabolism/cytology ; Animals ; Humans ; Mice ; *CRISPR-Cas Systems ; Insulin Resistance ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; },
abstract = {The beiging of white adipocytes enhances energy expenditure by utilizing fatty acids and glucose, offering therapeutic potential against obesity and type 2 diabetes. However, the genetic mechanisms driving this process remain unclear. Here, we performed multiple fluorescence-activated cell sorting (FACS)-based genome-wide CRISPR loss-of-function screens in beige adipocytes with or without insulin resistance (IR) induction, and systematically identified functional regulators of beige adipocyte adipogenesis and glucose metabolism. We further integrated transcriptomics and human genetics data to pinpoint key genes for adipogenesis and glucose metabolism in beige adipocytes. Moreover, we validated SULT2B1 and ATP1B2 as key adipogenesis genes for beige adipocytes, and COMMD7 gene as important regulator for glucose uptake against IR. These findings not only provide a comprehensive and valuable resource for cataloguing candidate functional genes underlying lipid and glucose homeostasis in beige adipocytes, but also offer potential therapeutic targets against metabolic disorders.},
}
@article {pmid40446205,
year = {2025},
author = {Oikemus, S and Hu, K and Shin, M and Idrizi, F and Goodman-Khan, A and Kolb, A and Ghanta, KS and Lee, J and Wagh, A and Wolfe, SA and Zhu, LJ and Watts, JK and Lawson, ND},
title = {Identifying optimal conditions for precise knock-in of exogenous DNA into the zebrafish genome.},
journal = {Development (Cambridge, England)},
volume = {152},
number = {12},
pages = {},
doi = {10.1242/dev.204571},
pmid = {40446205},
issn = {1477-9129},
support = {R21OD030004//NIH Office of the Director/ ; R35HL171534/HL/NHLBI NIH HHS/United States ; 1UG3TR002668/NH/NIH HHS/United States ; R21OD030004/NH/NIH HHS/United States ; 1UG3TR002668/NH/NIH HHS/United States ; R35HL171534/NH/NIH HHS/United States ; //University of Massachusetts Medical School/ ; },
mesh = {Animals ; *Zebrafish/genetics ; *Genome/genetics ; *Gene Editing/methods ; CRISPR-Cas Systems/genetics ; *Gene Knock-In Techniques/methods ; *DNA/genetics ; DNA Breaks, Double-Stranded ; Recombinational DNA Repair/genetics ; },
abstract = {CRISPR nucleases can be used to insert exogenous DNA into the zebrafish genome by homology-dependent repair (HDR), although germline transmission rates for precise edits remain quite low. Comparative studies to optimize HDR parameters for introducing base pair changes using short-read deep sequencing have been successful, but similar analysis for insertions is challenging due to read-length constraints. Here, we quantified editing outcomes using long-read sequencing to identify optimal template and CRISPR parameters for precise targeted insertion in zebrafish. Through side-by-side comparisons, we found that chemically modified templates out-perform those released in vivo from a plasmid, while Cas9 and Cas12a nucleases performed similarly for targeted insertion. Consistent with previous studies, precise editing rates were dependent on the distance between a double-strand break and the inserted sequence. We further found that non-homologous base pairs in homology templates significantly reduced precise editing rates. Using optimized parameters, we consistently achieved germline founder rates of greater than 20% for precise insertions across four loci. Together, our quantitative analyses identified optimal conditions for precise insertion of exogenous DNA into the zebrafish genome.},
}
@article {pmid40446005,
year = {2025},
author = {Shi, L and Zhang, Y and Duan, Y and Sun, M and Yuan, C and Cao, L and Kong, X and Zhang, W and Zheng, H and Wang, Q},
title = {Genome-scale CRISPR screen identifies TMEM198 driving double membrane vesicle formation in swine alphacoronavirus and murine betacoronavirus infected cells.},
journal = {PLoS pathogens},
volume = {21},
number = {5},
pages = {e1013211},
pmid = {40446005},
issn = {1553-7374},
mesh = {Animals ; Mice ; Swine ; *Membrane Proteins/genetics/metabolism ; CRISPR-Cas Systems ; Virus Replication ; *Alphacoronavirus/physiology/genetics ; Humans ; *Coronavirus Infections/virology/metabolism/genetics ; SARS-CoV-2 ; COVID-19/virology ; },
abstract = {COVID-19 pandemic caused by the SARS-CoV-2 which is well-publicized cross-species transmissibility. SARS-CoV-2 belongs to genus Betacoronavirus, several pathogenic alphacoronaviruses have shown similar patterns of emergence. Much less attention paid to host factors required for alphacoronavirus replication compared to those of betacoronaviruses. Here, we utilized a genome-wide CRISPR-Cas9-based screen to identify TMEM198 as a critical host protein for double-membrane vesicle (DMVs) formation during the replication of swine alphacoronavirus. Gene deletion of TMEM198 led to a reduction in the levels of viral infection in cells, whereas the ectopic expression of TMEM198 correspondingly resulted in an increase in infection levels. At the mechanistic level, TMEM198 directly binds to the C-terminal of nonstructural protein 3 (nsp3c) and nonstructural protein 4 (nsp4) to participate in the formation of DMVs. The first 35 amino acids at the N-terminal of TMEM198 are critical for the formation of DMVs and viral replication. Moreover, mice with a gene deletion of TMEM198 exhibit reduced susceptibility to the Betacoronavirus MHV. These results identify the function of TMEM198 in the formation of DMVs during the replication of swine alphacoronavirus and murine betacoronavirus.},
}
@article {pmid40445760,
year = {2025},
author = {Lamperis, SM and McMahon, KM and Calvert, AE and Rink, JS and Vasan, K and Pandkar, MR and Crentsil, EU and Chalmers, ZR and McDonald, NR and Kosmala, CJ and Bonini, MG and Matei, D and Gordon, LI and Chandel, NS and Thaxton, CS},
title = {CRISPR screen reveals a simultaneous targeted mechanism to reduce cancer cell selenium and increase lipid oxidation to induce ferroptosis.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {122},
number = {22},
pages = {e2502876122},
pmid = {40445760},
issn = {1091-6490},
support = {T32GM142604//HHS | NIH (NIH)/ ; R01 ES035353/ES/NIEHS NIH HHS/United States ; T2022-018//V Foundation for Cancer Research (VFCR)/ ; T32 GM142604/GM/NIGMS NIH HHS/United States ; R01ES035353//HHS | NIH (NIH)/ ; P30 CA060553/CA/NCI NIH HHS/United States ; R01 CA216882/CA/NCI NIH HHS/United States ; R01CA216882//HHS | NIH (NIH)/ ; },
mesh = {*Ferroptosis/genetics/drug effects ; Humans ; Cell Line, Tumor ; Phospholipid Hydroperoxide Glutathione Peroxidase/metabolism/genetics ; *Selenium/metabolism ; Oxidation-Reduction ; Female ; CRISPR-Cas Systems ; Lipid Peroxidation ; Scavenger Receptors, Class B/metabolism/genetics ; Lipoproteins, HDL/metabolism ; *Ovarian Neoplasms/metabolism/genetics/pathology ; Lipid Metabolism ; Nanoparticles/chemistry ; Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {Ferroptosis is a cell death mechanism distinguished by its dependence on iron-mediated lipid oxidation. Cancer cells highly resistant to conventional therapies often demonstrate lipid metabolic and redox vulnerabilities that sensitize them to cell death by ferroptosis. These include a unique dependency on the lipid antioxidant selenoenzyme, glutathione peroxidase 4 (GPx4), that acts as a ferroptosis inhibitor. Synthetic high-density lipoprotein-like nanoparticle (HDL NP) targets the high-affinity HDL receptor scavenger receptor class B type 1 (SR-B1) and regulates cell and cell membrane lipid metabolism. Recently, we reported that targeting cancer cell SR-B1 with HDL NP depleted cell GPx4, which is accompanied by increased cell membrane lipid peroxidation and cancer cell death. These data suggest that HDL NP may induce ferroptosis. Thus, we conducted an unbiased CRISPR-based positive selection screen and target validation studies in ovarian clear cell carcinoma (OCCC) cell lines to ascertain the mechanism through which HDL NP regulates GPx4 and kills cancer cells. The screen revealed two genes, acyl-CoA synthetase long chain family member 4 (ACSL4) and thioredoxin reductase 1 (TXNRD1), whose loss conferred resistance to HDL NP. Validation of ACSL4 supports that HDL NP induces ferroptosis as the predominant mechanism of cell death, while validation of TXNRD1 revealed that HDL NP reduces cellular selenium and selenoprotein production, most notably, GPx4. Accordingly, we define cancer cell metabolic targets that can be simultaneously actuated by a multifunctional, synthetic HDL NP ligand of SR-B1 to kill cancer cells by ferroptosis.},
}
@article {pmid40445465,
year = {2025},
author = {Hashemi, M and Khanaghah, XM and Nahand, JS},
title = {The CRISPR-Cas revolution in head and neck cancer: a new era of targeted therapy.},
journal = {Functional &amp; integrative genomics},
volume = {25},
number = {1},
pages = {113},
pmid = {40445465},
issn = {1438-7948},
mesh = {Humans ; *Head and Neck Neoplasms/therapy/genetics ; *CRISPR-Cas Systems ; *Gene Editing/methods ; *Genetic Therapy/methods ; Molecular Targeted Therapy ; Signal Transduction ; },
abstract = {Head and neck cancer (HNC) encompasses a diverse array of malignancies impacting the anatomical structures of the head and neck region, ranking as the seventh most prevalent cancer type globally. The occurrence and advancement of HNC are intricately linked to mutations and disruptions within critical signaling pathways, accentuating the imperative for targeted therapeutic interventions to rectify these genetic aberrations. Traditional treatment modalities, including surgical intervention and adjuvant chemotherapy or radiotherapy, frequently culminate in considerable morbidity and suboptimal prognoses. Recently, the CRISPR-Cas system has emerged as a revolutionary gene-editing platform, poised to redefine therapeutic approaches in gene therapy and oncological research. Despite its potential, CRISPR-Cas faces challenges such as off-target effects, delivery inefficiencies, and immunogenicity, which must be addressed for clinical success. This review meticulously evaluates the progress in CRISPR-Cas technologies aimed at targeting essential signaling pathways implicated in HNC, addressing current challenges while highlighting optimal targets, Cas nucleases, and innovative delivery mechanisms, thereby elucidating the therapeutic potential and clinical applicability of the CRISPR-Cas paradigm in the management of HNC.},
}
@article {pmid40442371,
year = {2025},
author = {Nugent, PJ and Park, H and Wladyka, CL and Yelland, JN and Sinha, S and Chen, KY and Bynum, C and Quarterman, G and Lee, SC and Hsieh, AC and Subramaniam, AR},
title = {Decoding post-transcriptional regulatory networks by RNA-linked CRISPR screening in human cells.},
journal = {Nature methods},
volume = {22},
number = {6},
pages = {1237-1246},
pmid = {40442371},
issn = {1548-7105},
support = {GM119835//U.S. Department of Health &amp; Human Services | National Institutes of Health (NIH)/ ; GM008268//U.S. Department of Health &amp; Human Services | National Institutes of Health (NIH)/ ; CA230617//U.S. Department of Health &amp; Human Services | National Institutes of Health (NIH)/ ; CA276308//U.S. Department of Health &amp; Human Services | National Institutes of Health (NIH)/ ; GM135362//U.S. Department of Health &amp; Human Services | National Institutes of Health (NIH)/ ; 1846521//National Science Foundation (NSF)/ ; },
mesh = {Humans ; *CRISPR-Cas Systems ; *Gene Regulatory Networks ; RNA, Guide, CRISPR-Cas Systems/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats ; HEK293 Cells ; Ribosomes/metabolism ; RNA, Messenger/genetics/metabolism ; *RNA/genetics/metabolism ; *RNA Processing, Post-Transcriptional ; },
abstract = {RNAs undergo a complex choreography of metabolic processes that are regulated by thousands of RNA-associated proteins. Here we introduce ReLiC, a scalable and high-throughput RNA-linked CRISPR approach to measure the responses of diverse RNA metabolic processes to knockout of 2,092 human genes encoding all known RNA-associated proteins. ReLiC relies on an iterative strategy to integrate genes encoding Cas9, single-guide RNAs (sgRNAs) and barcoded reporter libraries into a defined genomic locus. Combining ReLiC with polysome fractionation reveals key regulators of ribosome occupancy, uncovering links between translation and proteostasis. Isoform-specific ReLiC captures differential regulation of intron retention and exon skipping by SF3B complex subunits. Chemogenomic ReLiC screens decipher translational regulators upstream of messenger RNA (mRNA) decay and identify a role for the ribosome collision sensor GCN1 during treatment with the anti-leukemic drug homoharringtonine. Our work demonstrates ReLiC as a powerful framework for discovering and dissecting post-transcriptional regulatory networks in human cells.},
}
@article {pmid40442121,
year = {2025},
author = {Ma, S and Liao, K and Chen, K and Cheng, T and Yang, X and Chen, P and Li, S and Li, M and Zhang, X and Zhang, Y and Huang, T and Wang, X and Wang, L and Lin, Y and Rong, Z},
title = {hpCasMINI: An engineered hypercompact CRISPR-Cas12f system with boosted gene editing activity.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {5001},
pmid = {40442121},
issn = {2041-1723},
support = {82370078//National Natural Science Foundation of China (National Science Foundation of China)/ ; 82200072//National Natural Science Foundation of China (National Science Foundation of China)/ ; 82304008//National Natural Science Foundation of China (National Science Foundation of China)/ ; 82303995//National Natural Science Foundation of China (National Science Foundation of China)/ ; 82370003//National Natural Science Foundation of China (National Science Foundation of China)/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; Animals ; Humans ; Mice ; HEK293 Cells ; Fibroblast Growth Factors/genetics/metabolism ; Genetic Therapy/methods ; DNA Cleavage ; Tumor Suppressor Protein p53/genetics ; Dependovirus/genetics ; *CRISPR-Associated Proteins/genetics/metabolism ; Liver/metabolism ; PTEN Phosphohydrolase/genetics ; },
abstract = {Compact CRISPR-Cas systems have demonstrated potential for effective packaging into adeno-associated viruses (AAVs) for use in gene therapy. However, their applications are currently limited due to modest gene-editing activity. Here we introduce an engineered compact CRISPR-Cas12f (hpCasMINI, 554 aa), with hyper editing efficiency in mammalian cells via adding an α-helix structure to the N-terminus of an Un1Cas12f1 variant CasMINI (529 aa). The hpCasMINI system boosts gene activation and DNA cleavage activity with about 1.4-3.0-fold and 1.1-19.5-fold, respectively, and maintains the high specificity when compared to CasMINI. In addition, the system can activate luciferase reporter gene and endogenous Fgf21 gene in adult mouse liver, as well as construct liver tumorigenesis model via disrupting Trp53 and Pten genes and inserting oncogenic Kras[G12D] into the Trp53 locus. When compared to SpCas9 and LbCas12a, hpCasMINI displays higher gene activation and exhibits higher DNA cleavage specificity, although with lower activity, at the tested sites. Moreover, with a similar strategy, we engineer compact versions of hpOsCas12f1 (458 aa) from enOsCas12f1 and hpAsCas12f1 (447 aa) from AsCas12f1-HKRA, both of which display increased DNA cleavage activity, with hpAsCas12f1 also showing improved gene activation capability. Therefore, we develop activity-increased miniature hpCasMINI, hpOsCas12f1 and hpAsCas12f1 nucleases, which hold great potential for gene therapy in the future.},
}
@article {pmid40440869,
year = {2025},
author = {Zhang, R and Zhou, Q and Huang, S and Zhang, N and Sun, D},
title = {Advancements in CRISPR-Cas-based strategies for combating antimicrobial resistance.},
journal = {Microbiological research},
volume = {298},
number = {},
pages = {128232},
doi = {10.1016/j.micres.2025.128232},
pmid = {40440869},
issn = {1618-0623},
mesh = {*CRISPR-Cas Systems ; *Bacteria/genetics/drug effects ; Gene Editing/methods ; *Anti-Bacterial Agents/pharmacology ; *Drug Resistance, Multiple, Bacterial/genetics ; Humans ; *Drug Resistance, Bacterial/genetics ; Bacterial Infections/drug therapy/microbiology ; Plasmids/genetics ; Genome, Bacterial ; },
abstract = {Multidrug resistance (MDR) in bacteria presents a significant global health threat, driven by the widespread dissemination of antibiotic-resistant genes (ARGs). The CRISPR-Cas system, known for its precision and adaptability, holds promise as a tool to combat antimicrobial resistance (AMR). Although previous studies have explored the use of CRISPR-Cas to target bacterial genomes or plasmids harboring resistance genes, the application of CRISPR-Cas-based antimicrobial therapies is still in its early stages. Challenges such as low efficiency and difficulties in delivering CRISPR to bacterial cells remain. This review provides an overview of the CRISPR-Cas system, highlights recent advancements in CRISPR-Cas-based antimicrobials and delivery strategies for combating AMR. The review also discusses potential challenges for the future development of CRISPR-Cas-based antimicrobials. Addressing these challenges would enable CRISPR therapies to become a practical solution for treating AMR infections in the future.},
}
@article {pmid40440223,
year = {2025},
author = {Bachler, A and Padovan, A and Anderson, CJ and Wei, Y and Wu, Y and Pearce, S and Downes, S and James, B and Tessnow, AE and Sword, GA and Williams, M and Tay, WT and Gordon, KHJ and Walsh, TK},
title = {Disruption of HaVipR1 confers Vip3Aa resistance in the moth crop pest Helicoverpa armigera.},
journal = {PLoS biology},
volume = {23},
number = {5},
pages = {e3003165},
pmid = {40440223},
issn = {1545-7885},
mesh = {Animals ; *Moths/genetics/drug effects ; *Insecticide Resistance/genetics ; *Bacterial Proteins/pharmacology/genetics ; Insect Proteins/genetics/metabolism ; CRISPR-Cas Systems ; Crops, Agricultural/genetics/parasitology ; Pest Control, Biological/methods ; Plants, Genetically Modified/genetics ; Bacillus thuringiensis/genetics ; Helicoverpa armigera ; },
abstract = {The global reliance on Bacillus thuringiensis (Bt) proteins for controlling lepidopteran pests in cotton, corn, and soybean crops underscores the critical need to understand resistance mechanisms. Vip3Aa, one of the most widely deployed and currently effective Bt proteins in genetically modified crops, plays a pivotal role in pest management. This study investigates the molecular basis of Vip3Aa resistance in Australian Helicoverpa armigera through genetic crosses, and integrated genomic and transcriptomic analyses. We identified a previously uncharacterized gene, LOC110373801 (designated HaVipR1), as potentially important in Vip3Aa resistance in two field-derived resistant lines. Functional validation using CRISPR/Cas9 knockout in susceptible lines confirmed the gene's role in conferring high-level resistance to Vip3Aa. Despite extensive laboratory selection of Vip3Aa-resistant colonies in Lepidoptera, the biochemical mechanisms underlying resistance have remained elusive. Our research identifies HaVipR1 as a potential contributor to resistance, adding to our understanding of how insects may develop resistance to this important Bt protein. The identification of HaVipR1 contributes to our understanding of potential resistance mechanisms and may inform future resistance management strategies. Future work should explore the biochemical pathways influenced by HaVipR1 and assess its interactions with other resistance mechanisms. The approach utilized here underscores the value of field-derived resistant lines for understanding resistance in agricultural pests and highlights the need for targeted approaches to manage resistance sustainably.},
}
@article {pmid40439284,
year = {2025},
author = {Ukita, Y and Suzuki, R and Miyoshi, K and Saito, K and Okumura, M and Chihara, T},
title = {Generation of Odorant Receptor-QF2 Knock-In Drivers for Improved Analysis of Olfactory Circuits in Drosophila.},
journal = {Genes to cells : devoted to molecular &amp; cellular mechanisms},
volume = {30},
number = {4},
pages = {e70028},
pmid = {40439284},
issn = {1365-2443},
support = {21H02479//Japan Society for the Promotion of Science/ ; 24K02062//Japan Society for the Promotion of Science/ ; 20K15903//Japan Society for the Promotion of Science/ ; JP23KJ1645//Japan Society for the Promotion of Science/ ; },
mesh = {Animals ; *Receptors, Odorant/genetics/metabolism ; *Drosophila melanogaster/genetics/metabolism ; *Drosophila Proteins/genetics/metabolism ; Gene Knock-In Techniques/methods ; CRISPR-Cas Systems ; Animals, Genetically Modified ; Olfactory Receptor Neurons/metabolism ; *Olfactory Pathways/metabolism ; },
abstract = {Drosophila melanogaster has provided numerous insights into the olfactory system, primarily relying on a series of transgenic Gal4 drivers. The combined use of Gal4/UAS and a second binary expression system, such as the QF/QUAS system, provides the opportunity to manipulate the two distinct cell populations, thereby accelerating the elucidation of the olfactory neural mechanisms. However, resources apart from the Gal4/UAS system have been poorly developed. In this study, we generated a series of odorant receptor (Or)-QF2 knock-in driver (Or-QF2[KI]) lines for 23 Ors using the CRISPR/Cas9 knock-in method. In these lines, the QF2 protein is cotranslated with each Or product. The expression pattern of the Or-QF2[KI] drivers mostly corresponded to that of the Or-Gal4 drivers. In addition, the Or42a-QF2[KI] driver identified the additional expression pattern of Or42a, which is consistent with the data of single-nucleus RNA sequencing and is attributed to the Or-QF2[KI] drivers' ability to reflect the endogenous expression of the Or genes. Thus, these Or-QF2[KI] drivers can be used as valuable genetic tools for olfactory research in Drosophila.},
}
@article {pmid40438494,
year = {2025},
author = {Shao, Q and Ndzie Noah, ML and Golubnitschaja, O and Zhan, X},
title = {Mitochondrial medicine: "from bench to bedside" 3PM-guided concept.},
journal = {The EPMA journal},
volume = {16},
number = {2},
pages = {239-264},
pmid = {40438494},
issn = {1878-5077},
abstract = {Mitochondria are the primary sites for aerobic respiration and play a vital role in maintaining physiologic function at the cellular and organismal levels. Physiologic mitochondrial homeostasis, functions, health, and any kind of mitochondrial impairments are associated with systemic effects that are linked to the human health and pathologies. Contextually, mitochondria are acting as a natural vital biosensor in humans controlling status of physical and mental health in a holistic manner. So far, no any disorder is known as happening to humans independently from a compromised mitochondrial health as the cause (primary mitochondrial dysfunction) or a target of collateral damage (secondary mitochondrial injury). This certainty makes mitochondrial medicine be the superior instrument to reach highly ambitious objectives of predictive, preventive, and personalized medicine (PPPM/3PM). 3PM effectively implements the paradigm change from the economically ineffective reactive medical services to a predictive approach, targeted prevention and treatments tailored to individualized patient profiles in primary (protection against health-to-disease transition) and secondary (protection against disease progression) healthcare. Mitochondrial DNA (mtDNA) properties differ significantly from those of nuclear DNA (nDNA). For example, mtDNA as the cell-free DNA molecule is much more stable compared to nDNA, which makes mtDNA be an attractive diagnostic target circulating in human body fluids such as blood and tear fluid. Further, genetic variations in mtDNA contribute to substantial individual differences in disease susceptibility and treatment response. To this end, the current gene editing technologies, such as clustered regularly interspaced short palindromic repeats (CRISPR)/Cas, are still immature in mtDNA modification, and cannot be effectively applied in clinical practice posing a challenge for mtDNA-based therapies. In contrast, comprehensive multiomics technologies offer new insights into mitochondrial homeostasis, health, and functions, which enables to develop more effective multi-level diagnostics and targeted treatment strategies. This review article highlights health- and disease-relevant mitochondrial particularities and assesses involvement of mitochondrial medicine into implementing the 3PM objectives. By discussing the interrelationship between 3PM and mitochondrial medicine, we aim to provide a foundation for advancing early and predictive diagnostics, cost-effective targeted prevention in primary and secondary care, and exemplify personalized treatments creating proof-of-concept approaches for 3PM-guided clinical applications.},
}
@article {pmid40437542,
year = {2025},
author = {Li, C and Zha, H and Jiao, Z and Wei, K and Gao, H and Lai, F and Zhou, Z and Luo, H and Li, P},
title = {Genetic engineering of E. coli K-12 for heterologous carbohydrate antigen production.},
journal = {Microbial cell factories},
volume = {24},
number = {1},
pages = {126},
pmid = {40437542},
issn = {1475-2859},
support = {CSTB2024NSCQ-MSX1157//Natural Science Foundation of Chongqing, China/ ; SWU-KT23010//Fundamental Research Funds for the Central Universities/ ; 32300736//National Natural Science Foundation of China/ ; },
mesh = {*Genetic Engineering/methods ; Animals ; *Escherichia coli K12/genetics/metabolism ; Mice ; Multigene Family ; Mice, Inbred BALB C ; },
abstract = {BACKGROUND: Carbohydrate-based vaccines have made a remarkable impact on public health over the past three decades. Efficient production of carbohydrate antigens is a crucial prerequisite for the development of such vaccines. The enzymes involved in the synthesis of bacterial surface carbohydrate antigens are usually encoded by large, uninterrupted gene clusters. Non-pathogenic E. coli glycoengineering starts with the genetic manipulation of these clusters. Heterologous gene cluster recombination through an expression plasmid has several drawbacks, including continuous antibiotic selection pressure, genetic instability, and metabolic burdens. In contrast, chromosome-level gene cluster expression can minimize the metabolic effects on the host and reduce industrial costs.<br><br>RESULTS: In this study, we employed the suicide vector-mediated allelic exchange method to directly replace the native polysaccharide gene clusters in E. coli with heterologous ones. Unlike previously strategies, this method does not rely on I-SceI endonuclease or CRISPR/Cas system to release the linearized DNA insert and &#x3bb;-red recombinase to promote its homologous recombination. Meanwhile, the vectors could be conveniently constructed by assembling multiple large DNA fragments in order in vitro. The scarless chromosomal insertions were confirmed by whole-genome sequencing and the polysaccharide phenotypes of all glycoengineered E. coli mutants were evaluated through growth curves, silver staining, western blot, and flow cytometry. The data indicated that there was no obvious metabolic burden associated with the insertion of large gene clusters into the E. coli W3110 O-antigen locus, and the glycoengineered E. coli can produce LPS with a recovery rate around 1% of the bacterial dry weight. Moreover, the immunogenicity of the heterologously expressed carbohydrate antigens was analyzed by mice immunization experiments. The ELISA data demonstrated the successful induction of anti-polysaccharide IgM or IgG antibodies.<br><br>CONCLUSIONS: We have provided a convenient and reliable genomic glycoengineering method to produce efficacious, durable, and cost-effective carbohydrate antigens in non-pathogenic E. coli. Non-pathogenic E. coli glycoengineering has great potential for the highly efficient synthesis of heterologous polysaccharides and can serve as a versatile platform to produce next-generation biomedical agents, including glycoconjugate vaccines, glycoengineered minicells or outer membrane vesicles (OMVs), polysaccharide-based diagnostic reagents, and more.},
}
@article {pmid40437234,
year = {2025},
author = {Vanhooydonck, M and De Neef, E and De Saffel, H and Boel, A and Willaert, A and Callewaert, B and Claes, KBM},
title = {Prime editing outperforms homology-directed repair as a tool for CRISPR-mediated variant knock-in in zebrafish.},
journal = {Lab animal},
volume = {54},
number = {6},
pages = {165-172},
pmid = {40437234},
issn = {1548-4475},
support = {BOF21/DOC/242//Universiteit Gent (UGent)/ ; BOF GOA019-21//Universiteit Gent (UGent)/ ; },
mesh = {Animals ; *Zebrafish/genetics ; *Gene Knock-In Techniques/methods ; *Gene Editing/methods ; *CRISPR-Cas Systems ; *Recombinational DNA Repair ; Animals, Genetically Modified ; },
abstract = {Zebrafish serve as a valuable model organism for studying human genetic diseases. While generating knockout lines is relatively straightforward, introducing precise disease-specific genetic variants by knock-in (KI) remains challenging. KI lines, however, enable more accurate studies of molecular and physiological consequences of genetic diseases. Their generation is often hampered by low editing efficiency (EE) and potential off-target effects. Here, we optimized conventional CRISPR-Cas9-mediated homology-directed repair (HDR) strategies for precise KI of genetic variants in zebrafish and compared their efficacy with prime editing, a recently developed technique that is not yet commonly used. Using next-generation sequencing, we determined KI EE by HDR for six unique base-pair substitutions in three different zebrafish genes. We assessed the effect of (1) varying Cas9 amounts, (2) HDR templates with chemical modifications to improve integration efficiency, (3) different microinjection procedures and (4) introduction of additional synonymous guide-blocking variants in the HDR template. Increasing Cas9 amounts augmented KI EE, with optimal injected amounts of Cas9 between 200 pg and 800 pg. The use of Alt-R HDR templates further increased KI EE, while guide-blocking modifications did not. Injecting components directly into the cell was not superior to injections into the yolk. Prime editing, however, increased EE up to fourfold and expanded the F0 founder pool for four targets compared with conventional HDR editing, with fewer off-target effects. Therefore, prime editing is a very promising methodology for improving the creation of precise genomic edits in zebrafish, facilitating the modeling of human diseases.},
}
@article {pmid40436684,
year = {2025},
author = {Jin, YY and Zhang, P and Liu, DP},
title = {Optimizing homology-directed repair for gene editing: the potential of single-stranded DNA donors.},
journal = {Trends in genetics : TIG},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.tig.2025.04.014},
pmid = {40436684},
issn = {0168-9525},
abstract = {CRISPR (clustered regularly interspaced short palindromic repeat) system-based precise genome editing remarkably impacts both scientific investigation and therapeutic practices. Among various techniques, DNA donor-mediated homology-directed repair (HDR) represents a promising method for precise gene editing. Although efficiency constraints have previously limited HDR, recent advancements have significantly enhanced its effectiveness. Therefore, it is essential to highlight the progress made in this field and to reassess the potential of the HDR approach. In this review, we explore the fundamental principles of HDR-dependent gene editing and evaluate current strategies to enhance HDR efficiency, with particular emphasis on single-stranded DNA (ssDNA) donor-mediated HDR. Finally, we discuss the prospects of high-efficiency ssDNA donor-mediated precise gene editing in laboratory research and clinical therapies.},
}
@article {pmid40436222,
year = {2025},
author = {Amiri, M and Shojaei, A and Mashayekhan, S and Saeedi, S and Kamali, B and Biabanaki, ZS and Mousazadeh, S and Kiani, J and Molabashi, ZA and Karimi, M},
title = {Synthesis of low molecular weight polyethylene imine-polyamidoamine hybrid and its modified derivatives as efficient nanocarriers for pDNA and CRISPR/Cas9 delivery.},
journal = {International journal of pharmaceutics},
volume = {680},
number = {},
pages = {125778},
doi = {10.1016/j.ijpharm.2025.125778},
pmid = {40436222},
issn = {1873-3476},
mesh = {Humans ; Animals ; HEK293 Cells ; *DNA/administration &amp; dosage ; *Dendrimers/chemistry/administration &amp; dosage ; CRISPR-Cas Systems ; *Polyethyleneimine/chemistry ; *Polyamines/chemistry ; Mice ; Molecular Weight ; *Gene Transfer Techniques ; *Nanoparticles/chemistry/administration &amp; dosage ; MCF-7 Cells ; *Plasmids/administration &amp; dosage ; HCT116 Cells ; Mice, Inbred C57BL ; *Polyethylenes/chemistry ; *Imines/chemistry ; Histidine/chemistry ; Mesenchymal Stem Cells/metabolism ; *Drug Carriers/chemistry ; Cell Survival/drug effects ; },
abstract = {A branched copolymer based on polyethylene imine (PEI) 1.8 kDa and the first generation of polyamidoamine dendrimer (PAMAM G1) was synthesized as a low toxicity nanocarrier. This hybrid material, designated here by G1PEI, was used as a parent nanostructure for the next step to synthesize a series of new nanocarriers by attaching L-arginine (Arg), L-histidine (His), and heptafluorobutyric anhydride (F7) on G1PEI; in both cases of single modifier or in combination together. Positive zeta accompanied by gel retardation assay confirmed the suitable gene loading capacity of the nanocarriers with a minimum of 0.8:1 wt ratio. For the sake of screening, the biological performance of the nanocarriers was first evaluated in HEK-293 T cell line at various weight ratios. Accordingly, the best nanovectors were selected for pDNA delivery in MCF-7 and HCT-116 cell lines, as well as for CRISPR/Cas9 delivery in mesenchymal stem cells derived from the bone marrow of C57BL/6 green laboratory mice. Flow cytometry and fluorescence microscopic imaging revealed that G1PEI grafted with 10 groups of F7 (G1PEI-10F7), 5 groups of ArgF7 (G1PEI-5ArgF7), and 3 groups of ArgF7 accompanied with 2 groups of His (G1PEI-3ArgF7-2His) exhibited higher gene transfection efficiency than PEI 25 kDa (known as a golden standard in biological systems) in plasmid delivery and knockout, with observed knockout efficiencies ranging between 54 % to 90 %. Moreover, MTT tests demonstrated the lower toxicity of the nanocarriers compared to PEI 25 kDa.},
}
@article {pmid40435597,
year = {2025},
author = {Kim, HJ and Park, DG and Choi, SJ and Cho, SD},
title = {PCSK9 is a passenger gene in head and neck cancer with minimal pathological influence.},
journal = {Archives of oral biology},
volume = {176},
number = {},
pages = {106302},
doi = {10.1016/j.archoralbio.2025.106302},
pmid = {40435597},
issn = {1879-1506},
mesh = {Humans ; *Proprotein Convertase 9/genetics/metabolism ; *Head and Neck Neoplasms/genetics/pathology ; Cell Proliferation/genetics ; Cell Line, Tumor ; Apoptosis/genetics ; Cell Movement ; CRISPR-Cas Systems ; Blotting, Western ; Gene Expression Regulation, Neoplastic ; },
abstract = {OBJECTIVES: In this study, we aimed to explore the pathological effects of PCSK9 in head and neck cancer (HNC) by ablating its expression using the CRISPR-Cas9 knockout system.<br><br>DESIGN: To investigate the clinical importance of PCSK9 in HNC, in silico analysis was performed using datasets from the GEO database and the UALCAN database. To evaluate the role of PCSK9 in HNC pathogenesis, both CRISPR-Cas9 knockout system and pharmacological inhibition were employed to ablate PCSK9 expression in HNC cell lines. The impact of PCSK9 on cellular growth and proliferation was assessed using Cell Counting Kit-8, soft agar, and clonogenic assays in a 2D culture model, as well as a hanging drop spheroid formation assay with live/dead staining in a 3D culture model. The involvement of PCSK9 in apoptosis induction was evaluated by detecting c-PARP expression through Western blotting, measuring the sub-G1 population via cell cycle assay, and verifying the Annexin V-positive population. Finally, changes in metastatic profiles associated with fluctuations in PCSK9 expression were examined using wound healing and transwell migration/invasion assays.<br><br>RESULTS: In in silico analysis results, PCSK9 appeared to be related to the progression of HNC. However, experimental results demonstrated that PCSK9 plays a minimal role in cancer cell proliferation, anchorage-independent growth, colony formation capacity, in 2D cultures, as well as spheroidal growth in 3D cultures, and apoptosis induction. Furthermore, PCSK9 marginally influenced wound closure, metastatic potential, and invasive ability.<br><br>CONCLUSION: Collectively, these data suggest that PCSK9 serves a neutral role in HNC, functioning as a passenger gene.},
}
@article {pmid40435438,
year = {2025},
author = {Shu, F and Huang, Y and Yang, F and Guo, Y and Xu, R and Xiao, L and Feng, Y and Li, N},
title = {Calcium-dependent protein kinases 2A involved in the growth of both asexual and sexual stages of Cryptosporidium parvum.},
journal = {PLoS neglected tropical diseases},
volume = {19},
number = {5},
pages = {e0013107},
pmid = {40435438},
issn = {1935-2735},
mesh = {*Cryptosporidium parvum/growth &amp; development/enzymology/genetics ; Animals ; Cryptosporidiosis/parasitology ; Mice ; *Protein Kinases/genetics/metabolism ; CRISPR-Cas Systems ; *Protozoan Proteins/genetics/metabolism ; Oocysts/growth &amp; development ; Female ; Humans ; Life Cycle Stages ; Sporozoites/growth &amp; development ; },
abstract = {BACKGROUND: Cryptosporidium parvum is a protozoan pathogen that causes moderate to severe diarrhea in both humans and animals. Calcium-dependent protein kinases (CDPKs) are attractive drug targets against cryptosporidiosis given their critical role in the life cycle of Cryptosporidium spp. and their absence in human and animal hosts.<br><br>We used CRISPR-Cas9 technology to endogenously tag the CpCDPK2A gene in C. parvum IIdA20G1-HLJ strain with the hemagglutinin (HA) epitope and to delete the CpCDPK2A gene. An immunofluorescence assay was performed to localize the CpCDPK2A expression in the tagged strain and a luciferase assay was performed to compare growth rates of the tagged and deletion strains in vitro. Oocyst shedding, parasite load, villus length/crypt height ratio and survival of infected mice were used to evaluate the function of CpCDPK2A in vivo. The results revealed that CpCDPK2A was expressed in all the intracellular developmental stages, especially in the motile stages of sporozoites and merozoites. While CpCDPK2A is dispensable, deletion of the gene significantly reduced the growth of late asexual and sexual stages in vitro. In an interferon-&#x3b3; knockout mouse model, gene deletion of CpCDPK2A reduced oocyst shedding by 25-fold and increased survival of infected mice.<br><br>CONCLUSIONS/SIGNIFICANCE: These observations suggest that CpCDPK2A may contribute to both asexual and sexual replication of C. parvum and may be a potential target to block the transmission of this important zoonotic pathogen.},
}
@article {pmid40434364,
year = {2025},
author = {Hecht, AD and Igoshin, OA},
title = {Kinetic Mechanism for Fidelity of CRISPR-Cas9 Variants.},
journal = {The journal of physical chemistry letters},
volume = {16},
number = {22},
pages = {5570-5578},
doi = {10.1021/acs.jpclett.5c01154},
pmid = {40434364},
issn = {1948-7185},
mesh = {Kinetics ; *CRISPR-Cas Systems ; Fluorescence Resonance Energy Transfer ; Substrate Specificity ; *CRISPR-Associated Protein 9/metabolism/chemistry/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; DNA/chemistry/metabolism ; },
abstract = {CRISPR-Cas9 is a nuclease creating DNA breaks at sites with sufficient complementarity to the RNA guide. Notably, Cas9 does not require exact RNA-DNA complementarity and can cleave off-target sequences. Various high-accuracy Cas9 variants have been developed, but the precise mechanism of how these variants achieve higher accuracy remains unclear. Here, we develop a kinetic model of Cas9 substrate selection and cleavage parametrized by data from the literature, including single-molecule Förster resonance energy transfer (FRET) measurements. Based on observed FRET transition statistics, we predict that the Cas9 substrate recognition and cleavage mechanism must allow for HNH domain transitions independent of substrate binding. Additionally, we show that the enhancement in Cas9 substrate specificity must be due to changes in kinetics rather than changes in substrate binding affinities. Finally, we use our model to identify kinetic parameters for HNH domain transitions that can be perturbed to enable high-accuracy cleavage while maintaining cleavage speeds.},
}
@article {pmid40432879,
year = {2025},
author = {Melchior, F and Angelidou, IA and Chorianopoulou, M and Teichmann, B},
title = {The genetic technologies questionnaire in the Greek-speaking population: the moral judgement of the lay public.},
journal = {Frontiers in genetics},
volume = {16},
number = {},
pages = {1594724},
pmid = {40432879},
issn = {1664-8021},
abstract = {INTRODUCTION: Advancements in life sciences have significantly boosted biomedical capabilities. Genetic testing forecasts hereditary traits and disease susceptibility, while CRISPR/Cas allows permanent genome alterations. However, ethical considerations arise regarding the morality of these capabilities, particularly concerning the moral status, autonomy, and privacy of living beings. The lack of valid instruments to assess moral judgment in genetic technologies highlights the need for this study, aiming to translate and validate the "Genetic Technologies Questionnaire" (GTQ) and the short version of the "Conventional Technologies Questionnaire" (CTQ5) into Greek. As the full version of the GTQ with 30 questions could be too extensive for some studies, we also tested other versions: The short versions GTQ20-GR and GTQ5-GR which were already presented in the original study, as well as a version which included questions solely about humans (GTQ-H-GR) and is intended for use in human research and therapy, and the GTQ-Moral Status (GTQ-MS-GR), which included questions about genetic testing and gene editing in different living beings to investigate differences in moral status.<br><br>METHODS: A cross-sectional study involved 250 participants who completed an online questionnaire, assessing internal consistency, structural validity, known-groups validity, floor/ceiling effects, and retest reliability (subset of 50 participants). Correlational analyses explored relationships with education, age, genetic knowledge, religiosity, and genetic testing experience. The study followed the STROBE checklist for reporting.<br><br>RESULTS: The GTQ-GR (Cronbach's &#x3b1; = 0.929) and GTQ20-GR (&#x3b1; = 0.935) exhibit high reliability and stability in assessing moral judgment among lay people, whereas the GTQ5-GR (&#x3b1; = 0.866) and CTQ5-GR (&#x3b1; = 0.758) displayed some weaknesses. Participants tended to rate conventional technologies more favorably than genetic technologies, with genetic testing perceived more positively than genome editing. The two additional derived versions, GTQ-H-GR (&#x3b1; = 0.859) and GTQ-MS-GR (&#x3b1; = 0.787), also demonstrated solid psychometric characteristics.<br><br>CONCLUSION: The GTQ-GR is a valid and reliable questionnaire with strong psychometric properties and is now available in Greek.},
}
@article {pmid40431732,
year = {2025},
author = {Meng, P and Ni, B and Li, C and Sha, Z and Liu, C and Ren, W and Wei, R and Liu, F and Li, J and Wang, Z},
title = {Establishment and Implementation of the Point-of-Care RT-RAA-CRISPR/Cas13a Diagnostic Test for Foot-And-Mouth Disease Virus Serotype O in Pigs.},
journal = {Viruses},
volume = {17},
number = {5},
pages = {},
pmid = {40431732},
issn = {1999-4915},
support = {2021YFD1800300//National Key R&amp;D Program of China/ ; },
mesh = {Animals ; Swine ; *Foot-and-Mouth Disease Virus/genetics/isolation &amp; purification/classification ; *Swine Diseases/diagnosis/virology ; *Foot-and-Mouth Disease/diagnosis/virology ; Serogroup ; *CRISPR-Cas Systems ; Sensitivity and Specificity ; *Point-of-Care Systems ; RNA, Viral/genetics ; *Molecular Diagnostic Techniques/methods ; },
abstract = {Foot and mouth disease virus (FMDV) is a highly pathogenic virus that mainly infects cloven hooved animals, such as pigs. The establishment of a rapid, sensitive and accurate point-of-care detection method is critical for the timely identification and elimination of infected pigs for controlling this disease. In this study, a RT-RAA-CRISPR/Cas13a method was developed for the detection of FMDV serotype O in pigs. Six pairs of RT-RAA primers were designed based on the conserved gene sequence of FMDV serotype O, and the optimal amplification primers and reaction temperatures were screened. The CRISPR-derived RNA (crRNA) was further designed based on the optimal target band sequence and the most efficient crRNA was screened. The results revealed that FMDV-O-F4/R4 was the optimal primer set, and the optimal temperature for the RT-RAA reaction was 37 °C. Moreover, crRNA4 exhibited the strongest detection signal among the six crRNAs. The established RT-RAA-CRISPR/Cas13a method demonstrated high specificity and no cross-reactivity with other common swine pathogens such as Senecavirus A (SVA), porcine reproductive and respiratory virus (PRRSV), porcine epidemic diarrhea virus (PEDV), porcine circovirus type 2 (PCV2), classical swine fever virus (CSFV), and pseudorabies virus (PRV), additionally, it was observed to be highly sensitive, with a detection limit of 19.1 copies/µL. The repeatability of this method was also observed to be good. This method could produce stable fluorescence and exhibited good repeatability when three independent experiments yielded the same results. A validation test using three types of simulated clinical samples (including swab, tissue, and serum samples) revealed a 100% concordance rate. The detection results could be visualized via a fluorescence reader or lateral flow strips (LFSs). Thus, a highly specific and sensitive RT-RAA-CRISPR/Cas13a detection method was developed and is expected to be applied for the rapid detection of FMDV serotype O in situ.},
}
@article {pmid40429982,
year = {2025},
author = {Leal, K and Machuca, J and Gajardo, H and Palma, M and Contreras, MJ and Nuñez-Montero, K and Gutiérrez, &#xc1; and Barrientos, L},
title = {Structural Characterisation of TetR/AcrR Regulators in Streptomyces fildesensis So13.3: An In Silico CRISPR-Based Strategy to Influence the Suppression of Actinomycin D Production.},
journal = {International journal of molecular sciences},
volume = {26},
number = {10},
pages = {},
pmid = {40429982},
issn = {1422-0067},
support = {1210563//Fondecyt Regular/ ; 11230475//Fondecyt Iniciaci&#xf3;n/ ; 11240632//Fondecyt Iniciaci&#xf3;n/ ; },
mesh = {*Streptomyces/genetics/metabolism ; *Dactinomycin/biosynthesis ; *Bacterial Proteins/genetics/metabolism/chemistry ; Gene Expression Regulation, Bacterial ; Multigene Family ; Molecular Dynamics Simulation ; *Transcription Factors/genetics/metabolism/chemistry ; Anti-Bacterial Agents/biosynthesis ; *CRISPR-Cas Systems ; Biosynthetic Pathways/genetics ; },
abstract = {The growing threat of antimicrobial resistance has intensified the search for new bioactive compounds, particularly in extreme environments such as Antarctica. Streptomyces fildesensis So13.3, isolated from Antarctic soil, harbours a biosynthetic gene cluster (BGC) associated with actinomycin D production, an antibiotic with biomedical relevance. This study investigates the regulatory role of TetR/AcrR transcription factors encoded within this biosynthetic gene cluster (BGC), focusing on their structural features and expression under different nutritional conditions. Additionally, we propose that repressing an active pathway could lead to the activation of silent biosynthetic routes, and our in-silico analysis provides a foundation for selecting key mutations and experimentally validating this strategy. Expression analysis revealed that TetR-279, in particular, was upregulated in ISP4 and IMA media, suggesting its participation in nutrient-dependent BGC regulation. Structural modelling identified key differences between TetR-206 and TetR-279, with the latter containing a tetracycline-repressor-like domain. Molecular dynamics simulations confirmed TetR-279's structural stability but showed that the S166P CRISPy-web-guided mutation considerably affected its flexibility, while V167A and V167I had modest effects. These results underscore the importance of integrating omics, structural prediction, and gene editing to evaluate and manipulate transcriptional regulation in non-model bacteria. Targeted disruption of TetR-279 may derepress actinomycin biosynthesis, enabling access to silent or cryptic secondary metabolites with potential pharmaceutical applications.},
}
@article {pmid40429922,
year = {2025},
author = {Sheng, H and Gao, P and Yang, C and Quilichini, TD and Kochian, LV and Datla, R and Xiang, D},
title = {Advances in Genome Editing Through Haploid Induction Systems.},
journal = {International journal of molecular sciences},
volume = {26},
number = {10},
pages = {},
pmid = {40429922},
issn = {1422-0067},
mesh = {*Gene Editing/methods ; *Haploidy ; CRISPR-Cas Systems ; *Crops, Agricultural/genetics ; Plant Breeding/methods ; *Genome, Plant ; Diploidy ; Plants, Genetically Modified/genetics ; },
abstract = {Groundbreaking advances in gene editing technologies are transforming modern plant breeding by enabling precise genetic modifications that dramatically accelerate crop improvement. Haploid and diploid induction systems have emerged as particularly powerful tools in this landscape, offering both efficient gene editing capabilities and rapid production of homozygous lines while seamlessly integrating with the advanced genome-editing platforms such as CRISPR-Cas systems. This review synthesizes the current state of knowledge regarding the mechanisms, applications, and recent progress in haploid and diploid induction systems for gene editing. We examine their transformative potential for enhancing genetic gains and compressing breeding timelines, with significant implications for global food security. Additionally, we provide a critical analysis of emerging challenges of genome editing in crops and outline promising future directions for research and development.},
}
@article {pmid40429745,
year = {2025},
author = {Nilsri, N and Mekchaaum, R and Kalasin, S and Jongjitwimol, J and Daowtak, K},
title = {CRISPR/Cas9-Based Modeling of JAK2 V617F Mutation in K562 Cells Reveals Enhanced Proliferation and Sensitivity to Therapeutic Agents.},
journal = {International journal of molecular sciences},
volume = {26},
number = {10},
pages = {},
pmid = {40429745},
issn = {1422-0067},
support = {R2564C032 and AH-64-01-002//Naresuan University research funding and Faculty of Allied Health Sciences, Naresuan University research funding/ ; },
mesh = {Humans ; *Janus Kinase 2/genetics/metabolism ; Cell Proliferation/drug effects/genetics ; *CRISPR-Cas Systems/genetics ; K562 Cells ; Interferon-alpha/pharmacology ; *Mutation ; Arsenic Trioxide/pharmacology ; Myeloproliferative Disorders/genetics/drug therapy ; Antineoplastic Agents/pharmacology ; Point Mutation ; },
abstract = {The Janus kinase 2 (JAK2) protein fulfills an important role in hematopoiesis via the Janus kinase/signal transducer and activator of transcription (JAK/STAT) pathway, as it provides the genetic driver of BCR::ABL1-negative myeloproliferative neoplasms (MPNs), which are clinically manifested as polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF). The most common cause of MPNs is the mutation of JAK2 V617F in the JAK2 gene, which results in increased cell proliferation. However, both the pathogenesis and treatment regimen of BCR::ABL1-negative MPNs remain poorly understood. The aim of the present study was to establish K562 cell lines with a point mutation in exon 14 (JAK2p.V617F) using CRISPR/Cas9 technology. The modified JAK2 V617F cell lines were examined for the gene mutation using droplet digital PCR (DDPCR), and the presence of the mutation was confirmed by DNA sequencing. Modified cells were characterized by measuring JAK2 gene expression and the extent of cell proliferation. Interferon α2a (IFN-α2a) and arsenic trioxide were also administered to the cells to explore their potential effects. The JAK2 V617F-mutated cells were found to exhibit a higher level of JAK2 gene expression compared with the wild type. Interestingly, a significant increase in the proliferation rate was observed with the modified cells compared with the wild type cells (p < 0.001), as assessed from the JAK2 gene expression levels. Furthermore, the treatments with IFN-α2a and arsenic trioxide led to the preferential suppression of the cell proliferation rate of the K562 expressing mutant JAK2 cells compared with the wild type cells, and this suppression occurred in a dose-dependent manner(p < 0.01). Moreover, the modified cells were able to differentiate into megakaryocyte-like cells following stimulation with phorbol 12 myristate 13 acetate (PMA). Taken together, the results of the present study have shown that the CRISPR/Cas9-modified JAK2 V617F model may be used as a disease model in the search of novel therapies for MPNs.},
}
@article {pmid40429744,
year = {2025},
author = {Mikhaylova, E},
title = {Virus-Induced Genome Editing (VIGE): One Step Away from an Agricultural Revolution.},
journal = {International journal of molecular sciences},
volume = {26},
number = {10},
pages = {},
pmid = {40429744},
issn = {1422-0067},
support = {24-76-10065//Russian Science Foundation/ ; },
mesh = {*Gene Editing/methods ; CRISPR-Cas Systems ; *Plant Viruses/genetics ; *Genome, Plant ; Agriculture/methods ; *Crops, Agricultural/genetics/virology ; Plants, Genetically Modified/genetics ; Genetic Vectors/genetics ; },
abstract = {There is currently a worldwide trend towards deregulating the use of genome-edited plants. Virus-induced genome editing (VIGE) is a novel technique that utilizes viral vectors to transiently deliver clustered regularly interspaced short palindromic repeat (CRISPR) components into plant cells. It potentially allows us to obtain transgene-free events in any plant species in a single generation without in vitro tissue culture. This technology has great potential for agriculture and is already being applied to more than 14 plant species using more than 20 viruses. The main limitations of VIGE include insufficient vector capacity, unstable expression of CRISPR-associated (Cas) protein, plant immune reaction, host specificity, and reduced viral activity in meristem. Various solutions to these problems have been proposed, such as fusion of mobile elements, RNAi suppressors, novel miniature Cas proteins, and seed-borne viruses, but the final goal has not yet been achieved. In this review, the mechanism underlying the ability of different classes of plant viruses to transiently edit genomes is explained. It not only focuses on the latest achievements in virus-induced editing of crops but also provides suggestions for improving the technology. This review may serve as a source of new ideas for those planning to develop new approaches in VIGE.},
}
@article {pmid40429730,
year = {2025},
author = {Fu, L and Li, P and Rui, Z and Sun, J and Yang, J and Wang, Y and Jia, D and Hu, J and Li, X and Ma, R},
title = {CRISPR/Cas9-Mediated Knockout of the White Gene in Agasicles hygrophila.},
journal = {International journal of molecular sciences},
volume = {26},
number = {10},
pages = {},
pmid = {40429730},
issn = {1422-0067},
support = {202304051001006//Science and Technology Innovation Teams of Shanxi Province/ ; 20210302123386//Fundamental Research Program of Shanxi Province/ ; 2016YJ03//Science and Technology Innovation Fund of Shanxi Agricultural University/ ; ZBXY23A-10//Cultivation and Innovation Program for Scientific research, College of Plant Protection, Shanxi Agricultural University/ ; },
mesh = {*CRISPR-Cas Systems ; Gene Editing/methods ; *Gene Knockout Techniques ; *Amaranthaceae/genetics ; },
abstract = {Agasicles hygrophila is the most effective natural enemy for the control of the invasive weed Alternanthera philoxeroides (Mart.) Griseb. However, research on the gene function and potential genetic improvement of A. hygrophila is limited due to a lack of effective genetic tools. In this study, we employed the A. hygrophila white (AhW) gene as a target gene to develop a CRISPR/Cas9-based gene editing method applicable to A. hygrophila. We showed that injection of Cas9/sgRNA ribonucleoprotein complexes (RNPs) of the AhW gene into pre-blastoderm eggs induced genetic insertion and deletion mutations, leading to white eyes. Our results demonstrate that CRISPR/Cas9-mediated gene editing is possible in A. hygrophila, offering a valuable tool for studies of functional genomics and genetic improvement of A. hygrophila, which could potentially lead to more effective control of invasive weeds through the development of improved strains of this biocontrol agent. In addition, the white-eyed mutant strain we developed could potentially be useful for other transgenic research studies on this species.},
}
@article {pmid40428391,
year = {2025},
author = {Ziółkowska-Suchanek, I and Rozwadowska, N},
title = {Advancements in Gene Therapy for Non-Small Cell Lung Cancer: Current Approaches and Future Prospects.},
journal = {Genes},
volume = {16},
number = {5},
pages = {},
pmid = {40428391},
issn = {2073-4425},
mesh = {Humans ; *Carcinoma, Non-Small-Cell Lung/therapy/genetics ; *Genetic Therapy/methods/trends ; *Lung Neoplasms/therapy/genetics ; Gene Editing/methods ; CRISPR-Cas Systems ; Immunotherapy, Adoptive/methods ; },
abstract = {Non-small cell lung cancer (NSCLC) is the leading cause of cancer-related death worldwide, characterized by late diagnosis and resistance to conventional therapies. Gene therapy has emerged as a promising alternative for NSCLC therapy, especially for patients with advanced disease who have exhausted conventional treatments. This article delved into the current developments in gene therapy for NSCLC, including gene replacement and tumor suppressor gene therapy, gene silencing, CRISPR/Cas9 gene editing, and immune modulation with CAR-T cell therapy. In addition, the challenges and future prospects of gene-based therapies for NSCLC were discussed.},
}
@article {pmid40427606,
year = {2025},
author = {Gutiérrez-Hurtado, IA and García-Acéves, ME and Puga-Carrillo, Y and Guardado-Estrada, M and Becerra-Loaiza, DS and Carrillo-Rodríguez, VD and Plazola-Zamora, R and Godínez-Rubí, JM and Rangel-Villalobos, H and Aguilar-Velázquez, JA},
title = {Past, Present and Future Perspectives of Forensic Genetics.},
journal = {Biomolecules},
volume = {15},
number = {5},
pages = {},
pmid = {40427606},
issn = {2218-273X},
mesh = {Humans ; *Forensic Genetics/trends/methods/history ; Machine Learning ; CRISPR-Cas Systems ; },
abstract = {Forensic genetics has experienced remarkable advancements over the past decades, evolving from the analysis of a limited number of DNA segments to comprehensive genome-wide investigations. This progression has significantly improved the ability to establish genetic profiles under diverse conditions and scenarios. Beyond individual identification, forensic genetics now enables the inference of physical traits (e.g., eye, hair, and skin color, as well as body composition), biogeographic ancestry, lifestyle habits such as alcohol and tobacco use, and even the transfer of genital microbiomes post-coitus, among other characteristics. Emerging trends point to a future shaped by the integration of cutting-edge technologies, including CRISPR-Cas systems, artificial intelligence, and machine learning, which promise to further revolutionize the field. This review provides a thorough exploration of forensic genetics, tracing its evolution from its foundational methods (past) to its diverse modern applications (present) and offering insights into its potential future directions.},
}
@article {pmid40427601,
year = {2025},
author = {Marin-Quilez, A and García-Tuñón, I and Benito, R and Ordoñez, JL and Díaz-Ajenjo, L and Lama-Villanueva, A and Guerrero, C and Pérez-Losada, J and González-Porras, JR and Hernández-Rivas, JM and Del Rey, M and Bastida, JM},
title = {Examining the Effects of the RUNX1 p.Leu43Ser Variant on FPD/AML Phenotypes Using a CRISPR/Cas9-Generated Knock-In Murine Model.},
journal = {Biomolecules},
volume = {15},
number = {5},
pages = {},
pmid = {40427601},
issn = {2218-273X},
mesh = {Animals ; *Core Binding Factor Alpha 2 Subunit/genetics/metabolism ; Mice ; *CRISPR-Cas Systems/genetics ; Disease Models, Animal ; Gene Knock-In Techniques ; *Leukemia, Myeloid, Acute/genetics/pathology ; Phenotype ; *Blood Platelet Disorders/genetics/pathology ; Male ; Homozygote ; },
abstract = {Germline heterozygous variants in RUNX1 lead to Familial Platelet Disorder with Myeloid Leukemia Predisposition (FPD/AML). Cellular and/or animal models are helpful to uncovering the role of a variant in disease progression. Twenty-five mice per genotype (RUNX1[WT/WT], RUNX1[WT/L43S], RUNX1[L43S/L43S]), previously generated by CRISPR/Cas9, and nine sub-lethally irradiated mice per genotype were investigated. Peripheral blood (PB), bone marrow (BM), and spleen samples were analyzed by flow cytometry and histopathology. Deregulated genes were analyzed by RNA-seq in BM. An aberrant myeloid Mac1[+]Sca1[+]ckit[-] population in the PB, BM, and spleen of two homozygous and one heterozygous mouse was observed, as well as BM hypercellularity. No Mac1[+]Sca1[+]ckit[-] cells were detected in any RUNX1[WT/WT] mice. Moreover, the spleen of both homozygous mice showed destruction of the white/red pulp and the presence of apoptotic cells. The aberrant population was also detected in four irradiated mice, two heterozygous and two homozygous, in their PB, BM, and spleen. RNA-seq studies showed 698 genes significantly deregulated in the three non-irradiated Mac1[+]Sca1[+]ckit[-] mice vs. six healthy mice, highlighting the alteration of genes involved in apoptosis and DNA repair. These results indicate that the homozygous form of the variant p.Leu43Ser may contribute to the pathogenesis of aberrant cells.},
}
@article {pmid40427577,
year = {2025},
author = {Palit, P and Minkara, M and Abida, M and Marwa, S and Sen, C and Roy, A and Pasha, MR and Mosae, PS and Saha, A and Ferdoush, J},
title = {PlastiCRISPR: Genome Editing-Based Plastic Waste Management with Implications in Polyethylene Terephthalate (PET) Degradation.},
journal = {Biomolecules},
volume = {15},
number = {5},
pages = {},
pmid = {40427577},
issn = {2218-273X},
support = {Start-up fund//University of Tennessee at Chattanooga/ ; },
mesh = {*Polyethylene Terephthalates/metabolism/chemistry ; *Gene Editing/methods ; *Waste Management/methods ; *Plastics/metabolism/chemistry ; Biodegradation, Environmental ; CRISPR-Cas Systems ; Humans ; },
abstract = {Plastic pollution has become a significant environmental issue worldwide, with global plastic production expected to reach 1800 million tons by 2050. One of the most commonly used plastics in the world is polyethylene terephthalate (PET), a synthetic polymer that is extremely durable but difficult to degrade. Thus, PET is dangerous to human health. To address this crisis, innovative approaches are being developed, including genome editing technologies. One of the recently advanced genome editing technologies is PlastiCRISPR, a novel concept that applies CRISPR-based genome editing to transform plastic waste management. PlastiCRISPR utilizes microorganisms to degrade plastic, generating valuable bioproducts like biofuels and biochemicals. Thus, this technology offers a sustainable solution because of its simple design, adequacy, and low cost, which can be integrated into existing waste management systems. Importantly, this review focuses on the PlastiCRISPR-based management of PET because it could drastically lower plastic waste, sustain natural resources by decreasing the requirement for plastic production, minimize energy intake, etc. Overall, this review provides an overview of the principles, applications, challenges, and future prospects of PlastiCRISPR in combating plastic pollution and shaping a more sustainable future.},
}
@article {pmid40426207,
year = {2025},
author = {Wang, ZC and Stegall, H and Miyazawa, T and Keatinge-Clay, AT},
title = {A CRISPR-Cas9 system for knock-out and knock-in of high molecular weight DNA enables module-swapping of the pikromycin synthase in its native host.},
journal = {Microbial cell factories},
volume = {24},
number = {1},
pages = {125},
pmid = {40426207},
issn = {1475-2859},
support = {R01 GM145992/GM/NIGMS NIH HHS/United States ; GM145992/GM/NIGMS NIH HHS/United States ; },
mesh = {*CRISPR-Cas Systems ; *Streptomyces/genetics/metabolism/enzymology ; *Polyketide Synthases/genetics/metabolism ; Macrolides/metabolism ; Gene Editing/methods ; Gene Knockout Techniques ; Anti-Bacterial Agents/biosynthesis ; *Gene Knock-In Techniques ; Molecular Weight ; },
abstract = {BACKGROUND: Engineers seeking to generate natural product analogs through altering modular polyketide synthases (PKSs) face significant challenges when genomically editing large stretches of DNA.<br><br>RESULTS: We describe a CRISPR-Cas9 system that was employed to reprogram the PKS in Streptomyces venezuelae ATCC 15439 that helps biosynthesize the macrolide antibiotic pikromycin. We first demonstrate its precise editing ability by generating strains that lack megasynthase genes pikAI-pikAIV or the entire pikromycin biosynthetic gene cluster but produce pikromycin upon complementation. We then employ it to replace 4.4-kb modules in the pikromycin synthase with those of other synthases to yield two new macrolide antibiotics with activities similar to pikromycin.<br><br>CONCLUSION: Our gene-editing tool has enabled the efficient replacement of extensive and repetitive DNA regions within streptomycetes.},
}
@article {pmid40425955,
year = {2025},
author = {Chen, Y and Zhang, X and Huang, W and Jin, Z and Wei, X and Li, J},
title = {Ultrasensitive detection of atrazine by Schottky junction photoelectrochemical aptamer sensor based on signal amplification by cascade catalysis of CRISPR/Cas12a and G-quadruplex/hemin DNAzyme.},
journal = {Mikrochimica acta},
volume = {192},
number = {6},
pages = {376},
pmid = {40425955},
issn = {1436-5073},
mesh = {*G-Quadruplexes ; *DNA, Catalytic/chemistry/metabolism ; *Aptamers, Nucleotide/chemistry ; Hemin/chemistry/metabolism ; *Biosensing Techniques/methods ; *Electrochemical Techniques/methods ; Limit of Detection ; *Atrazine/analysis/chemistry ; CRISPR-Cas Systems ; Electrodes ; CRISPR-Associated Proteins/metabolism/chemistry ; Photochemical Processes ; Catalysis ; Indoles/chemistry ; Bacterial Proteins ; Endodeoxyribonucleases ; },
abstract = {Atrazine (ATZ) is used extensively, resulting in residues in food and the environment, posing a serious threat to human health. Herein, Cd0.5Zn0.5S/Ti3C2 photoelectric material was synthesized and immobilized on a FTO electrode as a photoanode. A photoelectrochemical (PEC) aptamer sensor was constructed for the highly sensitive and selective determination of ATZ based on signal amplification via cascade catalysis of CRISPR/Cas12a and G-quadruplex/hemin DNAzyme (G4/hemin DNAzyme). G4/hemin DNAzyme catalyses the oxidation reaction between H2O2 and dopamine (DA) to form polydopamine (PDA) deposit. This process, in turn, inhibits the photocurrent at the photoanode, leading to a decrease in photocurrent. Concurrently, the depletion of DA as an electron donor for the PEC reaction at the photoelectrode further contributes to the decrease in photocurrent. ATZ can hybridize with ATZ aptamer (Apt) in Apt/cDNA to release activation strand (cDNA), which activates the activity of CRISPR/Cas12a and triggers cleavage of G4, causing the cleaving of G4/hemin DNAzyme immobilized on the electrode surface. This process leads to a decrease of G4/hemin DNAzymes amount on the electrode, consequently reducing both the PDA generation and the DA consumption. As a result, the photocurrent is restored. The cascade catalysis of CRISPR/Cas12a and G4/hemin DNAzyme has been demonstrated to result in photocurrent amplification. The photocurrent change was linear with the logarithmic value of ATZ concentration in the range 1.00 × 10[-12] to 1.00 × 10[-5] mol/L. The limit of detection was 3.47 × 10[-13] mol/L. The sensor has been successfully applied to the determination of trace ATZ in environmental and food samples.},
}
@article {pmid40425635,
year = {2025},
author = {Imai, Y and Ozaki, S and Noda, T and Kobayashi, I and Sugitani, K and Kasashima, S and Morishita, E and Araiso, Y},
title = {Real-time imaging of blood coagulation and angiogenesis during development in a zebrafish model of type I antithrombin deficiency.},
journal = {Scientific reports},
volume = {15},
number = {1},
pages = {18538},
pmid = {40425635},
issn = {2045-2322},
support = {JPMJSP2135//Japan Science and Technology Agency/ ; JPMJFR200F//Japan Science and Technology Agency/ ; },
mesh = {Animals ; *Zebrafish/genetics/embryology ; *Blood Coagulation/genetics ; Disease Models, Animal ; *Neovascularization, Physiologic ; Zebrafish Proteins/genetics ; Animals, Genetically Modified ; Thrombosis/genetics ; CRISPR-Cas Systems ; Angiogenesis ; },
abstract = {Severe type I antithrombin (AT) deficiency is considered to cause embryonic lethality. Although several pathological analyses using mice or zebrafish have been attempted, the previous studies did not unveil the detailed mechanism leading to lethality in the early developmental stage. In order to solve this problem, we established type I AT deficient zebrafish by the CRISPR/Cas9 system into Tg(gata1:dsRed) and Tg(fli1a:GFP) lines, so that we could conduct real-time imaging of thrombosis and angiogenesis using fluorescence stereo zoom microscopy. The established zebrafish AT (zAT) mutants harbored frameshift mutations which resulted to be type I AT deficient, unable to secrete zAT protein into blood. Both heterozygous (zAT[+/-]) and homozygous (zAT[-/-]) mutants showed reduced survival rate and diverse thrombosis up to 9 days post fertilization. In addition, blood vessel formation was delayed at 30 hpf in zAT[-/-], which was recovered normally by 5 dpf and had little effect on survival. Notably, we analyzed the differences in gene expression profiles under AT-depleted conditions by real-time quantitative PCR, and zAT[-/-] juvenile zebrafish showed increased PLG gene expression and decreased F2 gene expression. Our in vivo study revealed the effects of AT deficiency on embryos during development from the aspects of coagulation and vascular formation.},
}
@article {pmid40425580,
year = {2025},
author = {Sharma, P and Kim, CY and Keys, HR and Imada, S and Joseph, AB and Ferro, L and Kunchok, T and Anderson, R and Sun, Y and Yilmaz, &#xd6;H and Weng, JK and Jain, A},
title = {Genetically encoded fluorescent reporter for polyamines.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {4921},
pmid = {40425580},
issn = {2041-1723},
support = {R35 GM151111/GM/NIGMS NIH HHS/United States ; R35GM151111//U.S. Department of Health &amp; Human Services | NIH | National Institute of General Medical Sciences (NIGMS)/ ; },
mesh = {*Polyamines/metabolism/analysis ; Humans ; *Genes, Reporter ; Mitochondria/metabolism ; Parkinson Disease/metabolism/genetics ; CRISPR-Cas Systems ; HEK293 Cells ; },
abstract = {Polyamines are abundant and evolutionarily conserved metabolites that are essential for life. Dietary polyamine supplementation extends life-span and health-span. Dysregulation of polyamine homeostasis is linked to Parkinson's disease and cancer, driving interest in therapeutically targeting this pathway. However, measuring cellular polyamine levels, which vary across cell types and states, remains challenging. We introduce a genetically encoded polyamine reporter for real-time measurement of polyamine concentrations in single living cells. This reporter utilizes the polyamine-responsive ribosomal frameshift motif from the OAZ1 gene. We demonstrate broad applicability of this approach and reveal dynamic changes in polyamine levels in response to genetic and pharmacological perturbations. Using this reporter, we conduct a genome-wide CRISPR screen and uncover an unexpected link between mitochondrial respiration and polyamine import, which are both risk factors for Parkinson's disease. By offering a lens to examine polyamine biology, this reporter may advance our understanding of these ubiquitous metabolites and accelerate therapy development.},
}
@article {pmid40425554,
year = {2025},
author = {Borot, F and Humbert, O and Ehmsen, JT and Fields, E and Kohli, S and Radtke, S and Swing, K and Pande, D and Enstrom, MR and Laszlo, GS and Mayuranathan, T and Ali, AM and Weiss, MJ and Yen, JS and Newby, GA and Walter, RB and Liu, DR and Mukherjee, S and Kiem, HP},
title = {Multiplex base editing to protect from CD33 directed drugs for immune and gene therapy.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {4899},
pmid = {40425554},
issn = {2041-1723},
support = {R01 HL156647/HL/NHLBI NIH HHS/United States ; K99 HL163805/HL/NHLBI NIH HHS/United States ; P51 OD010425/OD/NIH HHS/United States ; R00 HL163805/HL/NHLBI NIH HHS/United States ; R50 CA274319/CA/NCI NIH HHS/United States ; R35 GM118062/GM/NIGMS NIH HHS/United States ; U01 AI142756/AI/NIAID NIH HHS/United States ; R01 CA266556/CA/NCI NIH HHS/United States ; P01 HL053749/HL/NHLBI NIH HHS/United States ; R01 HL136135/HL/NHLBI NIH HHS/United States ; RM1 HG009490/HG/NHGRI NIH HHS/United States ; R01 HL151765/HL/NHLBI NIH HHS/United States ; U42 OD011123/OD/NIH HHS/United States ; },
mesh = {Humans ; Animals ; *Gene Editing/methods ; *Sialic Acid Binding Ig-like Lectin 3/genetics/metabolism/immunology ; *Genetic Therapy/methods ; Hematopoietic Stem Cells/metabolism/drug effects ; Gemtuzumab/pharmacology ; *Immunotherapy/methods ; Polymorphism, Single Nucleotide ; CRISPR-Cas Systems ; Mice ; },
abstract = {The selection of genetically engineered immune or hematopoietic cells in vivo after gene editing remains a clinical problem and requires a method to spare on-target toxicity to normal cells. Here, we develop a base editing approach exploiting a naturally occurring CD33 single nucleotide polymorphism leading to removal of full-length CD33 surface expression on edited cells. CD33 editing in human and nonhuman primate hematopoietic stem and progenitor cells protects myeloid progeny from CD33-targeted therapeutics without affecting normal hematopoiesis in vivo, thus demonstrating potential for improved immunotherapies with reduced off-leukemia toxicity. For broader application to gene therapies, we demonstrate highly efficient (>70%) multiplexed adenine base editing of the CD33 and gamma globin genes, resulting in long-term persistence of dual gene-edited cells with HbF reactivation in nonhuman primates. Using the CD33 antibody-drug conjugate Gemtuzumab Ozogamicin, we show resistance of engrafted, multiplex edited human cells in vivo, and a 2-fold enrichment for edited cells in vitro. Together, our results highlight the potential of adenine base editors for improved immune and gene therapies.},
}
@article {pmid40425072,
year = {2025},
author = {Nair, A and Rao, AS and Surabhi, MA and Gnanika, M and More, SS},
title = {Unravelling fungal pathogenesis: Advances in CRISPR-Cas9 for understanding virulence and adaptation.},
journal = {Fungal genetics and biology : FG &amp; B},
volume = {179},
number = {},
pages = {104006},
doi = {10.1016/j.fgb.2025.104006},
pmid = {40425072},
issn = {1096-0937},
mesh = {*CRISPR-Cas Systems ; *Fungi/pathogenicity/genetics ; Virulence/genetics ; Gene Editing ; Genome, Fungal ; *Adaptation, Physiological/genetics ; Humans ; Mycoses/microbiology ; Virulence Factors/genetics ; },
abstract = {Fungi, with their billion-year evolutionary history, have adapted to diverse ecological niches, including pathogenic roles that threaten global health, agriculture, and ecosystems. Fungal pathogenicity is shaped by the dynamic evolution of genetic traits that enable fungi to infect hosts, evade immune defenses, and develop resistance to antifungal treatments. Despite their significant clinical and ecological impact, the evolutionary processes underlying fungal virulence and adaptation remain incompletely understood. This review emphasizes the transformative role of CRISPR-Cas9 genome editing in revealing these mechanisms. By allowing precise manipulation of fungal genomes, CRISPR technologies have provided key insights into virulence factors, stress response mechanisms, immune evasion, and antifungal resistance pathways. These advances demonstrate how fungi adapt to selective pressures, repurpose conserved genetic pathways, and exploit genomic plasticity to thrive in host environments. This review explores the intersection of CRISPR technology and fungal biology, shedding light on its implications for understanding fungal pathogenesis and the potential to develop innovative therapeutic strategies against fungal infections. The integration of CRISPR applications into mycology holds promise for furthering our understanding of fungal evolutionary trajectories and enhancing the development of novel therapeutic approaches.},
}
@article {pmid40425009,
year = {2025},
author = {Shang, K and Huang, D and Liu, J and Yu, Z and Bian, W and Chen, J and Zhao, Y and Liu, L and Jiang, J and Wang, Y and Duan, Y and Ge, J and Zhang, S and Zhou, C and Han, Y and Hu, Y and Zheng, W and Sun, J and Huang, H and Pei, S and Qian, P and Sun, J},
title = {CD97-directed CAR-T cells with enhanced persistence eradicate acute myeloid leukemia in diverse xenograft models.},
journal = {Cell reports. Medicine},
volume = {6},
number = {6},
pages = {102148},
doi = {10.1016/j.xcrm.2025.102148},
pmid = {40425009},
issn = {2666-3791},
mesh = {*Leukemia, Myeloid, Acute/therapy/immunology/pathology ; Humans ; Animals ; Xenograft Model Antitumor Assays ; *Receptors, Chimeric Antigen/metabolism/immunology ; Mice ; *Immunotherapy, Adoptive/methods ; Cell Line, Tumor ; *Antigens, CD/metabolism/immunology/genetics ; *T-Lymphocytes/immunology ; Mice, Inbred NOD ; CRISPR-Cas Systems ; Hematopoietic Stem Cells ; Mice, SCID ; },
abstract = {Chimeric antigen receptor (CAR)-T therapy on acute myeloid leukemia (AML) is hindered by the absence of a suitable tumor-specific antigen. Here, we propose CD97 as a potential target for CAR-T therapy against AML based on its broader and higher expression on AML cells compared to normal hematopoietic stem and progenitor cells (HSPCs). To resolve the fratricide problem caused by CD97 expression on T cells, we knock out CD97 in CAR-T cells using CRISPR-Cas9. Our CD97[KO] CAR-T cells eliminate both AML cell lines and primary AML cells effectively while showing tolerable toxicity to HSPCs. Furthermore, we mutate the CD3ζ domain of the CAR and find that the optimized CD97 CAR-T cells exhibit persistent anti-tumor activity both in vitro and in multiple xenograft models. Mechanistically, transcriptional profiles reveal that the optimized CAR-T cells delay differentiation and resist exhaustion. Collectively, our study supports CD97 as a promising target for CAR-T therapy against AML.},
}
@article {pmid40424180,
year = {2025},
author = {Krishnaprasad, VH and Nayak, V and Kumar, S},
title = {World Health Organisation's Bacterial Pathogen Priority List (BPPL) 2017 and BPPL 2024 to combat global antimicrobial resistance crisis: 'challenges and opportunities'.},
journal = {The Journal of antimicrobial chemotherapy},
volume = {},
number = {},
pages = {},
doi = {10.1093/jac/dkaf167},
pmid = {40424180},
issn = {1460-2091},
abstract = {Antibiotic resistance, in a broader perspective, antimicrobial resistance (AMR) presents a formidable global health challenge, threatening the effectiveness of antibiotics and other antimicrobial agents. As a result, AMR has become more challenging or even impossible to treat, leading to increased morbidity and mortality. The World Health Organisation (WHO) has been at the forefront of international efforts to combat AMR by sensitizing the world about the pressing need to tackle AMR to save the future of the human race. This article analyses WHO's efforts to combat AMR, including creating the Bacterial Pathogen Priority List (BPPL), developing a global action plan to address AMR and promoting surveillance and stewardship programmes. This article also examines the progress achieved by BPPL 2017 and the challenges ahead for BPPL 2024. Additionally, this article explores various efforts to combat AMR through two major approaches, like 'research and development' and 'the policy and regulation-based' approach. This article underscores various emerging strategies to tackle AMR, for example, biofilm disruption, nanotechnology, antibiotic resistance breakers, antibody-antibiotic conjugates, rapid detection tools and alternative therapies like phage therapy, antimicrobial peptides, CRISPR-Cas system, probiotics and microbiota modulations. This article also highlights the importance of coordinated actions and sustained commitment to safeguarding public health and ensuring the continued effectiveness of antimicrobial therapies.},
}
@article {pmid40424009,
year = {2025},
author = {Wei, L and Wang, Z and She, Y and Fu, H},
title = {CRISPR/Cas Multiplexed Biosensing: Advances, Challenges, and Perspectives.},
journal = {Analytical chemistry},
volume = {97},
number = {23},
pages = {11943-11958},
doi = {10.1021/acs.analchem.4c04428},
pmid = {40424009},
issn = {1520-6882},
mesh = {*Biosensing Techniques/methods ; *CRISPR-Cas Systems/genetics ; Humans ; },
abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) protein systems are renowned for their high sensitivity and specificity, enabling them as a powerful diagnostic toolbox. Multiplexed detection of panels of targets, as opposed to single targets, is imperative for reliable and conclusive disease diagnostics. However, multiplex application of the CRISPR/Cas system has long been hindered by indistinguishable signals from specific targets due to nonspecific chaotic trans-cleavage. To make a breakthrough, substantial efforts have been devoted to CRISPR/Cas-powered multiplexed biosensing strategies, which consequently experienced rapid development over the past five years. This review systematically summarizes recent advances in CRISPR/Cas multiplexed detection encompassing Cas9, Cas12, and Cas13. Key focus issues include multiplex biosensing strategies and their respective advantages and limitations, sensing mechanisms, and detection performance of novel validated examples. Finally, the status and challenges of CRISPR/Cas multiplexed biosensing are critically discussed, and future outlooks are proposed for their potential practical application. This Perspective aims to inspire significant research and promote the development of the next generation of CRISPR/Cas multiplexed biosensing.},
}
@article {pmid40422684,
year = {2025},
author = {Ma, B and Li, Y and Wang, T and Li, D and Jia, S},
title = {Advances in CRISPR/Cas9-Based Gene Editing in Filamentous Fungi.},
journal = {Journal of fungi (Basel, Switzerland)},
volume = {11},
number = {5},
pages = {},
pmid = {40422684},
issn = {2309-608X},
support = {32160143//National Science Foundation of China/ ; },
abstract = {As an important class of microorganisms, filamentous fungi have crucial roles in protein secretion, secondary metabolite production and environmental pollution control. However, characteristics such as apical growth, heterokaryon, low homologous recombination (HR) efficiency and the scarcity of genetic markers mean that the application of traditional gene editing technology in filamentous fungi faces great challenges. The introduction of the RNA-mediated CRISPR/Cas (clustered regularly interspaced short palindromic repeat/CRlSPR-associated protein) system in filamentous fungi in recent years has revolutionized gene editing in filamentous fungi. In addition, the continuously expressed CRISPR system has significantly improved the editing efficiency, while the optimized sgRNA design and reduced cas9 concentration have effectively reduced the off-target effect, further enhancing the safety and reliability of the technology. In this review, we systematically analyze the molecular mechanism and regulatory factors of CRISPR/Cas9, focus on the optimization of its expression system and the improvement of the transformation efficiency in filamentous fungi, and reveal the core regulatory roles of HR and non-homologous end-joining (NHEJ) pathways in gene editing. Based on the analysis of various filamentous fungi applications, this review reveals the outstanding advantages of CRISPR/Cas9 in the enhancement of protein secretion, addresses the reconstruction of secondary metabolic pathways and pollutant degradation in the past decade, and provides a theoretical basis and practical guidance for the optimization of the technology and engineering applications.},
}
@article {pmid40422214,
year = {2025},
author = {Gibson, J and Dhungana, A and Pokhrel, M and Arthur, B and Suresh, P and Adebayo, O and Cottle, RN},
title = {Validation of Clinical-Grade Electroporation Systems for CRISPR-Cas9-Mediated Gene Therapy in Primary Hepatocytes for the Correction of Inherited Metabolic Liver Disease.},
journal = {Cells},
volume = {14},
number = {10},
pages = {},
pmid = {40422214},
issn = {2073-4409},
support = {P20 GM146584/GM/NIGMS NIH HHS/United States ; R01 HL168093/HL/NHLBI NIH HHS/United States ; 2021000920//American Association for the Study of Liver Diseases Foundation/ ; 1R01HL168093-01A1/GF/NIH HHS/United States ; },
mesh = {*Hepatocytes/transplantation/metabolism ; *Electroporation/methods ; Animals ; *CRISPR-Cas Systems/genetics ; *Genetic Therapy/methods ; Mice ; Gene Editing/methods ; *Tyrosinemias/therapy/genetics ; Humans ; *Liver Diseases/therapy/genetics ; Disease Models, Animal ; Male ; Hydrolases ; },
abstract = {Hepatocyte transplantation (HTx) combined with ex vivo gene therapy has garnered significant interest due to its potential for treating many inherited metabolic liver diseases. The biggest obstacle for HTx is achieving sufficient engraftment levels to rescue diseased phenotypes, which becomes more challenging when combined with ex vivo gene editing techniques. However, recent technological advancements have improved electroporation delivery efficiency, cell viability, and scalability for cell therapy. We recently demonstrated the impacts of electroporation for cell-based gene therapy in a mouse model of hereditary tyrosinemia type 1 (HT1). Here, we explore the use of the clinical-grade electroporator, the MaxCyte ExPERT GTx, utilized in the first FDA-approved CRISPR therapy, Casgevy, and evaluate its potential in primary hepatocytes in terms of delivery efficiency and cell viability. We assessed the gene editing efficiency and post-transplantation engraftment of hepatocytes from mTmG mice electroporated with CRISPR-Cas9-ribonucleoproteins (RNPs) targeting 4-hydroxyphenylpyruvate dioxygenase (Hpd) in a fumarylacetoacetate hydrolase (Fah)-deficient mouse model of HT1. After surgery, Fah[-/-] graft recipients were cycled off and on nitisinone to achieve independence from drug-induced Hpd inhibition, an indicator of HT1 disease correction. Transplanted hepatocytes subjected to electroporation using the GTx system had a cell viability of 89.9% and 100% on-target gene editing efficiency. Recipients transplanted with GTx-electroporated cells showed a smaller weight reduction than controls transplanted with untransfected cells (7.9% and 13.8%, respectively). Further, there were no mortalities in the GTx-recipient mice, whereas there was 25% mortality in the control recipients. Mean donor cell engraftment was significantly higher in GTx-recipient mice compared to untransfected control recipients (97.9% and 81.6%, respectively). Our results indicate that the GTx system does not negatively impact hepatocyte functionality and engraftment potential, thereby demonstrating the promise of GTx electroporation in hepatocytes as a viable cell therapy for treating genetic diseases that affect the liver.},
}
@article {pmid40422195,
year = {2025},
author = {Orlova, NA and Sinegubova, MV and Kolesov, DE and Khodak, YA and Tatarskiy, VV and Vorobiev, II},
title = {Genomic and Phenotypic Characterization of CHO 4BGD Cells with Quad Knockout and Overexpression of Two Housekeeping Genes That Allow for Metabolic Selection and Extended Fed-Batch Culturing.},
journal = {Cells},
volume = {14},
number = {10},
pages = {},
pmid = {40422195},
issn = {2073-4409},
support = {25-24-00140//Russian Science Foundation/ ; },
mesh = {CHO Cells ; Animals ; Cricetulus ; *Batch Cell Culture Techniques/methods ; CRISPR-Cas Systems/genetics ; *Gene Knockout Techniques ; Gene Editing ; Apoptosis/genetics ; *Genes, Essential/genetics ; Phenotype ; *Genomics ; },
abstract = {Re-engineering of CHO cells using genome editing and the overexpression of multiple helper genes is the central track for obtaining better cell lines for the production of biopharmaceuticals. Using two subsequent rounds of genome editing of the CHO S cells, we have developed the cell line CHO 4BGD with four knockouts of two pro-apoptotic genes bak1 and bax, and two common selection markers genes-glul (GS) and dhfr, and additional copies of genes bcl-2 and beclin-1 used for enhancement of macroautophagy. The NGS sequencing of 4BGD cells revealed that all eight targeted alleles were successfully disrupted. Two edited loci out of eight contained large inserts of non-relevant DNA. Further data analysis shows that cells have no off-target DNA editing events, and all known CHO genes are preserved. The cells obtained are completely resistant to the induction of apoptosis, and they are suitable for the generation of stably transfected cell lines with the dhfr selection marker. They also properly undergo the target gene amplification. The 4BGD-derived clonal cell line that secretes the monoclonal antibody retains the ability for prolonged fed-batch culturing. The method of obtaining multiply edited CHO cells using the multiplex CRISPR/Cas9 editing and simultaneous stable transfection of plasmids, coding for the housekeeping genes, is suitable for the rapid generation of massively edited CHO cells.},
}
@article {pmid40422075,
year = {2025},
author = {Guo, L and Duan, X and Li, J and Hao, Z and Bi, Y and Chen, Y and Du, X and Wu, S},
title = {YIPF5 is an essential host factor for porcine epidemic diarrhea virus double-membrane vesicle formation.},
journal = {Journal of virology},
volume = {99},
number = {6},
pages = {e0032025},
pmid = {40422075},
issn = {1098-5514},
support = {2023YFC3404300, 2023YFF0724700, 2023YFF1000200, 2021YFA0805900//National Key Research and Development Program of China/ ; 323QN261//the Natural Science Foundation for Youths of Hainan province, China/ ; 32425053//the National Natural Science Foundation of China (NSFC) Distinguished Young Scholar/ ; },
mesh = {Animals ; *Porcine epidemic diarrhea virus/physiology ; Swine ; Virus Replication ; *Coronavirus Infections/virology/metabolism/veterinary ; Cell Line ; *Swine Diseases/virology/metabolism ; CRISPR-Cas Systems ; *Host-Pathogen Interactions ; Viral Nonstructural Proteins/metabolism/genetics ; },
abstract = {UNLABELLED: Porcine epidemic diarrhea virus (PEDV) is a major coronavirus in swine, causing substantial economic losses in the industry. To deepen our understanding of the PEDV-host cell interactions, we performed whole-genome CRISPR/Cas9 screens on porcine IPEC-J2 and IPI-2I cell lines to identify key host factors essential for PEDV infection. Our study identified the Yip family 5 (YIPF5) protein as a critical host factor, where its knockout suppressed PEDV infection by specifically affecting the virus replication stage. YIPF5 interacts with viral non-structural protein (nsp) 3, 4, and 6, facilitating the formation of double-membrane vesicles (DMVs), essential for replication organelle biogenesis. The knockout of YIPF5 interferes with the interaction between nsp3 and nsp4, consequently impacting the formation of DMVs mediated by these proteins. These findings establish YIPF5 as a key host factor involved in DMV formation during PEDV infection, highlighting its potential as a therapeutic target.<br><br>IMPORTANCE: Coronaviruses pose serious health threats to both humans and animals. Identifying host genes critical for porcine epidemic diarrhea virus (PEDV) infection can uncover new therapeutic targets and enhance our understanding of coronavirus pathogenesis. In this study, we conducted genome-scale CRISPR/Cas9 screens in two porcine cell lines (IPEC-J2 and IPI-2I) and identified YIPF5 as an essential host factor for PEDV replication. Our results demonstrate that YIPF5 plays a pivotal role in the formation of PEDV-induced double-membrane vesicles (DMVs), which are crucial for viral replication. These findings shed new light on the molecular mechanisms of PEDV and suggest YIPF5 as a therapeutic target.},
}
@article {pmid40419487,
year = {2025},
author = {Byrne, SM and Burleigh, SM and Fragoza, R and Jiang, Y and Savva, Y and Pabon, R and Kania, E and Rainaldi, J and Portell, A and Mali, P and Briggs, AW},
title = {An engineered U7 small nuclear RNA scaffold greatly increases ADAR-mediated programmable RNA base editing.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {4860},
pmid = {40419487},
issn = {2041-1723},
mesh = {*Adenosine Deaminase/metabolism/genetics ; Animals ; *RNA Editing/genetics ; Humans ; Mice ; *RNA, Small Nuclear/genetics/metabolism/chemistry ; *Gene Editing/methods ; *RNA-Binding Proteins/metabolism/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; HEK293 Cells ; },
abstract = {Custom RNA base editing exploiting the human Adenosine Deaminase Acting on RNA (ADAR) enzyme may enable therapeutic gene editing without DNA damage or use of foreign proteins. ADAR's adenosine-to-inosine (effectively A-to-G) deamination activity can be targeted to transcripts using an antisense guide RNA (gRNA), but efficacy is challenged by limits of in vivo delivery. Embedding gRNAs into a U7 small nuclear RNA (snRNA) framework greatly enhances RNA editing with endogenous ADAR, and a 750-plex single-cell mutagenesis screen further improved the framework. An optimized scaffold with a stronger synthetic U7 promoter enables 76% RNA editing in vitro from a single DNA construct per cell, and 75% editing in a Hurler syndrome mouse brain after one systemic AAV injection, surpassing circular gRNA approaches. The technology also improves published DMD exon-skipping designs 25-fold in differentiated myoblasts. Our engineered U7 framework represents a universal scaffold for ADAR-based RNA editing and other antisense RNA therapies.},
}
@article {pmid40419416,
year = {2025},
author = {Wachholz Junior, D and Pontes, RG and Hryniewicz, BM and Kubota, LT},
title = {Exploring a CRISPR/Cas12a-powered impedimetric biosensor for amplification-free detection of a pathogenic bacterial DNA.},
journal = {Biosensors &amp; bioelectronics},
volume = {285},
number = {},
pages = {117607},
doi = {10.1016/j.bios.2025.117607},
pmid = {40419416},
issn = {1873-4235},
mesh = {*Biosensing Techniques/methods ; *Staphylococcus aureus/genetics/isolation &amp; purification/pathogenicity ; *CRISPR-Cas Systems/genetics ; *DNA, Bacterial/genetics/isolation &amp; purification ; Humans ; Limit of Detection ; *Staphylococcal Infections/microbiology/diagnosis ; },
abstract = {Timely and precise detection of bacterial infections is essential for improving patient outcomes and reducing healthcare costs, especially for sepsis, where delayed diagnosis increases mortality. Traditional culture- and PCR-based methods are time consuming and require complex sample processing, making them unsuitable for rapid diagnostics in resource-limited settings. CRISPR/Cas-based methods, particularly when combined with electrochemical sensing, offer a promising alternative for rapid point-of-care (POC) diagnostics of bacterial infections due to their simplicity and specificity. This study proposes a label-free impedimetric biosensor using the CRISPR/Cas12a system for rapid and amplification-free detection of Staphylococcus aureus DNA, a primary pathogen responsible for sepsis. By leveraging CRISPR/Cas12a's target-activated collateral cleavage on non-specific DNA reporters we investigated the impact of using a protospacer adjacent motif (PAM) sequence on detection sensitivity and specificity. Our biosensor demonstrated ultra-sensitive detection, with limit of detection as low as 20 aM for dsDNA targets in buffer and without any pre-amplification steps. The study also confirmed CRISPR specificity's dependence on the PAM sequence, showing that mismatches on targeting sequences reduces cleavage efficiency, with a drastic reduction in trans-cleavage activity for single mismatch in PAM-containing sequences. Additionally, we examined how the DNA reporter affects performance, noting reduced cleavage efficiency when a ssDNA target was paired with a dsDNA reporter. Furthermore, validation experiments using human serum samples confirmed the biosensor's accuracy for bacterial DNA detection in clinical settings. This work advances CRISPR-powered electrochemical biosensors, providing a detailed discussion on developing a highly sensitive, fast and amplification-free tool for early detection of sepsis-causing bacteria.},
}
@article {pmid40418657,
year = {2025},
author = {Johnson, MJ and DeFeo, AP and Slipek, NJ and Folsom, TD and Henley, T and Choudhry, MS and Webber, BR and Moriarity, BS},
title = {Non-Viral Engineering of Primary Human T Cells via Homology-Mediated End-Joining Targeted Integration of Large DNA Templates.},
journal = {Journal of visualized experiments : JoVE},
volume = {},
number = {219},
pages = {},
doi = {10.3791/68150},
pmid = {40418657},
issn = {1940-087X},
mesh = {Humans ; *T-Lymphocytes/cytology/immunology ; CRISPR-Cas Systems ; *DNA/genetics ; Animals ; *Cell Engineering/methods ; Mice ; *DNA End-Joining Repair ; *Genetic Engineering/methods ; },
abstract = {Many current adoptive cellular therapies rely on lenti- or retroviral vectors to engineer T cells for the expression of a chimeric antigen receptor (CAR) or exogenous T cell receptor (TCR) to target a specific tumor-associated antigen. Reliance on viral vectors for the production of therapeutic T cells significantly increases the timeline, cost, and complexity of manufacturing while limiting the translation of new therapies, particularly in the academic setting. A process is presented for efficient non-viral engineering of T cells using CRISPR/Cas9 and homology-mediated end joining to achieve targeted integration of large, multicistronic DNA cargo. This approach has achieved integration frequencies comparable to those of viral vectors while yielding highly functional T cells capable of potent anti-tumor efficacy both in vitro and in vivo. Notably, this method is rapidly adaptable to current good manufacturing practices (cGMP) and clinical scale-up, providing a near-term option for the manufacturing of therapeutic T cells for use in clinical trials.},
}
@article {pmid40417002,
year = {2025},
author = {Gallala, M},
title = {Application of CRISPR/Cas gene editing for infectious disease control in poultry.},
journal = {Open life sciences},
volume = {20},
number = {1},
pages = {20251095},
pmid = {40417002},
issn = {2391-5412},
abstract = {The poultry industry faces multifaceted challenges, including escalating demand for poultry products, climate change impacting feed availability, emergence of novel avian pathogens, and antimicrobial resistance. Traditional disease control measures are costly and not always effective, prompting the need for complementary methods. Gene editing (GE, also called genome editing) technologies, particularly CRISPR/Cas9, offer promising solutions. This article summarizes recent advancements in utilizing CRISPR/Cas GE to enhance infectious disease control in poultry. It begins with an overview of modern GE techniques, highlighting CRISPR/Cas9's advantages over other methods. The potential applications of CRISPR/Cas in poultry infectious disease prevention and control are explored, including the engineering of innovative vaccines, the generation of disease-resilient birds, and in vivo pathogen targeting. Additionally, insights are provided regarding regulatory frameworks and future perspectives in this rapidly evolving field.},
}
@article {pmid40415627,
year = {2025},
author = {Li, H and Zhu, Q and Zhu, J and Min, J},
title = {[Research progress in the developmental process of non-viral CAR-T technology].},
journal = {Xi bao yu fen zi mian yi xue za zhi = Chinese journal of cellular and molecular immunology},
volume = {41},
number = {5},
pages = {461-467},
pmid = {40415627},
issn = {1007-8738},
mesh = {Humans ; *Immunotherapy, Adoptive/methods ; *Receptors, Chimeric Antigen/genetics/immunology ; Animals ; Gene Transfer Techniques ; Genetic Vectors/genetics ; Gene Editing ; CRISPR-Cas Systems/genetics ; DNA Transposable Elements/genetics ; *T-Lymphocytes/immunology ; Neoplasms/therapy/immunology ; },
abstract = {Chimeric antigen receptor T (CAR-T) lymphocytes are at the forefront of adoptive immunotherapy research, and this technology has significantly advanced the prospects of tumor immunotherapy. CAR-T therapy has demonstrated remarkable efficacy in haematological tumours of lymphoid origin and provided therapeutic possibility for solid tumours. Currently, CAR-T cell preparation predominantly involves transfection of T cells with viral vectors. However, the production of viral vectors is time-consuming, expensive, and the vectors have low loading capacity, along with insertion instability. Consequently, there is a pressing need to develop more convenient and precise non-viral gene delivery methods. This paper reviews the most promising non-viral gene delivery technologies, including CRISPR/Cas9 gene editing, transposon systems such as Sleeping Beauty (SB) and PiggyBac (PB), and mRNA, and anticipates the future development of non-viral vector-based CAR-T therapies.},
}
@article {pmid40414878,
year = {2025},
author = {Lobel, JH and Ingolia, NT},
title = {Precise measurement of molecular phenotypes with barcode-based CRISPRi systems.},
journal = {Genome biology},
volume = {26},
number = {1},
pages = {142},
pmid = {40414878},
issn = {1474-760X},
support = {F32 GM148044/GM/NIGMS NIH HHS/United States ; R01 GM135233/GM/NIGMS NIH HHS/United States ; R21 HG012991/HG/NHGRI NIH HHS/United States ; },
mesh = {*CRISPR-Cas Systems ; Phenotype ; Humans ; Promoter Regions, Genetic ; },
abstract = {Genome-wide CRISPR-Cas9 screens have untangled regulatory networks driving diverse biological processes. Their success relies on interrogating specific molecular phenotypes and distinguishing key regulators from background effects. Here, we realize these goals by optimizing CRISPR interference with barcoded expression reporter sequencing (CiBER-seq) to dramatically improve the sensitivity and scope of genome-wide screens. We systematically address technical factors that distort phenotypic measurements by normalizing expression reporters against closely matched promoters. We use our improved CiBER-seq to accurately capture known components of well-studied RNA and protein quality control systems. These results demonstrate the precision and versatility of CiBER-seq for dissecting cellular pathways.},
}
@article {pmid40414079,
year = {2025},
author = {Lyu, H and Haag, C and Schwarz, N and Löffler, H and Mau-Holzmann, UA and Lerche, H and Rosa, F},
title = {Generation of an induced pluripotent stem cell (iPSC) line carrying a KCNA2 homozygous (p.Arg294His, R294H) mutation related to hereditary spastic paraplegia.},
journal = {Stem cell research},
volume = {86},
number = {},
pages = {103737},
doi = {10.1016/j.scr.2025.103737},
pmid = {40414079},
issn = {1876-7753},
mesh = {*Induced Pluripotent Stem Cells/metabolism/cytology ; Humans ; *Spastic Paraplegia, Hereditary/genetics/pathology ; Homozygote ; Cell Line ; *Mutation ; CRISPR-Cas Systems ; Kv1.2 Potassium Channel ; },
abstract = {The KCNA2 gene encodes the voltage-gated potassium channel Kv1.2, which is essential for repolarization of action potential. The R294H variant in KCNA2 represents the only potassium channel gene variant linked to hereditary spastic paraplegia (HSP) to date. However, this variant has been observed exclusively in heterozygous individuals. Here, we generated a homozygous KCNA2 R294H (c.881G > A) induced pluripotent stem cell (iPSC) line from a healthy individual iPSC line. The variant was introduced into both alleles using the CRISPR/Cas9 system. The resulting iPSC line has a normal karyotype, expresses key pluripotency markers, and is able to differentiate into all three germ layers.},
}
@article {pmid40414010,
year = {2025},
author = {Cao, X and Gao, Z and Yin, P and Wang, H and Yang, L},
title = {Crystal structure and inhibition mechanism of AcrIIA11.},
journal = {Biochemical and biophysical research communications},
volume = {772},
number = {},
pages = {152073},
doi = {10.1016/j.bbrc.2025.152073},
pmid = {40414010},
issn = {1090-2104},
mesh = {Streptococcus pyogenes/enzymology/genetics ; Crystallography, X-Ray ; Staphylococcus aureus/enzymology ; *Bacterial Proteins/chemistry/metabolism/antagonists &amp; inhibitors ; *CRISPR-Cas Systems ; *CRISPR-Associated Protein 9/antagonists &amp; inhibitors/chemistry/metabolism ; Models, Molecular ; Protein Conformation ; Protein Binding ; },
abstract = {Anti-CRISPR (Acr) proteins are naturally evolved inhibitors that precisely target and suppress CRISPR-Cas systems, representing a sophisticated molecular arms race between bacteriophages and their bacterial hosts. While Class 1 systems dominate among sequenced prokaryotic genomes, Class 2 systems remain primary sources of editing tools. Here, we report the structural and mechanistic characterization of AcrIIA11, an anti-CRISPR protein that simultaneously inhibits Streptococcus pyogenes (SpyCas9) and Staphylococcus aureus Cas9 (SauCas9). The 3.2 Å crystal structure reveals a compact α/β fold with distinct electropositive clefts implicated in DNA binding. While DALI analysis identified structural homology to transcriptional regulators and the RecA inhibitor PsiB (RMSD 3.3 Å), functional studies established that AcrIIA11 forms stable ternary complexes with both Cas9 orthologs and sgRNA. Biochemical assays demonstrated stronger inhibition of SauCas9 compared to SpyCas9, with EMSA revealing a critical dichotomy: AcrIIA11 maintains SauCas9-sgRNA binding to specific target DNA while completely blocking cleavage activity. Computational docking localizes AcrIIA11 at the HNH-RuvC interface without obstructing DNA-binding channels in SauCas9, suggesting allosteric inhibition through HNH domain displacement. This work establishes AcrIIA11 as a dual-purpose Cas9 inhibitor that preserves target recognition while inactivating nuclease function-a mechanism with potential applications in precision CRISPR control.},
}
@article {pmid40413996,
year = {2025},
author = {Duan, M and Li, G and Shen, J and Dai, R and Li, X and Liu, Z and Jia, F},
title = {A CRISPR/Cas12a biosensor for portable and accessible detection of Salmonella typhimurium via multi-indicator pH millidisc colorimetry and smartphone imaging platform.},
journal = {Biosensors &amp; bioelectronics},
volume = {286},
number = {},
pages = {117611},
doi = {10.1016/j.bios.2025.117611},
pmid = {40413996},
issn = {1873-4235},
mesh = {*Biosensing Techniques/instrumentation ; *Salmonella typhimurium/isolation &amp; purification/genetics ; Smartphone ; *Colorimetry/instrumentation ; CRISPR-Cas Systems/genetics ; Animals ; Chickens/microbiology ; Hydrogen-Ion Concentration ; Food Microbiology ; Limit of Detection ; },
abstract = {Conventional colorimetric CRISPR/Cas methods rely on a single chromogenic substrate and bulky and specialized signal detection instrument, which hinder their practical application. Herein, a portable and accessible CRISPR/Cas12a biosensor was for the first time reported to sensitively quantify Salmonella enterica serovar typhimurium (S. typhimurium), utilizing a multi-indicator pH millimeter disc (millidisc) for signal visualization, combined with a smartphone-based imaging platform for signal readout. The pH millidisc, composed of multiple indicators, possessed sensitive pH responsiveness and exhibited diverse color changes. The self-developed RGB mini-program, named DeepFood, was designed for portable smartphone use, featuring user-friendly operation and trend visualization for preliminary result analysis. Based on the RGB signal variation pattern with S. typhimurium, a distinct Senh signal type was designed, enhancing the signal-to-noise ratio from 3.38 to 7.11. Compared to the R signal type, the Senh signal type improved detection sensitivity by 36.23-fold (7.26 CFU/mL) in 0.01 M PBS buffer and 15.53-fold (1.41 × 10[2] CFU/mL) in chicken. The proposed biosensor offers significant improvements in detection sensitivity and practical applicability, with potential in food safety and environmental protection.},
}
@article {pmid40413994,
year = {2025},
author = {Liu, Y and Liu, Y and Wu, S and Cao, R and Pan, Y and Zhou, F},
title = {Engineered Cas12a-based one-tube detection of DNMT3A R882 H/C mutation in acute myeloid leukemia.},
journal = {Biosensors &amp; bioelectronics},
volume = {286},
number = {},
pages = {117609},
doi = {10.1016/j.bios.2025.117609},
pmid = {40413994},
issn = {1873-4235},
mesh = {DNA Methyltransferase 3A ; *Leukemia, Myeloid, Acute/genetics/diagnosis ; Humans ; *DNA (Cytosine-5-)-Methyltransferases/genetics ; *CRISPR-Cas Systems/genetics ; Mutation ; *Biosensing Techniques/methods ; *Endodeoxyribonucleases/genetics/chemistry ; Bacterial Proteins ; CRISPR-Associated Proteins ; },
abstract = {Advances in sequencing technologies have identified numerous genetic alterations associated with acute myeloid leukemia (AML), many of which play critical roles in diagnosis, classification, and prognosis. Among these, mutations in the DNA methyltransferase 3 alpha (DNMT3A) gene are particularly prevalent, with the R882H and R882C variants being the most common. Accurate and sensitive detection of DNMT3A mutations is crucial for prognosis, treatment guidance, and early intervention in AML. However, existing detection methods often fail to achieve an optimal balance among sensitivity, turnaround time, and operational simplicity. To address this limitation, we aimed to develop a rapid and highly sensitive method for detecting DNMT3A mutations. The CRISPR/Cas12a system shows promise for genetic detection due to its high sensitivity and single-base specificity. Here, we established a Cas12a-based one-tube assay for the detection of DNMT3A R882 H/C mutations. We utilized the mismatch tolerance of enAsU-R Cas12a to design crRNA for DNMT3A R882 H/C mutation and integrated CRISPR/Cas12a system with ERA. The entire detection process can be completed within 1 h at 37 °C. The optimized detection system demonstrated a sensitivity of 0.1 % when analyzing genomic DNA. To validate its clinical applicability, we tested samples from 49 AML patients and successfully identified all DNMT3A R882H/C-positive cases, including one with a mutation rate as low as 0.24 %. These results highlight the potential of our Cas12a-based one-tube detection system as a rapid, sensitive, and cost-effective method for detecting DNMT3A R882 H/C mutation. This approach could serve as a valuable tool for both diagnostic and therapeutic monitoring.},
}
@article {pmid40413357,
year = {2025},
author = {Jiang, B and An, Z and Niu, L and Qin, D},
title = {Precise genome editing process and its applications in plants driven by AI.},
journal = {Functional &amp; integrative genomics},
volume = {25},
number = {1},
pages = {109},
pmid = {40413357},
issn = {1438-7948},
support = {BLX202316//Fundamental Research Funds for the Central Universities/ ; },
mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems ; *Genome, Plant ; *Plants/genetics ; *Artificial Intelligence ; Plant Breeding ; },
abstract = {Genome editing technologies have emerged as the keystone of biotechnological research, enabling precise gene modification. The field has evolved rapidly through revolutionary advancements, transitioning from early explorations to the breakthrough of the CRISPR-Cas system. The emergence of the CRISPR-Cas system represents a huge leap in genome editing, prompting the development of advanced tools such as base and prime editors, thereby enhancing precise genomic engineering capabilities. The rapid integration of AI across disciplines is now driving another transformative phase in genome editing, streamlining workflows and enhancing precision. The application prospects of genome editing technology are extensive, particularly in plant breeding, where it has already presented unparalleled opportunities for improving plant traits. Here, we review early genome editing technologies, including meganucleases, ZFNs, TALENs, and CRISPR-Cas systems. We also provide a detailed introduction to next-generation editing tools-such as base editors and prime editors-and their latest applications in plants. At the same time, we summarize and prospect the cutting-edge developments and future trends of genome editing technologies in combination with the rapidly rising AI technology, including optimizing editing systems, predicting the efficiency of editing sites and designing editing strategies. We are convinced that as these technologies progress and their utilization expands, they will provide pioneering solutions to global challenges, ushering in an era of health, prosperity, and sustainability.},
}
@article {pmid40412199,
year = {2025},
author = {Shen, Y and Tang, X and Wang, J and Dai, H and Cui, Y and Hu, Q and Wu, Y and Jia, F and Hao, G},
title = {Dual nuclease-amplified sensitive biosensor for enrofloxacin detection using a DNase I-assisted CRISPR/Cas12a (CRISPR-DNase I) system.},
journal = {Talanta},
volume = {295},
number = {},
pages = {128367},
doi = {10.1016/j.talanta.2025.128367},
pmid = {40412199},
issn = {1873-3573},
abstract = {Recent years have witnessed the flourishing of CRISPR/Cas-based biosensors in various fields. However, most of them were developed for nucleic acid detection because non-nucleic acid targets are unable to unleash the cleavage activity of the CRISPR/Cas system directly. To circumvent this problem, activator DNA and deoxyribonuclease I (DNase I) were introduced in this research to render the CRISPR/Cas12a system as a new powerful tool for the detection of enrofloxacin (ENR), a common veterinary drug. In this biosensor, target ENR competed with DNase I- and bovine serum albumin-ENR composite-modified gold nanoparticles (DNase I-AuNPs-BSA-ENR) for the binding sites on the surface of antibody-modified magnetic nanoparticles (immuno-MNPs). Then, the captured DNase I-AuNPs-BSA-ENR degraded the activator DNA in the solution, which inhibited the activation of the CRISPR/Cas12a system. Finally, the fluorescence released by the activated CRISPR/Cas12a system was measured for the quantitative detection of ENR. The ingenious use of activator DNA and DNase I helped transduce the target recognition event into the cleavage activity of the CRISPR/Cas12a system. Moreover, the dual enzymatic amplification from DNase I and the CRISPR/Cas12a system guaranteed the sensitivity of this method with a low detection limit of 0.04 ng/mL. The developed biosensor extended the application of the CRISPR/Cas12a system for the sensitive detection of non-nucleic acid targets, providing a powerful tool in various fields such as environmental monitoring, food safety and clinical diagnosis.},
}
@article {pmid40412122,
year = {2025},
author = {Wang, R and Yang, Y and Wang, Z and Ma, C and Wu, M and Du, Y and Zhang, X and Cao, M and Xu, H},
title = {Stimuli-responsive peptide nanocarriers for tumor-specific CRISPR/Cas9 delivery and precision genome editing.},
journal = {Journal of colloid and interface science},
volume = {697},
number = {},
pages = {137932},
doi = {10.1016/j.jcis.2025.137932},
pmid = {40412122},
issn = {1095-7103},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Gene Editing ; *Nanoparticles/chemistry ; *Peptides/chemistry ; HeLa Cells ; *Drug Carriers/chemistry ; Particle Size ; *Ribonucleoproteins/chemistry/genetics ; Cell Survival/drug effects ; *Neoplasms/genetics ; Surface Properties ; },
abstract = {CRISPR/Cas9 ribonucleoprotein (RNP) delivery remains a critical challenge due to its large size, instability, and off-target effects. Here, we report a stimuli-responsive cationic amphiphilic peptide, (CR3)3C, designed for cancer-targeted delivery of CRISPR/Cas9 RNP. The peptide integrates three functional domains: (1) a naphthyl-diphenylalanine (Nap-FF) motif enabling self-assembly into stable nanoparticles via aromatic interactions, (2) a matrix metalloproteinase-7 (MMP7)-cleavable linker (GPLGLA) for tumor microenvironment-specific activation, and (3) a redox-responsive cationic domain ((CRRR)3-C) for electrostatic RNP binding and glutathione (GSH)-triggered intracellular release. The (CR3)3C/RNP nanocomplexes (108.8 nm diameter, ζ = +10.89 mV) demonstrate exceptional stability and cellular uptake efficiency. Mechanistic studies reveal caveolae-mediated endocytosis and lipid raft-associated pathways, proton sponge effect-driven endosomal escape, and nuclear localization facilitated by Cas9's nuclear localization signal. In HeLa-EGFP cells, (CR3)3C/RNP shows 33.8 % gene editing efficiency at 100 nM RNP with >90 % cell viability. This work establishes a programmable, non-viral platform that synergizes enzymatic and redox responsiveness for tumor-targeted genome editing, addressing critical barriers in CRISPR therapeutics.},
}
@article {pmid40411834,
year = {2025},
author = {Guo, Y and Guo, W and Li, C and Xu, H and Zhang, X and Zou, X and Sun, Z},
title = {Fe3O4@Au Nanoparticle-Enabled Magnetic Separation Coupled with CRISPR/Cas12a for Ultrasensitive Detection of Foodborne Pathogens.},
journal = {Journal of agricultural and food chemistry},
volume = {73},
number = {22},
pages = {13949-13959},
doi = {10.1021/acs.jafc.5c04580},
pmid = {40411834},
issn = {1520-5118},
mesh = {*Biosensing Techniques/instrumentation/methods ; Gold/chemistry ; CRISPR-Cas Systems ; *Staphylococcus aureus/isolation &amp; purification/genetics ; Animals ; Milk/microbiology ; *Salmonella/isolation &amp; purification/genetics ; Food Contamination/analysis ; Metal Nanoparticles/chemistry ; Food Microbiology ; Electrochemical Techniques/instrumentation/methods ; Limit of Detection ; *Endodeoxyribonucleases/genetics ; Bacterial Proteins ; CRISPR-Associated Proteins ; },
abstract = {The rapid detection of foodborne pathogens, such as Staphylococcus aureus and Salmonella, is critical for ensuring food safety. Herein, we present a magnetically controlled electrochemical biosensor integrating CRISPR/Cas12a with Fe3O4@Au nanoparticles designed to achieve ultrasensitive and multiplexed detection. By utilization of the magnetic separation of CRISPR-cleaved ssDNA from Fe3O4@Au nanoparticles, the sensor circumvents intricate electrode modifications, enabling direct signal readout. This approach expedites the workflow to 65 min while achieving a detection limit of 2 CFU/mL. Additionally, the sensor exhibits signal stability over 45 days and demonstrates its versatility by enabling the separate detection of both Gram-positive (S. aureus) and Gram-negative (Salmonella) pathogens. With validation in milk samples with high interference resistance, this platform bridges CRISPR programmability with practical deployability, offering a robust solution for on-site monitoring. The innovation lies in its simplified design, enhanced stability, and clinical versatility, setting a new benchmark for rapid, low-cost pathogen detection in resource-limited environments.},
}
@article {pmid40411798,
year = {2025},
author = {Shi, K and Zhang, Y and Tao, Y and Wang, Y and Yang, J and Deng, R and Yang, H},
title = {Preamplification-Free Detection of RNA N6-Methyladenosine Modification at Single-Base Resolution Using the CRISPR Tandem Assay.},
journal = {Analytical chemistry},
volume = {97},
number = {22},
pages = {11454-11461},
doi = {10.1021/acs.analchem.4c06782},
pmid = {40411798},
issn = {1520-6882},
mesh = {*Adenosine/analogs &amp; derivatives/analysis/metabolism ; Humans ; *CRISPR-Cas Systems ; RNA, Long Noncoding/genetics ; *RNA ; Lung Neoplasms/diagnosis/genetics ; },
abstract = {N6-Methyladenosine (m[6]A) ranks among the most prevalent modifications in RNA, which serves as a biomarker for diseases, such as lung cancer. Herein, we developed a CRISPR/Cas13a-Csm6 tandem assay (termed CRISPRm[6]A assay) allowing for preamplification-free, sensitive, and rapid detection of RNA m[6]A modifications. The coupling of Cas13a-Csm6 tandem with MazF endoribonuclease enables the assay to identify m[6]A RNA with single-base resolution. Compared to the CRISPRm[6]A assay using Cas13a alone, the tandem CRISPRm[6]A assay yielded an improved sensitivity for RNA detection by ∼22 times, thus enabling preamplification-free detection of RNA m[6]A. Particularly, the proposed assay enabled quantification of m[6]A abundance down to 0.5% at the picomole level in lncRNA MALAT1 and demonstrated a 100% correlation in diagnosing nonsmall cell lung cancer. In summary, the CRISPRm[6]A assay supports two key applications in biological samples: (1) precise determination of m[6]A sites and (2) quantitative measurement of m[6]A fractions. Therefore, the CRISPR tandem method presents a promising tool for RNA epigenetics-based diagnostics.},
}
@article {pmid40411669,
year = {2025},
author = {Hassan, HM and Zubair, A and Helal, MH and Almagharbeh, WT and Elmagzoub, RM},
title = {New hope and promise with CRISPR-Cas9 technology for the treatment of HIV.},
journal = {Functional &amp; integrative genomics},
volume = {25},
number = {1},
pages = {108},
pmid = {40411669},
issn = {1438-7948},
mesh = {*CRISPR-Cas Systems ; Humans ; *HIV Infections/therapy/genetics/virology ; *Gene Editing/methods ; *HIV-1/genetics ; Genetic Therapy/methods ; },
abstract = {The commencement of Highly Active Antiretroviral Therapy almost completely stopped viral replication, enabling the immune system to restore its full functionality. The rise in life expectancy has resulted in a decrease in the incidence of classical infections and HIV-associated cancers. HAART has raised concerns, including its exorbitant cost (which hinders its implementation in developing nations), the need for strict adherence, and the potential for both immediate and prolonged ill effects. Lipodystrophy is a significant long-term consequence of HIV that may result in central fat accumulation and severe peripheral fat depletion. Current initiatives to tackle these difficulties include the global expansion of access to HAART, the development of novel drugs that mitigate early side effects, and the introduction of once-daily drug combinations that enhance adherence. The CRISPR-Cas9 system has facilitated the creation of a powerful instrument for precise gene editing. This method has lately established itself as the gold standard for efficient HIV-1 genome editing in HIV therapy, owing to progress in related disciplines. CRISPR may be customized to cleave specific sequences by altering Cas9. This article offers a concise overview of promising CRISPR-Cas9 technology. This technique has the potential to halt the transmission of HIV-1 and alleviate its symptoms. CRISPR-Cas9 technology will be significant in the fight against HIV-1 in the future.},
}
@article {pmid40411467,
year = {2025},
author = {Wang, X and Zhong, L and Zhang, W and Wu, P and Wang, M and Li, D and Dong, L and Wang, G},
title = {CRISPR Digital Sensing: From Micronano-Collaborative Chip to Biomolecular Detection.},
journal = {ACS nano},
volume = {19},
number = {22},
pages = {20427-20451},
doi = {10.1021/acsnano.5c03474},
pmid = {40411467},
issn = {1936-086X},
mesh = {Humans ; *COVID-19/diagnosis/genetics/virology ; *CRISPR-Cas Systems/genetics ; SARS-CoV-2/genetics/isolation &amp; purification ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; *Biosensing Techniques/methods ; *Lab-On-A-Chip Devices ; },
abstract = {The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) sensing technology proved to be valuable during the COVID-19 pandemic through its sensitivity, specificity, robustness, and versatility. However, issues such as overreliance on amplification, susceptibility to false positives, lack of quantification strategies, and complex operation procedures have hindered its broader application in bioanalysis and clinical diagnostics. The collision between micronano-collaborative chips and CRISPR technology has effectively addressed these bottlenecks, offering innovative solutions for diagnosis and treatment. Unlike conventional micronano chips, micronano digital chips enhance CRISPR's response to trace amounts of target molecules by leveraging highly controllable local environments and compartmentalized microreactors. This advancement improves detection efficiency and revolutionizes traditional in vitro bioanalytical processes. First, the working principles, fabrication techniques, and performance metrics of CRISPR-based digital droplet microfluidics and microarray chips are examined. Then, the applications of CRISPR digital sensing chips in bioassays are reviewed, emphasizing their importance in advancing in vitro detection systems for gene editing. Finally, the prospects of CRISPR digital sensing technology are explored, particularly its potential for body surface biomonitoring and its broader development opportunities in the biomedical field.},
}
@article {pmid40410287,
year = {2025},
author = {Wei, J and Shao, Y and Liang, Y and Bu, X and Zhou, W and Liang, Y and Li, Y},
title = {A type III-associated Cas6 functions as a negative regulator of type I-B CRISPR-Cas system in Thermus thermophilus.},
journal = {Communications biology},
volume = {8},
number = {1},
pages = {793},
pmid = {40410287},
issn = {2399-3642},
support = {32170096//National Natural Science Foundation of China (National Science Foundation of China)/ ; },
mesh = {*Thermus thermophilus/genetics/metabolism/enzymology ; *CRISPR-Cas Systems ; *Bacterial Proteins/metabolism/genetics ; *CRISPR-Associated Proteins/metabolism/genetics ; RNA, Bacterial/metabolism/genetics ; Gene Expression Regulation, Bacterial ; },
abstract = {CRISPR-Cas systems are small RNA-guided immune systems in prokaryotes. CRISPR RNA (crRNA) provides sequence specificity and programmability, guiding the effector complex to cleave target nucleic acids. Cas6 family ribonucleases can cleave precursor crRNA to generate functional crRNAs in most type I and type III CRISPR-Cas systems. Most existing studies of Cas6 functions are mainly focused on nuclease activity in vitro and Cas6-processed product characterization in vivo. However, in hosts harboring multiple CRISPR systems, the biological functions of the co-occurrence of various Cas6 proteins and their cross-cleavage activity toward different types of crRNAs remain largely unexplored. In this study, we biochemically characterized the cross-cleavage activity of two Cas6 proteins in Thermus thermophilus HB27 and first found that Cas6 could anchor the mature crRNA and interact with Cas5 subunit of type I-B system, revealing the functions of Cas6 to mediate the assembly of type I Cascade complex. We further demonstrated that the type III-associated Cas6 protein could act as a negative regulator by competing with the I-B Cas6 protein during the assembly of type I-B Cascade complex, significantly suppressing the interference activity of type I-B system. Our findings provide an insight into the functional coupling and regulation mechanisms underlying multiple CRISPR-Cas systems.},
}
@article {pmid40409866,
year = {2025},
author = {Wang, L and Wu, Y and Pan, S and Pan, F and Chen, J},
title = {Rapid and sensitive detection of Karlodinium veneficum using RAA and CRISPR-Cas12a technologies.},
journal = {Harmful algae},
volume = {146},
number = {},
pages = {102864},
doi = {10.1016/j.hal.2025.102864},
pmid = {40409866},
issn = {1878-1470},
mesh = {*Dinoflagellida/genetics/isolation &amp; purification ; *CRISPR-Cas Systems ; *Nucleic Acid Amplification Techniques/methods ; Harmful Algal Bloom ; },
abstract = {The harmful algal species Karlodinium veneficum (K. veneficum) poses a significant threat to aquatic ecosystems, economic stability, and human health due to its toxin production and widespread occurrence. Rapid climatic changes and eutrophication have intensified harmful algal blooms (HABs), making the timely detection of K. veneficum critical. To address this need, we developed a rapid and accurate detection method of K. veneficum by combining Recombinase Aided Amplification (RAA) with CRISPR/LbCas12a. This method targets the internal transcribed spacer (ITS) sequence of K. veneficum and utilizes the "collateral activity" of CRISPR/LbCas12a for visualization. Our method can detect plasmid DNA as low as 5.9 × 10[2] copies/µL and genomic DNA as low as 3.6 × 10[-2] ng/µL, achieving a detection limit of 10 cells of K. veneficum through a simplified DNA extraction process. The entire detection process, from DNA crude extract to result visualization, can be completed in as fast as 90 min, making it suitable for field applications requiring a rapid response. In addition, our method was validated against a wide range of non-target microalgae species, confirming its specificity to K. veneficum and eliminating the risk of cross-reactivity. Overall, the RAA-CRISPR/LbCas12a system is simple, accurate, and sensitive, showing great potential for field applications in monitoring K. veneficum.},
}
@article {pmid40409549,
year = {2025},
author = {Okada, R and Sakaguchi, R and Komaki, T and Nonaka, R and Polat, OK and Kihara, T and Asanuma, K and Yamamoto, T and Isaka, Y and Mori, Y and Mori, MX},
title = {Delayed inactivation of TRPC6 as a determinative characteristic of FSGS-associated variants.},
journal = {The Journal of biological chemistry},
volume = {301},
number = {6},
pages = {110256},
doi = {10.1016/j.jbc.2025.110256},
pmid = {40409549},
issn = {1083-351X},
abstract = {Transient receptor potential canonical 6 (TRPC6) is a receptor-operated nonspecific cation channel. To date, more than 30 TRPC6 variants have been reported to focal segmental glomerulosclerosis (FSGS), which can present from infancy to adulthood and is characterized by proteinuria and often nephrotic syndrome leading to kidney failure. These variants may exhibit gain-of-function (e.g. K874X) or loss-of-function (e.g. L395A, G757D) phenotypes, making the role of TRPC6 in FSGS controversial. Here, we characterized Ca[2+]-dependent inactivation (CDI) of TRPC6 after the receptor activation and found that >85% of TRPC6 variants exhibit delayed CDI. Thus, prolonged TRPC6 channel opening due to impaired inactivation may be a common feature of FSGS-associated variants. This effect was confirmed in immortalized mouse podocytes (MPC-5) in which the coiled-coil (CC) domain was deleted from the channel (C6ΔCC). Podocytes expressing C6ΔCC exhibited delayed CDI and increased basal Ca[2+] levels as well as disruption of the F-actin cytoskeleton. Moreover, transcriptomic data from C6ΔCC-expressing podocytes showed weak expression of the podocyte markers Synpo and Magi2. These results indicate that CDI of TRPC6 is critical for maintaining proper podocyte function. Notably, we observed a correlation between the magnitude of the prolongation of TRPC6 channel activity and the age diagnosed with FSGS. Our findings thus demonstrate that delayed inactivation due to lack of CDI is a determinative characteristic of FSGS-associated TRPC6 variants, affecting both the structure and the function of glomerular podocytes.},
}
@article {pmid40409480,
year = {2025},
author = {Yi, B and Zhou, B and Zhou, D and Yang, L and Xu, H},
title = {CRISPR/Cas-powered nucleic acid amplification and amplification-free biosensors for public safety detection: Principles, advances and prospects.},
journal = {Biotechnology advances},
volume = {83},
number = {},
pages = {108609},
doi = {10.1016/j.biotechadv.2025.108609},
pmid = {40409480},
issn = {1873-1899},
abstract = {Rapid, accurate, cost-effective, and efficient ultrasensitive detection strategies are essential for public health safety (including food safety, disease prevention and environmental governance). The CRISPR/CRISPR-associated (Cas) detection is a cutting-edge technology that has been widely used in the detection of public health safety due to its targeted cleavage properties (signal amplification), attomolar level sensitivity, high specificity (recognizing single-base mismatches), and rapid turnover time. However, the current research about CRISPR/Cas-based biosensors is not clear, such as mechanism problem and application differences of integrating CRISPR/Cas system with other technologies, and how to further innovate and develop in the future. Therefore, further detailed analysis and comparative discussion of CRISPR/Cas-based biosensors is needed. Currently, CRISPR/Cas system powered biosensors can be mainly categorized into two types: CRISPR/Cas system powered nucleic acid amplification biosensors and CRISPR/Cas system powered nucleic acid amplification-free biosensors. The two biosensors have different characteristics and advantages. This paper first provides an in-depth investigation of the enzymatic mechanism of CRISPR/Cas system at the molecular level. Then, this paper summarizes the principles and recent advances of CRISPR/Cas system powered nucleic acid amplification biosensors and CRISPR/Cas system powered nucleic acid amplification-free biosensors and discusses their integration mechanisms in depth. More, the differences and application-oriented between the two biosensors are further discussed. Finally, the application orientation and future perspectives of the two biosensors are discussed, and unique insights into the future development of CRISPR/Cas system are provided.},
}
@article {pmid40409263,
year = {2025},
author = {Yang, Z and Yao, Y and Chen, X and Madigan, V and Pu, S and Fan, X and Pu, J and Bei, F},
title = {Cross-species tropism of AAV.CPP.16 in the respiratory tract and its gene therapies against pulmonary fibrosis and viral infection.},
journal = {Cell reports. Medicine},
volume = {6},
number = {6},
pages = {102144},
doi = {10.1016/j.xcrm.2025.102144},
pmid = {40409263},
issn = {2666-3791},
mesh = {*Dependovirus/genetics/physiology ; Animals ; *Genetic Therapy/methods ; Humans ; Mice ; Genetic Vectors/genetics ; *Viral Tropism ; Administration, Intranasal ; Lung/virology/pathology ; SARS-CoV-2/genetics ; CRISPR-Cas Systems ; Mice, Inbred C57BL ; *Respiratory System/virology/metabolism ; Gene Editing ; *Pulmonary Fibrosis/therapy/genetics ; Vascular Endothelial Growth Factor A/metabolism/genetics ; COVID-19/virology ; *Idiopathic Pulmonary Fibrosis/therapy ; *Virus Diseases/therapy ; Disease Models, Animal ; Female ; },
abstract = {Efficient gene delivery vectors are crucial for respiratory and lung disease therapies. We report that AAV.CPP.16, an engineered adeno-associated virus (AAV) variant derived from AAV9, efficiently transduces airway and lung cells in mice and non-human primates via intranasal administration. AAV.CPP.16 outperforms AAV6 and AAV9, two wild-type AAVs with demonstrated tropism for respiratory tissues, and efficiently targets key respiratory cell types. It supports gene supplementation and editing therapies in two clinically relevant mouse models of respiratory and lung diseases. A single intranasal dose of AAV.CPP.16 expressing a dual-target, vascular endothelial growth factor (VEGF)/transforming growth factor (TGF)-β1-neutralizing protein protected lungs from idiopathic pulmonary fibrosis, while a similar application of AAV.CPP.16 carrying an "all-in-one" CRISPR-Cas13d system inhibited transcription of the SARS-CoV-2-derived RNA-dependent RNA polymerase (Rdrp) gene. Our findings highlight AAV.CPP.16 as a promising vector for respiratory and lung gene therapy.},
}
@article {pmid40408898,
year = {2025},
author = {Li, M and Tao, C and Tang, Z and Li, K and Wang, Y and Zhao, C and Tao, C and Geng, J and Sun, K},
title = {Ultrasensitive clinical identification of hepatitis B surface antigen (HBsAg) by CRISPR-assisted nanopore sensing.},
journal = {Biosensors &amp; bioelectronics},
volume = {286},
number = {},
pages = {117579},
doi = {10.1016/j.bios.2025.117579},
pmid = {40408898},
issn = {1873-4235},
mesh = {*Biosensing Techniques/methods ; Humans ; *Hepatitis B Surface Antigens/blood/isolation &amp; purification/genetics ; *Nanopores/ultrastructure ; *Hepatitis B virus/isolation &amp; purification/genetics ; *CRISPR-Cas Systems/genetics ; *Hepatitis B/diagnosis/virology/blood ; Limit of Detection ; Aptamers, Nucleotide/chemistry ; },
abstract = {Hepatitis B virus (HBV) infection is a major global health issue. The underdiagnosis of HBV contributes to the increasing mortality from hepatitis B-related complications. Hepatitis B surface antigen is a biomarker guiding the clinical management of chronic hepatitis B, and its disappearance from the blood is a key sign of functional cure. There is a need for highly sensitive detection methods for early intervention and prevention of disease recurrence. We presented a new CRISPR-assisted nanopore sensing method for ultrasensitive detection of hepatitis B surface antigen. It uses the high specificity and turnover efficiency of the CRISPR-Cas12a system. The system is activated by the competitive binding between hepatitis B surface antigen and its aptamer, followed by restriction enzyme digestion. The products are detected by a nanopore for precise quantification at very low concentrations. The result achieves the limit of quantification (LOQ) of 10 fM, outperforming conventional assays. Clinical validation with patient samples confirms its superiority. This integrated technology is a powerful tool for HBV early diagnosis, treatment monitoring, and disease assessment, and paves the way for nanopore technology in clinical diagnostics.},
}
@article {pmid40408895,
year = {2025},
author = {Xu, Y and Deng, R and Liu, X and Zhou, Y},
title = {CRISPR/Cas12a-triggered electrochemiluminescence biosensor to ultrasensitive detect herpes simplex virus via self-enhanced near-infrared selenium-based polymer dots.},
journal = {Biosensors &amp; bioelectronics},
volume = {286},
number = {},
pages = {117597},
doi = {10.1016/j.bios.2025.117597},
pmid = {40408895},
issn = {1873-4235},
mesh = {*Biosensing Techniques/methods ; CRISPR-Cas Systems/genetics ; *Polymers/chemistry ; Humans ; Limit of Detection ; *Simplexvirus/isolation &amp; purification/genetics ; *Selenium/chemistry ; Luminescent Measurements/methods ; *Herpes Simplex/virology/diagnosis ; Electrochemical Techniques/methods ; *Quantum Dots/chemistry ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {Herpes simplex virus (HSV) as one of the most popular herpesviruses exhibits nearly identical clinical symptoms among the family of herpesvirus species and it is urgent to develop high specific biosensors to realize accurate detection of HSV. CRISPR/Cas12a systems with programmability and high specificity could serve an essential role in accurate diagnosis of HSV in complex samples. Herein, a novel near-infrared selenium-based polymer dots with self-enhanced effect were unprecedentedly designed and successfully synthesized in this work, and an ultrasensitive biosensor has been constructed together with the CRISPR/Cas12a system for the specific detection of HSV. Notably, the as-prepared selenium-based polymer dots exhibited near-infrared emission with a peak at 760 nm, which could significantly minimize background noise and achieve high sensitivity. The proposed biosensor demonstrated a wide linear range from 1 fM to 1 nM and the limit of detection as low as 0.1 fM (S/N = 3). Undoubtedly, the CRISPR/Cas12a-triggered ECL biosensor proposed in this work could provide a simple, sensitive, and environmentally friendly approach to detect HSV in clinical applications.},
}
@article {pmid40408893,
year = {2025},
author = {Kong, H and Yi, K and Zhu, X and Chen, L and Wang, H and Ju, E and Lv, S and Lao, YH and Shao, D and Xie, X and Cheng, D and Zhang, Y and Tao, Y and Li, M},
title = {Antifouling fusion-mediated diagnostic platform to detect viral DNA-positive extracellular vesicles for in situ blood-based liquid biopsy.},
journal = {Biosensors &amp; bioelectronics},
volume = {286},
number = {},
pages = {117568},
doi = {10.1016/j.bios.2025.117568},
pmid = {40408893},
issn = {1873-4235},
mesh = {Humans ; *Extracellular Vesicles/virology/chemistry/genetics ; Liquid Biopsy ; *DNA, Viral/blood/isolation &amp; purification/genetics ; *Biosensing Techniques/methods ; CRISPR-Cas Systems ; },
abstract = {In liquid biopsy, extracellular vesicles (EVs) have emerged as promising biomarkers due to their ability to carry protected nucleic acids. In particular, DNA enclosed within these vesicles shows great diagnostic potential for monitoring oncovirus-related disease progression. However, current methods still require labor-intensive procedures and bulk analysis. Additionally, in situ detection from blood is hindered by abundant serum proteins, interfering with the accuracy of diagnosis. To address these limitations, we developed an antifouling fusion-mediated CRISPR/Cas detector (AFFECTOR) as a user-friendly and efficient diagnostic platform for directly detecting EV-contained viral DNA in serum samples. Leveraging zwitterionic phosphatidylcholine to resist protein interference, the platform enables stable membrane fusion with intact EVs even in serum-containing environments, allowing highly specific and sensitive detection of internal DNA via the CRISPR/Cas12a sensing system, lasting just 2 h at 37 °C. In clinical samples from oncovirus-infected patients and healthy donors, the platform achieved one-step detection of viral DNA-positive EVs. Notably, viral DNA in circulating EVs was found for the first time to correlate with oncovirus infection stages. Overall, this platform provides a practical tool for diagnostic applications and expands the detection window in liquid biopsy.},
}
@article {pmid40405306,
year = {2025},
author = {Marei, HE},
title = {Stem cell therapy: a revolutionary cure or a pandora's box.},
journal = {Stem cell research &amp; therapy},
volume = {16},
number = {1},
pages = {255},
pmid = {40405306},
issn = {1757-6512},
mesh = {Humans ; *Stem Cell Transplantation/ethics ; *Stem Cell Research/ethics/legislation &amp; jurisprudence ; Gene Editing ; *Cell- and Tissue-Based Therapy/ethics ; Embryonic Stem Cells ; Animals ; CRISPR-Cas Systems ; },
abstract = {This review article examines how stem cell therapies can cure various diseases and injuries while also discussing the difficulties and moral conundrums that come with their application. The article focuses on the revolutionary developments in stem cell research, especially the introduction of gene editing tools like CRISPR-Cas9, which can potentially improve the safety and effectiveness of stem cell-based treatments. To guarantee the responsible use of stem cells in clinical applications, it is also argued that standardizing clinical procedures and fortifying ethical and regulatory frameworks are essential first steps. The assessment also highlights the substantial obstacles that still need to be addressed, such as the moral dilemmas raised by the use of embryonic stem cells, the dangers of unlicensed stem cell clinics, and the difficulties in obtaining and paying for care for patients. The study emphasizes how critical it is to address these problems to stop exploitation, guarantee patient safety, and increase the accessibility of stem cell therapy. The review also addresses the significance of thorough clinical trials, public education, and policy development to guarantee that stem cell research may fulfill its full potential. The review concludes by describing stem cell research as a promising but complicated topic that necessitates a thorough evaluation of both the hazards and the benefits. To overcome the ethical, legal, and accessibility obstacles and eventually guarantee that stem cell treatments may be safely and fairly included in conventional healthcare, it urges cooperation between the scientific community, legislators, and the general public.},
}
@article {pmid40404851,
year = {2025},
author = {Syed, M and Khan, RS and Nazir, S and Khan, S and Ul Islam, Z and Khan, S and Ikuo, N},
title = {Silencing of disease susceptibility genes: an effective disease resistance strategy against fungal pathogens.},
journal = {Plant cell reports},
volume = {44},
number = {6},
pages = {127},
pmid = {40404851},
issn = {1432-203X},
mesh = {*Plant Diseases/microbiology/genetics/immunology ; *Disease Resistance/genetics ; *Gene Silencing ; *Fungi/pathogenicity/physiology ; RNA Interference ; Host-Pathogen Interactions/genetics ; Genes, Plant ; CRISPR-Cas Systems ; },
abstract = {Silencing of target susceptibility (S) genes in plants exhibits a promising and durable strategy for enhanced resistance to fungal pathogens by causing disruption in the host mechanisms that the pathogens exploit, offering an alternative to the traditional resistance gene-based approaches. Devastating fungal diseases have significantly reduced crop productivity, posing a potential threat to global food security. Producing disease-resistant cultivars is the most effective strategy for protecting crops against these fungal pathogens. Typically, susceptibility (S) genes in host plants facilitate the penetration and proliferation of phytopathogens. Perturbation of these S genes can potentially impede the compatibility between the host and the fungal pathogens, thereby providing broad-spectrum and lasting resistance. Consequently, the identification and targeting of S-genes have gained increasing interest in enhancing disease resistance in plants. In this review, we describe three distinct categories of S genes that function during different stages of the infection process. We focus on various gene silencing technologies, including RNA interference (RNAi), virus-induced gene silencing (VIGS), and CRISPR-Cas9, to improve plant disease resistance against fungal pathogens. The numerous examples discussed here illustrate the potential of S-genes for use in plant disease-resistance breeding.},
}
@article {pmid40403777,
year = {2025},
author = {Wu, HJ and Yang, JP and Ma, WJ and Li, ZH and Feng, HY and Yang, ZN and Xu, HJ},
title = {A CRISPR/Cas9-induced point mutation on the GABA receptor subunit RDL confers high resistance to phenylpyrazole insecticides in the rice planthopper Laodelphax striatellus.},
journal = {Insect biochemistry and molecular biology},
volume = {181},
number = {},
pages = {104327},
doi = {10.1016/j.ibmb.2025.104327},
pmid = {40403777},
issn = {1879-0240},
mesh = {Animals ; *Hemiptera/genetics/drug effects/metabolism ; *Insecticide Resistance/genetics ; *Insecticides/pharmacology ; *Pyrazoles/pharmacology ; CRISPR-Cas Systems ; *Receptors, GABA/genetics/metabolism ; *Point Mutation ; *Insect Proteins/genetics/metabolism ; Female ; },
abstract = {The Delphacidae planthopper Laodelphax striatellus (Order: Hemiptera) is one of the most damaging insect pests of rice crops in Asia. The phenylpyrazole insecticide fipronil was introduced in China in the mid-1990s to control these pests, but its widespread use has led to the development of high levels of resistance. Field sampling coupled with in vitro assays indicated that an A2'N-point mutation in the gamma-aminobutyric acid receptor RDL has been linked to fipronil resistance; however, genetic evidence supporting this association has been lacking. Here, we generated an A2'N-point mutation (LsRDL[A/N]) in L. striatellus using CRISPR/Cas9-mediated homologous recombination. Bioassay revealed that the LsRDL[A/N] mutants conferred a 1211-fold higher resistance to fipronil compared to wild-type planthoppers. Furthermore, these mutants also showed significant resistance (>1000-fold) to ethiprole but not to imidacloprid, demonstrating that the A2'N mutation confers resistance specific to phenylpyrazole insecticides. However, the LsRDL[A/N] mutants displayed reduced fecundity, lower egg hatching rates, and prolonged egg and nymph stages, indicating that fipronil resistance carries a substantial fitness cost. These findings enhance our understanding of the genetic mechanisms underlying phenylpyrazole resistance and provide valuable insights for developing effective resistance management strategies for planthoppers.},
}
@article {pmid40403646,
year = {2025},
author = {Wu, Y and Kilgore, R and Hetzler, Z and Chu, W and Moore, B and Barbieri, E and Wei, Q and Menegatti, S},
title = {Purification of CRISPR Cas12a from E. coli cell lysates using peptide affinity ligands.},
journal = {Journal of chromatography. A},
volume = {1755},
number = {},
pages = {466065},
doi = {10.1016/j.chroma.2025.466065},
pmid = {40403646},
issn = {1873-3778},
mesh = {*Escherichia coli/chemistry/genetics/enzymology ; *Chromatography, Affinity/methods ; Ligands ; *CRISPR-Associated Proteins/isolation &amp; purification/metabolism/chemistry ; *Peptides/chemistry/metabolism ; CRISPR-Cas Systems ; *Bacterial Proteins/isolation &amp; purification/metabolism/chemistry ; *Endodeoxyribonucleases/isolation &amp; purification/metabolism/chemistry ; Acidaminococcus/enzymology ; },
abstract = {CRISPR Cas nucleases are revolutionizing gene therapy by providing a precise and efficient tool for editing the human genome, and are increasingly applied for engineering microorganisms for bioremediation, drought-resistant crops, and livestock with higher productivity. While Cas9 is currently the most widely utilized member of the CRISPR family, Cas12a stands is gaining prominence for its ability to produce staggered cuts in the target DNA while requiring a shorter guide RNA (crRNA). Current methods of Cas purification such as affinity tag, immunoaffinity, and ion exchange chromatography do not provide either the productivity or the purity needed to meet the demand of clinics and biotechnology industries. Responding to this need, this study presents the first affinity ligands for Cas12a purification via affinity chromatography. The ligands were initially designed in silico as peptide mimetics of anti-CRISPR protein inhibitors AcrVA1 and AcrVA4, and ranked experimentally by Cas12a dynamic binding. Selected ligands P5 and P9 were utilized for purifying Cas12a derived from Acidaminococcus sp. (AsCas12a) and Lachnospiraceae sp. (LbCas12a) from clarified Escherichia coli cell lysates. P5-functionalized resin afforded high yield (up to 80 %), purity (> 93 %), and DNA editing activity (∼72 %) of Cas12a from E. coli lysates featuring different Cas12a and host cell protein titers. The characterization of ligand P5 by surface plasmon resonance (SPR) indicated adsorption kinetics (ka ∼ 1.21·10[5] M[-1]s[-1]) and dissociation constant (KD ∼ 1.76·10[-6] M) that confirmed the ligand design criteria and are characteristic of peptide affinity ligands.},
}
@article {pmid40403622,
year = {2025},
author = {Choi, H and Yi, TG and Gho, YS and Kim, JH and Kim, S and Choi, YJ and Lim, S and Eom, SH and Jung, KH and Ha, SH},
title = {Augmenting carotenoid accumulation by multiplex genome editing of the redundant CCD family in rice.},
journal = {Plant physiology and biochemistry : PPB},
volume = {225},
number = {},
pages = {110008},
doi = {10.1016/j.plaphy.2025.110008},
pmid = {40403622},
issn = {1873-2690},
mesh = {*Oryza/genetics/metabolism ; *Gene Editing/methods ; *Carotenoids/metabolism ; *Plant Proteins/genetics/metabolism ; Gene Expression Regulation, Plant ; CRISPR-Cas Systems ; *Dioxygenases/genetics/metabolism ; Gene Knockout Techniques ; Plants, Genetically Modified ; },
abstract = {The biodegradation of carotenoid is carried out and regulated by a family of carotenoid cleavage dioxygenases (CCDs). In rice, potential redundancy of OsCCD1, OsCCD4a, and OsCCD4b, among multiple CCDs, was predicted through in silico protein-ligand docking simulations, which were based on the interactions of diverse carotene and xanthophyll substrates in the active sites. To elucidate the roles of the three CCDs in planta, we generated single, double, and triple knockout (KO) rice lines using CRISPR-Cas9 technology and confirmed their genetic stabilities. Triple KO (osccd1/osccd4a/osccd4b) lines exhibited enhanced carotenoid contents notably in both leaves after the harvesting stage and under dark-induced senescence, verifying that was caused by a simultaneously successful blocking of OsCCD1/OsCCD4a/OsCCD4b activities in these conditions. Transcriptional profiling of this triple KO line revealed downregulation of key genes involved in carotenoid biosynthesis, suggesting a feedback mechanism to regulate carotenoid levels. In order to explore and increase practical agricultural use of this triple KO line, we performed agronomic assessments that showed no adverse effects on major traits such as photosynthetic rate and seed productivity and then developed a T-DNA-free version of this triple KO rice line with high carotenoid content. Our study highlights the capacity of OsCCD1/OsCCD4a/OsCCD4b as promising targets for genome editing in biofortification strategies aimed at improving the functionality of rice and suggests their applicability to other forage crops.},
}
@article {pmid40403614,
year = {2025},
author = {Dong, H and Qin, Y and Zhang, P and Lv, L and Yu, C and Jia, P and Zhao, J and Du, F and Guo, Y and Sun, X},
title = {"Turn-on" aptamer-immune lateral flow assays for the detection of small molecule targets based on CHA-assisted and CRISPR/Cas12a mediated signal transduction and amplification.},
journal = {Biosensors &amp; bioelectronics},
volume = {285},
number = {},
pages = {117593},
doi = {10.1016/j.bios.2025.117593},
pmid = {40403614},
issn = {1873-4235},
mesh = {*Aptamers, Nucleotide/chemistry ; CRISPR-Cas Systems/genetics ; *Biosensing Techniques/methods ; Limit of Detection ; Signal Transduction ; },
abstract = {Lateral flow assays (LFAs) have emerged as crucial tools for on-site food safety detection due to their simple operation and intuitive detection results. Nevertheless, LFAs for small molecule targets such as pesticides often present a "Turn-off" signal output, which leads to their low sensitivity and the risk of false positives. In this study, a CRISPR/Cas12a system-mediated strategy was employed to convert aptamer signals into the signals of immune LFAs, achieving a "Turn-on" signal output for highly sensitive detection of small molecule targets. The binding of aptamers to targets released the trigger sequence to initiate the catalytic hairpin assembly (CHA) reaction, generating double-stranded DNA, which subsequently activated the CRISPR/Cas12a system to cleave the FAM-labeled Reporter. Eventually, the "Turn-on" visual output of the signal was realized through an anti-6-FAM immune LFAs. The experiment optimized the sample pool preparation, CHA reaction conditions, CRISPR/Cas12a activation parameters, and the assembly process of the LFAs. The limit of detection for procymidone was as low as 0.015 ng/mL, which was 52.67 times more sensitive than those of conventional aptamer-based LFAs without signal amplification strategies. This method exhibits high specificity for procymidone and a recovery rate ranging from 94.00% to 104.20% in vegetable samples, demonstrating excellent stability and practicability.},
}
@article {pmid40403612,
year = {2025},
author = {Lin, L and Xue, Y and Tan, L and Jiang, C and Liu, M and Li, X and Qiu, J and Zhang, H and Zhou, J and Shu, B},
title = {Micro-scale thermofluidics enable autonomous and scalable CRISPR diagnostics for sexually transmitted infections screening.},
journal = {Biosensors &amp; bioelectronics},
volume = {285},
number = {},
pages = {117591},
doi = {10.1016/j.bios.2025.117591},
pmid = {40403612},
issn = {1873-4235},
mesh = {Humans ; *Biosensing Techniques/instrumentation ; *Sexually Transmitted Diseases/diagnosis/microbiology ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Nucleic Acid Amplification Techniques/instrumentation ; Equipment Design ; CRISPR-Cas Systems ; Lab-On-A-Chip Devices ; },
abstract = {The development of clustered regularly interspaced short palindromic repeats (CRISPR)-based nucleic acid detection has recently been a center of interest for next-generation molecular diagnostics. Despite considerable advances, simple and effective strategies to harness the isothermal amplification reaction and CRISPR-based detection for maximal performance and minimal complexity are still desirable. Here, a thermofluidic approach leverages the micro-scale chemical and physical mechanism to perform autonomous and scalable CRISPR-based diagnostics (CRISPR-Dx) in a greatly simplified format, which was called "Thermofluidic CRISPR". Originating from the concept of convective PCR, it utilizes looped microchannel reactors to perform approximatively undisturbed isothermal amplification reaction at balanced temperature by virtue of the restricted molecular diffusion across the microchannel, in which the reagents of two reactions are compartmentalized virtually; then it creates circulatory flow within the loop channel to mix the amplificons and CRISPR reagents via Rayleigh-Bénard thermal convection, by simply warming up one side of the loop channel. Due to the simplicity and scalability, a low-cost, battery-powered, portable diagnostic platform, incorporating with smartphone-enabled real-time fluorescence readout, to perform rapid (<30 min), highly sensitive (2 copies per reaction), quantitative and multiplexed CRISPR-Dx was constructed. Its diagnostic performance in rapid screening of multiple pathogens from 196 clinical samples for syndromic testing of sexually transmitted infections was evaluated, exhibiting 97.4 % sensitivity and 100 % specificity benchmarked against the laboratory-based testing. Leveraging the micro-scale chemical and physical mechanism to simplify workflows for CRISPR-Dx may enhance their versatility and facilitate their broader applicability at the point of care.},
}
@article {pmid40403178,
year = {2025},
author = {Lee, Y and Lee, JH and Lee, T and Shin, M and Yoon, J},
title = {Amplification-Free CRISPR/Cas12a-Based Electrochemical Biosensor with Enhanced Sensitivity for Viral Detection.},
journal = {ACS sensors},
volume = {},
number = {},
pages = {},
doi = {10.1021/acssensors.5c00576},
pmid = {40403178},
issn = {2379-3694},
abstract = {To detect contagious viral nucleic acids, traditional biosensors often require target amplification steps or use fluorescence and Raman probes tagged on nucleic acids, which are time-consuming, complex, and expensive. Recently, the CRISPR/Cas12a has received the attraction for development of nucleic acid biosensors, beyond its conventional role-like gene editing, but the enhancement of the sensitivity of CRISPR/Cas-based biosensors is still required to simplify the biosensing steps. Here, we develop a CRISPR/Cas12a-based electrochemical biosensor for the detection of viral nucleic acids in a simple manner. The novel mismatch Ag probe (MAP), as a sensing probe, and the highly conductive gold electrode on indium tin oxide with a nano array (GELITION) are introduced that enable the amplification-free and ultrasensitive detection of nucleic acids using a CRISPR/Cas12a system. The biosensing ability of the developed biosensor is validated using human papillomavirus type 16 and 18 viral DNAs (HPV16 and HPV18), achieving a limit of detection (LOD) of 1 fM without amplification and complex steps. Our developed biosensor is expected to be applicable in detecting various viruses and could contribute to the early detection of future pandemics.},
}
@article {pmid40402294,
year = {2025},
author = {Hassane, AMA and Obiedallah, M and Karimi, J and Khattab, SMR and Hussein, HR and Abo-Dahab, Y and Eltoukhy, A and Abo-Dahab, NF and Abouelela, ME},
title = {Unravelling fungal genome editing revolution: pathological and biotechnological application aspects.},
journal = {Archives of microbiology},
volume = {207},
number = {7},
pages = {150},
pmid = {40402294},
issn = {1432-072X},
mesh = {*Gene Editing/methods ; *Genome, Fungal ; CRISPR-Cas Systems ; *Biotechnology/methods ; *Fungi/genetics/pathogenicity ; Humans ; Candida albicans/genetics/pathogenicity ; Aspergillus fumigatus/genetics/pathogenicity ; },
abstract = {Fungi represent a broad and evolutionarily unique group within the eukaryotic domain, characterized by extensive ecological adaptability and metabolic versatility. Their inherent biological intricacy is evident in the diverse and dynamic relationships they establish with various hosts and environmental niches. Notably, fungi are integral to disease processes and a wide array of biotechnological innovations, highlighting their significance in medical, agricultural, and industrial domains. Recent advances in genetic engineering have revolutionized fungal research, with CRISPR/Cas emerging as the most potent and versatile genome editing platform. This technology enables precise manipulation of fungal genomes, from silencing efflux pump genes in Candida albicans (enhancing antifungal susceptibility) to targeting virulence-associated sirtuins in Aspergillus fumigatus (attenuating pathogenicity). Its applications span gene overexpression, multiplexed mutagenesis, and secondary metabolite induction, proving transformative for disease management and biotechnological innovation. CRISPR/Cas9's advantages-unmatched precision, cost-effectiveness, and therapeutic potential-are tempered by challenges like off-target effects, ethical dilemmas, and regulatory gaps. Integrating nanoparticle delivery systems and multi-omics approaches may overcome technical barriers, but responsible innovation requires addressing these limitations. CRISPR-driven fungal genome editing promises to redefine solutions for drug-resistant infections, sustainable bioproduction, and beyond as the field evolves. In conclusion, genome editing technologies have enhanced our capacity to dissect fungal biology and expanded fungi's practical applications across various scientific and industrial domains. Continued innovation in this field promises to unlock the vast potential of fungal systems further, enabling more profound understanding and transformative biotechnological progress.},
}
@article {pmid40401996,
year = {2025},
author = {Pandey, N and Misra, CS and Rath, D},
title = {Cas11 augments Cascade functions in type I-E CRISPR system but is redundant for gene silencing and plasmid interference.},
journal = {The Biochemical journal},
volume = {482},
number = {12},
pages = {},
doi = {10.1042/BCJ20253056},
pmid = {40401996},
issn = {1470-8728},
mesh = {*CRISPR-Cas Systems ; *Plasmids/genetics/metabolism ; *Escherichia coli Proteins/genetics/metabolism ; *Gene Silencing ; *Escherichia coli/genetics/metabolism ; *CRISPR-Associated Proteins/genetics/metabolism ; Gene Editing ; *Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {The structural and mechanistic complexity of Escherichia coli's type I Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated (CRISPR-Cas) system, compared with the multidomain, single effector protein-based type II systems, limits its application in genome editing and silencing. Despite the higher prevalence of the type I endogenous systems in bacteria, significant research has focused on improving the type II systems. While the type-I CRISPR system possesses several advantages over others, it may benefit from further studies to simplify the system for ease of use. To enable this, the dispensability of the type-I Cascade components (Cas8, Cas11, Cas7, Cas5 and Cas6) for genome editing and silencing applications was evaluated in vivo. We created deletion variants of each of the Cascade components and investigated their effects on gene silencing and plasmid interference in two genetically distinct E. coli lineages: BW25113, a K-12 strain that bears an endogenous, albeit repressed type I-E CRISPR system; and BL21, a natural mutant lacking the type I-E CRISPR-Cas system. Cas8, Cas7 and Cas5 were found to be indispensable for gene silencing and plasmid interference. Dispensability of Cas6, which is involved in crRNA maturation, was strain-dependent. Notably, Cas11, which has no definitive function assigned to it, was found to be dispensable for gene silencing and plasmid interference.},
}
@article {pmid40401794,
year = {2025},
author = {Jiao, L and Zhou, Q and Sun, D},
title = {CRISPR-Based Regulation for High-Throughput Screening.},
journal = {ACS synthetic biology},
volume = {14},
number = {6},
pages = {1890-1904},
doi = {10.1021/acssynbio.5c00076},
pmid = {40401794},
issn = {2161-5063},
abstract = {CRISPR technology has revolutionized genome editing by enabling precise, permanent modifications to genetic material. To circumvent the irreversible alterations associated with traditional CRISPR methods and facilitate research on both essential and nonessential genes, CRISPR interference or inhibition (CRISPRi) and CRISPR activation (CRISPRa) were developed. The gene-silencing approach leverages an inactivated Cas effector protein paired with guide RNA to obstruct transcription initiation or elongation, while the gene-activation approach exploits the programmability of CRISPR to activate gene expression. Recent advances in CRISPRi technology, in combination with other technologies (e.g., biosensing, sequencing), have significantly expanded its applications, allowing for genome-wide high-throughput screening (HTS) to identify genetic determinants of phenotypes. These screening strategies have been applied in biomedicine, industry, and basic research. This review explores the CRISPR regulation mechanisms, offers an overview of the workflow for genome-wide CRISPR-based regulation for screens, and highlights its superior suitability for HTS across biomedical and industrial applications. Finally, we discuss the limitations of current CRISPRi/a HTS screening methods and envision future directions in CRISPR-mediated HTS research, considering its potential for broader application across diverse fields.},
}
@article {pmid40401239,
year = {2025},
author = {Mak, JK and Bendandi, A and Salim, JA and Mazoni, I and de Moraes, FR and Borro, L and Störtz, F and Rocchia, W and Neshich, G and Minary, P},
title = {Learning to utilize internal protein 3D nanoenvironment descriptors in predicting CRISPR-Cas9 off-target activity.},
journal = {NAR genomics and bioinformatics},
volume = {7},
number = {2},
pages = {lqaf054},
pmid = {40401239},
issn = {2631-9268},
mesh = {*CRISPR-Cas Systems ; *Machine Learning ; *RNA, Guide, CRISPR-Cas Systems/genetics/chemistry ; *CRISPR-Associated Protein 9/chemistry/genetics/metabolism ; Gene Editing ; Mutation ; },
abstract = {Despite advances in determining the factors influencing cleavage activity of a CRISPR-Cas9 single guide RNA (sgRNA) at an (off-)target DNA sequence, a comprehensive assessment of pertinent physico-chemical/structural descriptors is missing. In particular, studies have not yet directly exploited the information-rich internal protein 3D nanoenvironment of the sgRNA-(off-)target strand DNA pair, which we obtain by harvesting 634 980 residue-level features for CRISPR-Cas9 complexes. As a proof-of-concept study, we simulated the internal protein 3D nanoenvironment for all experimentally available single-base protospacer-adjacent motif-distal mutations for a given sgRNA-target strand pair. By determining the most relevant residue-level features for CRISPR-Cas9 off-target cleavage activity, we developed STING_CRISPR, a machine learning model delivering accurate predictive performance of off-target cleavage activity for the type of single-base mutations considered in this study. By interpreting STING_CRISPR, we identified four important Cas9 residue spatial hotspots and associated structural/physico-chemical descriptor classes influencing CRISPR-Cas9 (off-)target cleavage activity for the sgRNA-target strand pairs covered in this study.},
}
@article {pmid40400348,
year = {2025},
author = {Khatri, M and Shanmugam, NRS and Zhang, X and Patel, RSKR and Yin, Y},
title = {AcrDB update: Predicted 3D structures of anti-CRISPRs in human gut viromes.},
journal = {Protein science : a publication of the Protein Society},
volume = {34},
number = {6},
pages = {e70177},
pmid = {40400348},
issn = {1469-896X},
support = {R01GM140370/NH/NIH HHS/United States ; R21AI171952/NH/NIH HHS/United States ; 58-8042-9-089//U.S. Department of Agriculture (USDA)/ ; },
mesh = {Humans ; *Bacteriophages/chemistry/genetics ; Protein Conformation ; Databases, Protein ; *Viral Proteins/chemistry/genetics/metabolism ; *Gastrointestinal Microbiome ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Models, Molecular ; },
abstract = {Anti-CRISPR (Acr) proteins play a key role in phage-host interactions and hold great promise for advancing genome-editing technologies. However, finding new Acrs has been challenging due to their low sequence similarity. Recent advances in protein structure prediction have opened new pathways for Acr discovery by using 3D structure similarity. This study presents an updated AcrDB, with the following new features not available in other databases: (1) predicted Acrs from human gut virome databases, (2) Acr structures predicted by AlphaFold2, (3) a structural similarity search function to allow users to submit new sequences and structures to search against 3D structures of experimentally known Acrs. The updated AcrDB contains predicted 3D structures of 795 candidate Acrs with structural similarity (TM-score ≥0.7) to known Acrs supported by at least two of the three non-sequence similarity-based tools (TM-Vec, Foldseek, AcrPred). Among these candidate Acrs, 121 are supported by all three tools. AcrDB also includes 3D structures of 122 experimentally characterized Acr proteins. The 121 most confident candidate Acrs were combined with the 122 known Acrs and clustered into 163 sequence similarity-based Acr families. The 163 families were further subject to a structure similarity-based hierarchical clustering, revealing structural similarity between 44 candidate Acr (cAcr) families and 119 known Acr families. The bacterial hosts of these 163 Acr families are mainly from Bacillota, Pseudomonadota, and Bacteroidota, which are all dominant gut bacterial phyla. Many of these 163 Acr families are also co-localized in Acr operons. All the data and visualization are provided on our website: https://pro.unl.edu/AcrDB.},
}
@article {pmid40400168,
year = {2025},
author = {Jiang, M and Zhao, X and Zhang, C and Liu, R and Hu, J and Lv, Y},
title = {Thermus thermophilus Argonaute-Mediated Single Particle Counting Platform for Multiplex Cancer-Related Biomarkers Detection.},
journal = {Analytical chemistry},
volume = {97},
number = {22},
pages = {11907-11916},
doi = {10.1021/acs.analchem.5c02118},
pmid = {40400168},
issn = {1520-6882},
mesh = {Humans ; *Thermus thermophilus/metabolism/chemistry ; *Argonaute Proteins/metabolism/chemistry ; *MicroRNAs/analysis/blood/genetics ; *Biomarkers, Tumor/analysis/blood/genetics ; *Bacterial Proteins/metabolism/chemistry ; Mass Spectrometry ; *Colorectal Neoplasms/diagnosis ; },
abstract = {The clustered regularly interspaced short palindromic repeats-Cas (CRISPR-Cas) system has achieved remarkable success in the field of nucleic acid detection, while its Achilles' heel lies in the difficulties encountered in flexibility regarding the multiplex detection. As a sister system of CRISPR-Cas, prokaryotic Argonautes (pAgos) have precise recognition, multiturnover, and more importantly multiple specific cleavage characteristics, which is a potential candidate for the next generation of multiplex detection. Herein, a single particle counting platform was developed for the simultaneous detection of three colorectal cancer-related miRNAs (miR-141, miR-31, and miR-21) by combining single particle inductively coupled plasma mass spectrometry (SP-ICPMS) with the Thermus thermophilus Argonaute protein (TtAgo), with nanoparticles as signal probes for cleavage. The platform demonstrated high sensitivity (aM level) and specificity due to the dual-cycle mechanism of exponential isothermal amplification (EXPAR) and TtAgo cleavage, as well as the combination of TtAgo's specific cleavage capability and the multiplex detection advantages of metal stable isotope tagging. Additionally, the platform showed good robustness in human serum and cell extracts, indicating significant potential in clinical applications.},
}
@article {pmid40400140,
year = {2025},
author = {Jordt, LM and Gellert, M and Zelms, F and Bekeschus, S and Lillig, CH},
title = {The thioredoxin-like and one glutaredoxin domain are required to rescue the iron-starvation phenotype of HeLa GLRX3 knock out cells.},
journal = {FEBS letters},
volume = {},
number = {},
pages = {},
doi = {10.1002/1873-3468.70072},
pmid = {40400140},
issn = {1873-3468},
support = {Li984/3-1//Deutsche Forschungsgemeinschaft/ ; Li984/4-1//Deutsche Forschungsgemeinschaft/ ; },
abstract = {Glutaredoxin 3 (Grx3) is a multidomain protein (Trx-GrxA-GrxB) with a Trx-like domain and two Grx domains containing a CGFS motif for binding Fe2S2 clusters. To study the function of these domains, HeLa cells with GLRX3 knockout were generated via CRISPR/Cas. The knockout activated iron-regulatory protein 1, indicating iron starvation due to impaired iron metabolism. Transfection with constructs encoding wild-type or individual domains showed that only the Trx-GrxA construct could rescue the phenotype, matching the effect of full-length Grx3. The specific role of the second Grx domain in human Grx3, absent in simpler eukaryotes such as yeast, remains unclear. While the individual domains are insufficient to rescue the knockout of full-length Grx3, the Trx-GrxA module is functionally critical. Impact statement Glutaredoxin 3 (Grx3) contains a Trx-like domain and two Grx domains. The importance of the domains in higher eukaryotes has not previously been addressed in physiological or cellular contexts. Here, we report GLRX3 knockout results in activation of iron regulatory protein 1, and a Trx-GrxA construct could rescue the phenotype.},
}
@article {pmid40400023,
year = {2025},
author = {Kermanshahi, AZ and Ebrahimi, F and Taherpoor, A and Eslami, N and Baghi, HB},
title = {HPV-driven cancers: a looming threat and the potential of CRISPR/Cas9 for targeted therapy.},
journal = {Virology journal},
volume = {22},
number = {1},
pages = {156},
pmid = {40400023},
issn = {1743-422X},
mesh = {Humans ; *CRISPR-Cas Systems ; *Gene Editing/methods ; *Papillomavirus Infections/therapy/virology/complications ; *Neoplasms/therapy/virology ; *Papillomaviridae/genetics ; *Genetic Therapy/methods ; Oncogene Proteins, Viral/genetics ; Female ; *Uterine Cervical Neoplasms/therapy/virology ; },
abstract = {Cervical and other anogenital malignancies are largely caused by E6 and E7 oncogenes of high-risk human papillomaviruses (HPVs), which inhibit important tumor suppressors like p53 and pRb when they are persistently activated. The main goal of traditional treatments is to physically or chemically kill cancer cells, but they frequently only offer temporary relief, have serious side effects, and have a high risk of recurrence. Exploring the efficacy and accuracy of CRISPR-Cas9 gene editing in both inducing death in HPV-infected cancer cells and restoring the activity of tumor suppressors is our main goal. In this study, we propose a novel precision oncology strategy that targets and inhibits the detrimental effects of the E6 and E7 oncogenes using the CRISPR-Cas9 gene editing system. In order to do this, we create unique guide RNAs that target the integrated HPV DNA and reactivate p53 and pRb. Reactivation is meant to halt aberrant cell development and restart the cell's natural dying pathways. This review discusses the potential of CRISPR/Cas9 in targeting HPV oncogenes, with a focus on studies that have demonstrated its promise in cancer treatment. Given the absence of a definitive treatment for papillomavirus infection and its subsequent association with various cancers, future clinical trials and experimental investigations appear essential to establish and evaluate the therapeutic potential of CRISPR-based approaches. This approach provides a less invasive alternative to conventional treatments and opens the door to personalized care that considers the genetic makeup of each patient's tumor.},
}
@article {pmid40399682,
year = {2025},
author = {Panagopoulos, A and Stout, M and Kilic, S and Leary, P and Vornberger, J and Pasti, V and Galarreta, A and Lezaja, A and Kirschenbühler, K and Imhof, R and Rehrauer, H and Ziegler, U and Altmeyer, M},
title = {Multigenerational cell tracking of DNA replication and heritable DNA damage.},
journal = {Nature},
volume = {642},
number = {8068},
pages = {785-795},
pmid = {40399682},
issn = {1476-4687},
mesh = {*DNA Replication/genetics ; Humans ; *DNA Damage/genetics ; *Single-Cell Analysis ; *Cell Lineage/genetics ; *Cell Tracking/methods ; DNA Repair ; Gene Editing ; Polyploidy ; CRISPR-Cas Systems/genetics ; Cell Cycle/genetics ; Epigenesis, Genetic ; },
abstract = {Cell heterogeneity is a universal feature of life. Although biological processes affected by cell-to-cell variation are manifold, from developmental plasticity to tumour heterogeneity and differential drug responses, the sources of cell heterogeneity remain largely unclear[1,2]. Mutational and epigenetic signatures from cancer (epi)genomics are powerful for deducing processes that shaped cancer genome evolution[3-5]. However, retrospective analyses face difficulties in resolving how cellular heterogeneity emerges and is propagated to subsequent cell generations. Here, we used multigenerational single-cell tracking based on endogenously labelled proteins and custom-designed computational tools to elucidate how oncogenic perturbations induce sister cell asymmetry and phenotypic heterogeneity. Dual CRISPR-based genome editing enabled simultaneous tracking of DNA replication patterns and heritable endogenous DNA lesions. Cell lineage trees of up to four generations were tracked in asynchronously growing cells, and time-resolved lineage analyses were combined with end-point measurements of cell cycle and DNA damage markers through iterative staining. Besides revealing replication and repair dynamics, damage inheritance and emergence of sister cell heterogeneity across multiple cell generations, through combination with single-cell transcriptomics, we delineate how common oncogenic events trigger multiple routes towards polyploidization with distinct outcomes for genome integrity. Our study provides a framework to dissect phenotypic plasticity at the single-cell level and sheds light onto cellular processes that may resemble early events during cancer development.},
}
@article {pmid40399557,
year = {2025},
author = {Bourgade, B and Xie, H and Lindblad, P and Stensjö, K},
title = {Development of a CRISPR activation system for targeted gene upregulation in Synechocystis sp. PCC 6803.},
journal = {Communications biology},
volume = {8},
number = {1},
pages = {772},
pmid = {40399557},
issn = {2399-3642},
support = {2021-01669//Svenska Forskningsr&#xe5;det Formas (Swedish Research Council Formas)/ ; },
mesh = {*Synechocystis/genetics/metabolism ; *Up-Regulation ; *Gene Expression Regulation, Bacterial ; Metabolic Engineering/methods ; Butanols/metabolism ; Biofuels ; *CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {The photosynthetic cyanobacterium Synechocystis sp. PCC 6803 offers a promising sustainable solution for simultaneous CO2 fixation and compound bioproduction. While various heterologous products have now been synthesised in Synechocystis, limited genetic tools hinder further strain engineering for efficient production. Here, we present a versatile CRISPR activation (CRISPRa) system for Synechocystis, enabling robust multiplexed activation of both heterologous and endogenous targets. Following tool characterisation, we applied CRISPRa to explore targets influencing biofuel production, specifically isobutanol (IB) and 3-methyl-1-butanol (3M1B), demonstrating a proof-of-concept approach to identify key reactions constraining compound biosynthesis. Notably, individual upregulation of target genes, such as pyk1, resulted in up to 4-fold increase in IB/3M1B formation while synergetic effects from multiplexed targeting further enhanced compound production, highlighting the value of this tool for rapid metabolic mapping. Interestingly, activation efficacy did not consistently predict increases in compound formation, suggesting complex regulatory interactions influencing bioproduction. This work establishes a CRISPRa system for targeted upregulation in cyanobacteria, providing an adaptable platform for high-throughput screening, metabolic pathway optimisation and functional genomics. Our CRISPRa system provides a crucial advance in the genetic toolbox available for Synechocystis and will facilitate innovative applications in both fundamental research and metabolic engineering in cyanobacteria.},
}
@article {pmid40399309,
year = {2025},
author = {Schmerer, N and Janga, H and Aillaud, M and Hoffmann, J and Aznaourova, M and Wende, S and Steding, H and Halder, LD and Uhl, M and Boldt, F and Stiewe, T and Nist, A and Jerrentrup, L and Kirschbaum, A and Ruppert, C and Rossbach, O and Ntini, E and Marsico, A and Valasarajan, C and Backofen, R and Linne, U and Pullamsetti, SS and Schmeck, B and Schulte, LN},
title = {A searchable atlas of pathogen-sensitive lncRNA networks in human macrophages.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {4733},
pmid = {40399309},
issn = {2041-1723},
support = {LOEWE/2/13/519/03/06.001(0002)/74//Hessisches Ministerium f&#xfc;r Wissenschaft und Kunst (Hessen State Ministry of Higher Education, Research and the Arts)/ ; LOEWE/5/A004/519/06/00.005(0008)/E31//Hessisches Ministerium f&#xfc;r Wissenschaft und Kunst (Hessen State Ministry of Higher Education, Research and the Arts)/ ; LOEWE/2/13/519/03/06.001(0002)/74//Hessisches Ministerium f&#xfc;r Wissenschaft und Kunst (Hessen State Ministry of Higher Education, Research and the Arts)/ ; 10.21.2.024MN//Fritz Thyssen Stiftung (Fritz Thyssen Foundation)/ ; SFB-TR-84 (subproject C10)//Deutsche Forschungsgemeinschaft (German Research Foundation)/ ; BA 2168/25-1//Deutsche Forschungsgemeinschaft (German Research Foundation)/ ; TRR 167/2 NeuroMac//Deutsche Forschungsgemeinschaft (German Research Foundation)/ ; CIBSS - EXC-2189 - Project ID 390939984//Deutsche Forschungsgemeinschaft (German Research Foundation)/ ; SFB-1213 (Project A01)//Deutsche Forschungsgemeinschaft (German Research Foundation)/ ; SFB-TR-84 (subproject C1)//Deutsche Forschungsgemeinschaft (German Research Foundation)/ ; PerMed-COPD (01EK2203A)//Bundesministerium f&#xfc;r Bildung und Forschung (Federal Ministry of Education and Research)/ ; Deep Legion (031L0288A)//Bundesministerium f&#xfc;r Bildung und Forschung (Federal Ministry of Education and Research)/ ; },
mesh = {Humans ; *RNA, Long Noncoding/genetics/metabolism ; *Macrophages/metabolism/immunology ; *Gene Regulatory Networks ; GATA2 Transcription Factor/genetics/metabolism ; NF-kappa B/metabolism ; Macrophage Activation/genetics ; Gene Expression Regulation ; CRISPR-Cas Systems ; },
abstract = {Long noncoding RNAs (lncRNA) are crucial yet underexplored regulators of human immunity. Here we develop GRADR, a method integrating gradient profiling with RNA-binding proteome analysis, to map the protein interactomes of all expressed RNAs in a single experiment to study mechanisms of lncRNA-mediated regulation of human primary macrophages. Applying GRADR alongside CRISPR-multiomics, we reveal a network of NFκB-dependent lncRNAs, including LINC01215, AC022816.1 and ROCKI, which modulate distinct aspects of macrophage immunity, particularly through interactions with mRNA-processing factors, such as hnRNP proteins. We further uncover the function of ROCKI in repressing the messenger of the anti-inflammatory GATA2 transcription factor, thus promoting macrophage activation. Lastly, all data are consolidated in the SMyLR web interface, a searchable reference catalog for exploring lncRNA functions and pathway-dependencies in immune cells. Our results thus not only highlight the important functions of lncRNAs in immune regulation, but also provide a rich resource for lncRNA studies.},
}
@article {pmid40399255,
year = {2025},
author = {Riesenberg, S and Kanis, P and Karlic, R and Maricic, T},
title = {Robust prediction of synthetic gRNA activity and cryptic DNA repair by disentangling cellular CRISPR cleavage outcomes.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {4717},
pmid = {40399255},
issn = {2041-1723},
mesh = {*RNA, Guide, CRISPR-Cas Systems/genetics/metabolism ; Humans ; *DNA Repair/genetics ; *CRISPR-Cas Systems/genetics ; Gene Editing/methods ; INDEL Mutation ; CRISPR-Associated Protein 9/metabolism/genetics ; DNA Cleavage ; CRISPR-Associated Proteins/metabolism/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; HEK293 Cells ; Endodeoxyribonucleases/metabolism/genetics ; Bacterial Proteins/metabolism/genetics ; },
abstract = {The ability to robustly predict guide RNA (gRNA) activity is a long-standing goal for CRISPR applications, as it would reduce the need to pre-screen gRNAs. Quantification of formation of short insertions and deletions (indels) after DNA cleavage by transcribed gRNAs has been typically used to measure and predict gRNA activity. We evaluate the effect of chemically synthesized Cas9 gRNAs on different cellular DNA cleavage outcomes and find that the activity of different gRNAs is largely similar and often underestimated when only indels are scored. We provide a simple linear model that reliably predicts synthetic gRNA activity across cell lines, robustly identifies inefficient gRNAs across different published datasets, and is easily accessible via online genome browser tracks. In addition, we develop a homology-directed repair efficiency prediction tool and show that unintended large-scale repair events are common for Cas9 but not for Cas12a, which may be relevant for safety in gene therapy applications.},
}
@article {pmid40398759,
year = {2025},
author = {Zhao, S and Zhang, J and Yu, S and He, D and Li, B and Fan, Y and Liu, G and Tang, Y and Diao, Y},
title = {Integration of RPA and CRISPR-Cas13a collateral activity for one-step detection of DHAV-3: A biological macromolecule-enabled diagnostic platform.},
journal = {International journal of biological macromolecules},
volume = {314},
number = {},
pages = {144400},
doi = {10.1016/j.ijbiomac.2025.144400},
pmid = {40398759},
issn = {1879-0003},
mesh = {*CRISPR-Cas Systems/genetics ; Animals ; *Recombinases/metabolism ; RNA, Viral/genetics ; *Nucleic Acid Amplification Techniques/methods ; *Picornaviridae/genetics/isolation &amp; purification ; Ducks/virology ; },
abstract = {Duck hepatitis A virus type 3 (DHAV-3), which is families of the Picornaviridae, poses severe threats to waterfowl industries due to rapid antigenic evolution and limitations in conventional diagnostics. Herein, we engineered a CRISPR-Cas13a-mediated RNA detection system by leveraging the intrinsic HEPN domain-dependent collateral cleavage activity of Cas13a, synergistically integrated with recombinase polymerase amplification (RPA) to target DHAV-3 RNA. This biological macromolecule-driven platform achieved ultrasensitive detection (1 copies/μL) within 35 min through sequence-specific crRNA guidance and Cas13a-triggered fluorescent/lateral flow signal amplification. Rigorous validation against four avian pathogens (ARV, H9N2 AIV, TMUV, AstV) confirmed 100 % specificity, highlighting the precise macromolecular interactions between Cas13a and target RNA. Clinical evaluation of 30 field samples demonstrated complete concordance with RT-qPCR. By harnessing the programmable functionality of Cas13a and the thermostable enzymes in RPA, this study provides a novel paradigm for RNA-guided biological macromolecule applications in point-of-care diagnostics, bridging molecular mechanisms with agricultural biosecurity needs.},
}
@article {pmid40398569,
year = {2025},
author = {Zhou, Y and Duan, Y and Chen, L and Yang, Y and Ma, L and Chen, W and Liao, Z and Cai, J and Li, D},
title = {Engineering dispersed mycelium morphology in Aspergillus niger for enhanced mycoprotein production via CRISPR/Cas9-mediated genome editing.},
journal = {Bioresource technology},
volume = {432},
number = {},
pages = {132652},
doi = {10.1016/j.biortech.2025.132652},
pmid = {40398569},
issn = {1873-2976},
mesh = {*Aspergillus niger/genetics/metabolism/cytology ; *Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; *Mycelium/genetics/metabolism/cytology ; Fermentation ; *Fungal Proteins/biosynthesis/genetics/metabolism ; Biomass ; },
abstract = {Filamentous fungi are widely utilized in industrial fermentation processes due to their high productivity, with mycelial morphology directly influencing fermentation broth viscosity and target product yield, which is a critical parameter for process optimization. Aspergillus niger, an FDA-approved safe filamentous fungus, typically forms tightly packed mycelial pellets in submerged cultures, which severely restricts its industrial application potential by limiting mass transfer efficiency. To address this challenge, CRISPR/Cas9 mediated genome editing coupled with fermentation optimization enhanced microbial protein production in A. niger. Endogenous α-1,3-glucan synthase genes (agsA, agsB) and galactosaminogalactan (GAG) synthase genes (sph3, uge3) were disrupted using CRISPR/Cas9, achieving complete dispersion of filamentous pellets in liquid media. This morphological engineering strategy resulted in a 77.52 % increase in biomass and 39.98 % enhancement in mycelial protein content compared to the wild-type strain (A. niger Li2). Transcriptomic analysis revealed that the engineered strain (A. niger AnΔABSU) exhibited upregulated transporter proteins (ABC transporters, MFS transporters, sugar transporters), accelerating nutrient uptake and energy metabolism; altered cell wall integrity pathways, including activation of the MAPK signaling cascade and increased sensitivity to cell wall stressors; enhanced amino acid biosynthesis, driven by upregulated gene expression in key metabolic pathways. Furthermore, response surface methodology (RSM) with Box-Behnken design optimized the fermentation medium, yielding 16.67 g/L biomass and 45.91 % protein content, representing 115.37 % and 67.01 % improvements over the unoptimized wild-type control. This study establishes a novel paradigm for constructing high-efficiency microbial protein cell factories via integrated morphological-engineering and fermentation optimization.},
}
@article {pmid40398216,
year = {2025},
author = {Huang, Y and Li, H and Qi, L and Wang, Z and Liu, Z and Wu, R and Chen, Q and Zhu, C and Sun, D and Liu, L and Zhang, L and Feng, G},
title = {NanoCRISPR-assisted biomimetic tissue-equivalent patch regenerates the intervertebral disc by inhibiting endothelial-to-mesenchymal transition.},
journal = {Biomaterials},
volume = {322},
number = {},
pages = {123404},
doi = {10.1016/j.biomaterials.2025.123404},
pmid = {40398216},
issn = {1878-5905},
mesh = {Animals ; Humans ; *Intervertebral Disc/physiology ; *Regeneration ; Intervertebral Disc Degeneration/therapy/pathology ; Tissue Scaffolds/chemistry ; Vascular Endothelial Growth Factor A/metabolism ; *Biomimetic Materials/chemistry/pharmacology ; *Nanoparticles/chemistry ; CRISPR-Cas Systems ; Annulus Fibrosus ; Indoles/chemistry ; Polymers/chemistry ; },
abstract = {The integrity of the intervertebral disc (IVD), an immune-privileged organ protected by the blood-disc barrier, is compromised following annulus fibrosus (AF) injury. This breach facilitates angiogenesis, immune cell infiltration, and inflammation, accelerating intervertebral disc degeneration (IDD) and resulting in various clinical disorders. Current treatments fail to adequately address biological repair of AF defects and angiogenesis. Single-cell RNA sequencing analyses reveal that vascular endothelial growth factor (VEGF), secreted by IDD-associated fibrochondrocytes, is crucial in promoting angiogenesis by inducing endothelial-to-mesenchymal transition (EndoMT). This study proposes a nano-clustered regularly interspaced short palindromic repeats (CRISPR)-assisted AF patch with an aligned, polydopamine-modified nano-lamellae nanofibrous scaffold that replicates the hierarchical structure of natural AF, providing a conducive microenvironment for AF repair. A zeolitic imidazolate framework-8-based nanoCRISPR system encapsulates the CRISPR/CRISPR-associated protein 9 complex to target and eliminate VEGF-mediated angiogenic factors. In vitro studies demonstrate that the nanoCRISPR-assisted patch can enhance AF cell adhesion and migration, promote extracellular matrix deposition, knock out VEGF expression, and inhibit EndoMT. In vivo studies show its significant efficacy in promoting AF repair, inhibiting abnormal angiogenesis, and delaying IDD progression. This study presents a promising approach for structural and biological AF regeneration, addressing physical and angiogenic barriers in IVD regeneration.},
}
@article {pmid40397713,
year = {2025},
author = {Teng, H and Hang, Q and Zheng, C and Yan, Y and Liu, S and Zhao, Y and Deng, Y and Nie, L and Wu, W and Sheldon, M and Yu, Z and Shi, W and Gao, J and Meng, C and Martinez, C and Zhang, J and Yao, F and Sun, Y and Zhao, D and Gan, B and Meng, T and Ma, L},
title = {In vivo CRISPR activation screen identifies acyl-CoA-binding protein as a driver of bone metastasis.},
journal = {Science translational medicine},
volume = {17},
number = {799},
pages = {eado7225},
doi = {10.1126/scitranslmed.ado7225},
pmid = {40397713},
issn = {1946-6242},
mesh = {Animals ; *Bone Neoplasms/secondary/genetics/metabolism/pathology ; Humans ; Cell Line, Tumor ; Mice ; *Diazepam Binding Inhibitor/metabolism/genetics ; Lipid Metabolism ; Fatty Acids/metabolism ; Reactive Oxygen Species/metabolism ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Ferroptosis ; Oxidation-Reduction ; Neoplasm Metastasis ; Female ; *CRISPR-Cas Systems/genetics ; },
abstract = {One of the most common sites of cancer metastasis is to the bone. Bone metastasis is associated with substantial morbidity and mortality, and current therapeutic interventions remain largely palliative. Metastasizing tumor cells need to reprogram their metabolic states to adapt to the nutrient environment of distant organs; however, the role and translational relevance of lipid metabolism in bone metastasis remain unclear. Here, we used an in vivo CRISPR activation screening system coupled with positive selection to identify acyl-coenzyme A (CoA) binding protein (ACBP) as a bone metastasis driver. In nonmetastatic and weakly metastatic cancer cells, overexpression of wild-type ACBP, but not the acyl-CoA-binding deficient mutant, stimulated fatty acid oxidation (FAO) and bone metastasis. Conversely, knockout of ACBP in highly bone metastatic cancer cells abrogated metastatic bone colonization. Mechanistically, ACBP-mediated FAO increased ATP and NADPH production, reduced reactive oxygen species, and inhibited lipid peroxidation and ferroptosis. We found that ACBP expression correlated with metabolic signaling, bone metastatic ability, and poor clinical outcomes. In mouse models, pharmacological blockade of FAO or treatment with a ferroptosis inducer inhibited bone metastasis. Together, our findings reveal the role of lipid metabolism in tumor cells adapting and thriving in the bone and identify ACBP as a key regulator of this process. Agents that target FAO or induce ferroptosis represent a promising therapeutic approach for treating bone metastases.},
}
@article {pmid40397663,
year = {2025},
author = {Gast, K and Baker, S and Borges, AL and Ward, S and Banfield, JF and Barrangou, R},
title = {Metagenome-Derived CRISPR-Cas12a Mining and Characterization.},
journal = {The CRISPR journal},
volume = {8},
number = {3},
pages = {189-204},
doi = {10.1089/crispr.2024.0099},
pmid = {40397663},
issn = {2573-1602},
mesh = {*CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; *Metagenome/genetics ; Escherichia coli/genetics ; Animals ; Cattle ; *CRISPR-Associated Proteins/genetics/metabolism ; Clustered Regularly Interspaced Short Palindromic Repeats ; RNA, Guide, CRISPR-Cas Systems/genetics ; *Endodeoxyribonucleases/genetics/metabolism ; Bacterial Proteins/genetics ; },
abstract = {The advent of clustered regularly interspaced short palindromic repeats (CRISPR)-based technologies has revolutionized genome editing, with continued interest in expanding the CRISPR-associated proteins (Cas) toolbox with diverse, efficient, and specific effectors. CRISPR-Cas12a is a potent, programmable RNA-guided dual nickase, broadly used for genome editing. Here, we mined dairy cow microbial metagenomes for CRISPR-Cas systems, unraveling novel Cas12a enzymes. Using in silico pipelines, we characterized and predicted key drivers of CRISPR-Cas12a activity, encompassing guides and protospacer adjacent motifs for five systems. We next assessed their functional potential in cell-free transcription-translation assays with GFP-based fluorescence readouts. Lastly, we determined their genome editing potential in vivo in Escherichia coli by generating 1 kb knockouts. Unexpectedly, we observed natural sequence variation in the bridge-helix domain of the best-performing candidate and used mutagenesis to alter the activity of Cas12a orthologs, resulting in increased gene editing capabilities of a relatively inefficient candidate. This study illustrates the potential of underexplored metagenomic sequence diversity for the development and refinement of genome editing effectors.},
}
@article {pmid40396759,
year = {2025},
author = {Vergnes, J-B and Roger, B and Iggo, R and Wodrich, H},
title = {Advanced viral genome in vitro Cas9 editing (AdVICE): an overnight method for traceless and limitless manipulation of adenoviral and vector genomes with large transgenes.},
journal = {Journal of virology},
volume = {99},
number = {6},
pages = {e0226524},
pmid = {40396759},
issn = {1098-5514},
support = {ANR-19-CE15-0013//Agence Nationale de la Recherche/ ; ANR-21-CE14-0074//Agence Nationale de la Recherche/ ; 2021-0261/2022-0225//French Cancer Ligue Comite des Landes/ ; 2021-0174//French Cancer Ligue Comite des Pyrenees-Atlantiques/ ; },
mesh = {*Genome, Viral ; *Gene Editing/methods ; *CRISPR-Cas Systems ; *Adenoviridae/genetics ; *Genetic Vectors/genetics ; *Transgenes ; Humans ; },
abstract = {The size and complexity of large viral genomes limit the technical possibilities for genome manipulations in fundamental research and medical or technological applications. State-of-the-art recombineering in bacteria has partially overcome this limit but remains a time-consuming and complex procedure requiring specialist expertise. Here, we describe a simplified and highly efficient in vitro protocol for unlimited and traceless manipulation applicable to large viral genomes from DNA viruses using a combination of CRISPR/Cas9 cleavage and in vitro DNA assembly. We successfully used the protocol to manipulate adenovirus genomes, showing that genome rescue from viruses, insertions, deletions, and mutagenesis can be performed in a simple overnight procedure in a standard laboratory setting without the need for advanced knowledge of molecular biology. Finally, we use our approach to demonstrate the de novo, multi-step construction of an adenovirus vector suitable for delivering very large transgenes for gene editing.IMPORTANCEThe 36 kb size of the adenoviral genome has long been a deterrent to the construction of adenoviral mutants by scientists wishing to study the virus itself or to construct adenoviral vectors for cell biology and gene therapy. Most previous techniques, such as recombineering and yeast gap repair, impress more by their elegance than by their ease. In this paper, we use Cas9 ribonucleoprotein particles (RNPs) to target cleavage to specific sites in an adenoviral plasmid, then repair the break by Gibson assembly. Gibson assembly with synthetic DNA fragments has transformed basic cloning. Combining it with Cas9 RNPs, which act like highly specific restriction enzymes, makes adenoviral mutagenesis as easy as traditional plasmid cloning. We have used the approach to modify multiple sites in the adenoviral genome, but it could be applied to any large DNA virus for which the genome can be cloned in a plasmid.},
}
@article {pmid40394594,
year = {2025},
author = {Guo, J and Shan, Y and Hu, G and Zhu, X and Zhao, K and Wu, T and Qiao, Q and Chi, Y and Cui, L and Ge, Y},
title = {Rapid visual detection of Monkeypox virus by one-step LAMP-CRISPR/Cas12b assay.},
journal = {Virology journal},
volume = {22},
number = {1},
pages = {151},
pmid = {40394594},
issn = {1743-422X},
support = {2023YFC2605100, 2023YFC2605104,BK20231374, BK20221413//National Key R&amp;D Program of China&#xff1b;Natural science foundation of Jiangsu Province/ ; },
mesh = {*Nucleic Acid Amplification Techniques/methods ; *Molecular Diagnostic Techniques/methods ; Sensitivity and Specificity ; *CRISPR-Cas Systems ; *Monkeypox virus/isolation &amp; purification/genetics ; Humans ; *Mpox, Monkeypox/diagnosis/virology ; Limit of Detection ; },
abstract = {BACKGROUND: Monkeypox virus (MPXV) infection has garnered significant global attention due to its rising incidence and substantial public health implications. A rapid, sensitive, and accurate diagnostic method is urgently required to enable early intervention and effective management of MPXV outbreaks.<br><br>RESULTS: In this study, we developed a novel one-step assay that integrates loop-mediated isothermal amplification (LAMP) with CRISPR/Cas12b in one-pot for the detection of MPXV. The entire detection process did not require opening the lid of the reaction tube and could be completed within 40&#xa0;min using extracted viral nucleic acids, which is faster than real-time quantitative PCR (qPCR). And the results could be interpreted through either real-time fluorescence or naked-eye visualization. The limit of detection (LOD) of the assay was demonstrated to be 6.5 copies per reaction and no cross-reactivity with other pathogens such as HSV, EBV, CVA16, EV-A71, and MV was found. Furthermore, when evaluated using 113 clinical samples, the assay achieved 100% sensitivity (71/71) and 100% specificity (42/42) compared to the qPCR.<br><br>CONCLUSIONS: In resource-limited settings, our method requires only a portable heat block or water bath and a blue light or ultraviolet flashlight for visual detection of MPXV, making it highly accessible. The integration of LAMP and CRISPR/Cas12b provides a robust, user-friendly platform for point-of-care testing, with promising potential for the rapid molecular diagnosis of infectious diseases.},
}
@article {pmid40394064,
year = {2025},
author = {Gu, S and Bodai, Z and Anderson, RA and So, HYA and Cowan, QT and Komor, AC},
title = {Elucidating the genetic mechanisms governing cytosine base editing outcomes through CRISPRi screens.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {4685},
pmid = {40394064},
issn = {2041-1723},
support = {T32 GM146648/GM/NIGMS NIH HHS/United States ; T32GM146648//U.S. Department of Health &amp; Human Services | NIH | National Institute of General Medical Sciences (NIGMS)/ ; 27975//Research Corporation for Science Advancement (Research Corporation)/ ; T32GM008326//U.S. Department of Health &amp; Human Services | NIH | National Institute of General Medical Sciences (NIGMS)/ ; MCB-2048207//National Science Foundation (NSF)/ ; T32 GM008326/GM/NIGMS NIH HHS/United States ; },
mesh = {*Cytosine/metabolism ; *Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; Humans ; DNA Repair/genetics ; DNA Mismatch Repair/genetics ; HEK293 Cells ; Ubiquitin-Protein Ligases/metabolism/genetics ; },
abstract = {Cytosine base editors enable programmable and efficient genome editing using an intermediate featuring a U•G mismatch across from a DNA nick. This intermediate facilitates two major outcomes, C•G to T•A and C•G to G•C point mutations, and it is not currently well-understood which DNA repair factors are involved. Here, we couple reporters for cytosine base editing activity with knockdown of 2015 DNA processing genes to identify genes involved in these two outcomes. Our data suggest that mismatch repair factors facilitate C•G to T•A outcomes, while C•G to G•C outcomes are mediated by RFWD3, an E3 ubiquitin ligase. We also propose that XPF, a 3'-flap endonuclease, and LIG3, a DNA ligase, are involved in repairing the intermediate back to the original C•G base pair. Our results demonstrate that competition and collaboration among different DNA repair pathways shape cytosine base editing outcomes.},
}
@article {pmid40393960,
year = {2025},
author = {Liu, Z and Luan, X and Lu, Q and Qin, S and Zeng, F and Li, Z and He, B and Song, Y},
title = {Reactive oxygen species responsive nanomotors for gene edited metabolic disruption and immunotherapy.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {4708},
pmid = {40393960},
issn = {2041-1723},
mesh = {*Reactive Oxygen Species/metabolism ; *Immunotherapy/methods ; *Gene Editing/methods ; Humans ; Animals ; Mice ; CRISPR-Cas Systems ; Cell Line, Tumor ; Tumor Microenvironment ; Heme Oxygenase-1/metabolism ; *Neoplasms/therapy/immunology/genetics ; CD8-Positive T-Lymphocytes/immunology ; Glycolysis ; Nanoparticles/chemistry ; Female ; },
abstract = {While gene-editing-based tumor therapy holds promise, conventional passive-diffusion vectors face limited penetration in dense solid tumors. Here, we developed a ROS-driven gene editing nanomotor (RDN@PL), which takes hemin as the core and encapsulates CRISPR/Cas9 plasmids targeting LDHA (A glycolysis key enzyme). In tumor microenvironments, RDN@PL consumes extracellular ROS to fuel self-diffusiophoresis, achieving higher intratumoral accumulation than passive particles. Upon internalization, heme oxygenase-1 (HO-1) degrades RDN@PL, releasing CO and plasmids. LDHA knockout suppresses glycolysis while CO elevates mitochondrial ROS, which triggers apoptosis by disrupting metabolism and enhancing immunity. Simultaneously, extracellular ROS depletion by non-internalized nanomotors reverses immunogenic cell death (ICD) inhibition, enhancing CD8+ T cell infiltration in tumor. The Janus nanomotor enables extracellular ROS scavenging and intracellular ROS increment via HO-1-responsive cargo release and gene editing. This multi-level intervention strategy demonstrates 93.9 % tumor growth suppression in solid tumor models, providing a blueprint for engineering intelligent nanovesicles in precision oncology.},
}
@article {pmid40393243,
year = {2025},
author = {Li, H and Lian, S and Zhang, Z and Bi, W and Meng, Q and Ding, Q},
title = {The CRISPR/Cas13a-assisted electrochemiluminescence sensing device combined with entropy-driven and hybrid chain reaction nucleic acid amplification techniques for ultra-sensitive analysis of brain natriuretic peptide.},
journal = {Talanta},
volume = {295},
number = {},
pages = {128310},
doi = {10.1016/j.talanta.2025.128310},
pmid = {40393243},
issn = {1873-3573},
abstract = {Brain natriuretic peptide (BNP) is considered a reliable marker of heart failure disease, and its timely detection can provide important pathological information to prevent or treat heart failure. In this article, an electrochemiluminescence (ECL) sensing device based on a boron carbon nitride/gold nanoparticle (BCN/AuNPs) complex is developed to determine BNP. Prominently, the CRISPR/CAS 13a enzyme reverse cleavage mode, the entropy-driven and hammer hybridization chain reaction processes were involved in the entire detection scheme. Ultimately, with multiple reaction methods and amplification reactions of nucleic acids, this ECL sensing device is able to achieve a detection limit as low as of 0.03 pg/mL and linear range from 0.1 pg/mL to 30 ng/mL for BNP. In addition, the ECL sensing device based on BCN/AuNPs complex obtained satisfactory stability and specificity, and can also be extended to the detection of other pathological markers.},
}
@article {pmid40390640,
year = {2025},
author = {Zhang, C and Chen, Y and Chen, X and Lin, X and Huang, Z and Zhang, L and Liu, R and Lv, Y},
title = {Single nanoparticle analysis-based CRISPR/Cas12 bioassay for amplification-free HIV detection.},
journal = {Chemical communications (Cambridge, England)},
volume = {61},
number = {48},
pages = {8759-8762},
doi = {10.1039/d5cc01716e},
pmid = {40390640},
issn = {1364-548X},
mesh = {Humans ; *CRISPR-Cas Systems ; Limit of Detection ; *Nanoparticles/chemistry ; *Biological Assay/methods ; *HIV/isolation &amp; purification ; *Biosensing Techniques/methods ; *HIV-1/isolation &amp; purification ; *HIV Infections/diagnosis ; *Metal Nanoparticles/chemistry ; },
abstract = {To reduce the "window period" in HIV detection, most analytical methods require additional enzymes for signal amplification. Exempting challenges like primer interference and false positives in amplification strategies, we developed an amplification-free bioassay that uses CRISPR's potent cleavage activity and the competent sensitivity of single-nanoparticle analysis. An attomolar detection limit was achieved with adequate selectivity. Serum and cell tests confirm the bioassay's accurate and sensitive HIV detection.},
}
@article {pmid40389868,
year = {2025},
author = {Amoroso, CG and Andolfo, G},
title = {Hazelnut allergome overview and Cor a gRNAs identification.},
journal = {BMC plant biology},
volume = {25},
number = {1},
pages = {661},
pmid = {40389868},
issn = {1471-2229},
support = {E53D23020840001P//Ministero dell'Istruzione, dell'Universit&#xe0; e della Ricerca/ ; },
mesh = {*Corylus/genetics/immunology ; *Allergens/genetics/immunology ; *Plant Proteins/genetics/immunology ; Genome, Plant ; CRISPR-Cas Systems ; Phylogeny ; *Antigens, Plant/genetics/immunology ; },
abstract = {BACKGROUND: Corylus species (hazelnuts) are a valuable source of nutrients and are widely consumed worldwide. Nevertheless, Corylus avellana (Cor a) contains 13 allergens (Cor a 1, Cor a 2, Cor a 6, Cor a 8, Cor a 9, Cor a 10, Cor a 11, Cor a 12, Cor a 13, Cor a 14, Cor a 15, Cor a 16, and Cor a TLP) that have been deposited into the official database (WHO/IUIS) for allergen nomenclature. The recent availability of several Corylus genomes provided opportunities to explore allergome variability, and thus to develop hypoallergenic varieties using modern biotech approaches. Certainly, the identification of CRISPR-Cas9 guide RNA (gRNA) is a pivotal step in achieving this goal. User-friendly web tools include limited reference genomes to design CRISPR-Cas9 gRNAs, while bioinformatic software for custom analysis require advanced command-line skills.<br><br>RESULTS: This work explored the evolutionary trajectories of allergenic Cor a homologs in C. avellana, C. americana, C. heterophylla, and C. mandshurica genome assemblies. 52 Cor a orthologs were found in the analyzed species, and a recent tandem duplication of Cor a 1 was found in C. americana. Three new gene models were predicted in C. avellana and C. mandshurica for Cor a 16 and Cor a 10. Additionally, we identified 56 Cor a isoallergens, of which ten Cor a isoforms. Furthermore, phylogenetic analysis sheds light on the evolutionary dynamics of three hazelnut allergens revealing the evolutionary complexity of Cor a 1, Cor a 2, and Cor a TLP within the Corylus genus. A list of multiple gRNAs designed for the CRISPR-Cas9 system was provided for the singular and multiple silencing of Cor a homologs in each Corylus genome.<br><br>CONCLUSIONS: This study enhances our knowledge on the evolutionary path of Cor a allergens among Corylus species and provides highly accurate on-target guides targeting hazelnut allergome.},
}
@article {pmid40389577,
year = {2025},
author = {Lv, C and Zhang, F and Ren, L and Zhu, P and Cheng, X and Yang, X and Chen, C and Liu, B},
title = {Rapid visual detection assay for Bactrocera dorsalis (Hendel) using recombinase polymerase amplification and CRISPR/Cas12b.},
journal = {Scientific reports},
volume = {15},
number = {1},
pages = {17328},
pmid = {40389577},
issn = {2045-2322},
support = {no. 62272060 and 62272061//National Natural Science Foundation of China/ ; no. 62272060 and 62272061//National Natural Science Foundation of China/ ; no. 62272060 and 62272061//National Natural Science Foundation of China/ ; no. 62272060 and 62272061//National Natural Science Foundation of China/ ; },
mesh = {Animals ; *Tephritidae/genetics ; *CRISPR-Cas Systems ; *Recombinases/metabolism/genetics ; *Nucleic Acid Amplification Techniques/methods ; Sensitivity and Specificity ; },
abstract = {The oriental fruit fly Bactrocera dorsalis (Hendel) is considered as a quarantine pest in many countries and regions. Challenges remain in distinguishing this species with morphological similarities, especially in relevant development stages. In recent years, CRISPR/Cas12b genetic diagnostics has seen rapid advancements and offers an efficient tool for the identification of pathogens, viruses, and other genetic targets. Here we developed a new and rapid visual detection assay of B. dorsalis using recombinase polymerase amplification (RPA) and the CRISPR/Cas12b system. The system can detect different developmental stages of B. dorsalis within 30-35 min at 43 ℃ and the results are easily observed by the naked eye based on the color change in the tube during the reaction. The specificity and high sensitivity of this method was demonstrated, allowing for detection from 3.2 pg µL[- 1] of DNA. With crude DNA, this diagnostic system successfully identified B. dorsalis by holding the reaction tubes in the hand. Our study demonstrates that RPA-CRISPR/Cas12b visualization system is effective to detect B. dorsalis rapidly and accurately. This approach can be applied for monitoring and identification of other pests in border and relevant locations, preventing biological invasions and ensuring pest control.},
}
@article {pmid40389480,
year = {2025},
author = {Troester, S and Eder, T and Wukowits, N and Piontek, M and Fernández-Pernas, P and Schmoellerl, J and Haladik, B and Manhart, G and Allram, M and Maurer-Granofszky, M and Scheidegger, N and Nebral, K and Superti-Furga, G and Meisel, R and Bornhauser, B and Valent, P and Dworzak, MN and Zuber, J and Boztug, K and Grebien, F},
title = {Transcriptional and epigenetic rewiring by the NUP98::KDM5A fusion oncoprotein directly activates CDK12.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {4656},
pmid = {40389480},
issn = {2041-1723},
support = {P35628//Austrian Science Fund (Fonds zur F&#xf6;rderung der Wissenschaftlichen Forschung)/ ; P35298//Austrian Science Fund (Fonds zur F&#xf6;rderung der Wissenschaftlichen Forschung)/ ; TAI490//Austrian Science Fund (Fonds zur F&#xf6;rderung der Wissenschaftlichen Forschung)/ ; },
mesh = {Humans ; *Nuclear Pore Complex Proteins/metabolism/genetics ; *Oncogene Proteins, Fusion/metabolism/genetics ; *Leukemia, Myeloid, Acute/genetics/metabolism/pathology ; *Cyclin-Dependent Kinases/metabolism/genetics ; *Epigenesis, Genetic ; Cell Line, Tumor ; Animals ; Transcription, Genetic ; Mice ; Gene Expression Regulation, Leukemic ; DNA Damage ; CRISPR-Cas Systems ; Retinoblastoma-Binding Protein 2 ; },
abstract = {Nucleoporin 98 (NUP98) fusion oncoproteins are strong drivers of pediatric acute myeloid leukemia (AML) with poor prognosis. Here we show that NUP98 fusion-expressing AML harbors an epigenetic signature that is characterized by increased accessibility of hematopoietic stem cell genes and enrichment of activating histone marks. We employ an AML model for ligand-induced degradation of the NUP98::KDM5A fusion oncoprotein to identify epigenetic programs and transcriptional targets that are directly regulated by NUP98::KDM5A through CUT&amp;Tag and nascent RNA-seq. Orthogonal genome-wide CRISPR/Cas9 screening identifies 12 direct NUP98::KDM5A target genes, which are essential for AML cell growth. Among these, we validate cyclin-dependent kinase 12 (CDK12) as a druggable vulnerability in NUP98::KDM5A-expressing AML. In line with its role in the transcription of DNA damage repair genes, small-molecule-mediated CDK12 inactivation causes increased DNA damage, leading to AML cell death. Altogether, we show that NUP98::KDM5A directly regulates a core set of essential target genes and reveal CDK12 as an actionable vulnerability in AML with oncogenic NUP98 fusions.},
}
@article {pmid40389438,
year = {2025},
author = {Pandey, AC and Bezney, J and DeAscanis, D and Kirsch, EB and Ahmed, F and Crinklaw, A and Choudhary, KS and Mandala, T and Deason, J and Hamidi, JS and Siddique, A and Ranganathan, S and Brown, K and Armstrong, J and Head, S and Ordoukhanian, P and Steinmetz, LM and Topol, EJ},
title = {A CRISPR/Cas9-based enhancement of high-throughput single-cell transcriptomics.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {4664},
pmid = {40389438},
issn = {2041-1723},
support = {UL1TR002550//U.S. Department of Health &amp; Human Services | NIH | National Center for Advancing Translational Sciences (NCATS)/ ; 20CDA35310187//American Heart Association (American Heart Association, Inc.)/ ; KL2 TR002552/TR/NCATS NIH HHS/United States ; UL1 TR002550/TR/NCATS NIH HHS/United States ; KL2TR002552//U.S. Department of Health &amp; Human Services | NIH | National Center for Advancing Translational Sciences (NCATS)/ ; },
mesh = {*Single-Cell Analysis/methods ; *CRISPR-Cas Systems/genetics ; Humans ; *Transcriptome/genetics ; *Gene Expression Profiling/methods ; Animals ; High-Throughput Nucleotide Sequencing/methods ; Muscle, Smooth, Vascular/cytology/metabolism ; RNA-Seq/methods ; Sequence Analysis, RNA/methods ; Mice ; Myocytes, Smooth Muscle/metabolism ; },
abstract = {Single-cell RNA-seq (scRNAseq) struggles to capture the cellular heterogeneity of transcripts within individual cells due to the prevalence of highly abundant and ubiquitous transcripts, which can obscure the detection of biologically distinct transcripts expressed up to several orders of magnitude lower levels. To address this challenge, here we introduce single-cell CRISPRclean (scCLEAN), a molecular method that globally recomposes scRNAseq libraries, providing a benefit that cannot be recapitulated with deeper sequencing. scCLEAN utilizes the programmability of CRISPR/Cas9 to target and remove less than 1% of the transcriptome while redistributing approximately half of reads, shifting the focus toward less abundant transcripts. We experimentally apply scCLEAN to both heterogeneous immune cells and homogenous vascular smooth muscle cells to demonstrate its ability to uncover biological signatures in different biological contexts. We further emphasize scCLEAN's versatility by applying it to a third-generation sequencing method, single-cell MAS-Seq, to increase transcript-level detection and discovery. Here we show the possible utility of scCLEAN across a wide array of human tissues and cell types, indicating which contexts this technology proves beneficial and those in which its application is not advisable.},
}
@article {pmid40389165,
year = {2025},
author = {Öktem, M and Nguyen, TH and Bosman, EDC and Fens, MHAM and Caiazzo, M and Mastrobattista, E and Lei, Z and de Jong, OG},
title = {Lipopeptide-mediated delivery of CRISPR/Cas9 ribonucleoprotein complexes for gene editing and correction.},
journal = {Journal of controlled release : official journal of the Controlled Release Society},
volume = {383},
number = {},
pages = {113854},
doi = {10.1016/j.jconrel.2025.113854},
pmid = {40389165},
issn = {1873-4995},
mesh = {*Gene Editing/methods ; Animals ; *CRISPR-Cas Systems ; *Ribonucleoproteins/administration &amp; dosage/genetics/chemistry ; Humans ; *Lipopeptides/chemistry/administration &amp; dosage ; *CRISPR-Associated Protein 9/administration &amp; dosage/genetics ; Mice ; *Cell-Penetrating Peptides/chemistry/administration &amp; dosage ; HEK293 Cells ; Gene Transfer Techniques ; },
abstract = {CRISPR/Cas gene editing is a highly promising technology for the treatment and even potential cure of genetic diseases. One of the major challenges for its therapeutic use is finding safe and effective vehicles for intracellular delivery of the CRISPR/Cas9 ribonucleoprotein (RNP) complex. In this study, we tested and characterized a series of novel fatty acid-modified versions of a previously reported Cas9 RNP carrier, consisting of a complex of the cell-penetrating peptide (CPP) LAH5 with Cas9 RNP and homology-directed DNA repair templates. Comparative experiments demonstrated that RNP/peptide nanocomplexes showed various improvements depending on the type of fatty acid modification. These improvements included enhanced stability in serum, improved membrane disruption capability and increased transfection efficacy. Cas9 RNP/oleic acid LAH5 peptide nanocomplexes showed the overall best performance for both gene editing and correction. Moreover, Cas9 RNP/oleic acid LAH5 nanocomplexes significantly protected the Cas9 protein cargo from enzymatic protease digestion. In addition, in vivo testing demonstrated successful gene editing after intramuscular administration. Despite the inherent barriers of the tightly organized muscle tissues, we achieved approximately 10 % gene editing in the skeletal muscle tissues when targeting the CAG-tdTomato locus in the transgenic Ai9 Cre-LoxP reporter mouse strain and 7 % gene editing when targeting the Ccr5 gene, without any observable short-term toxicity. In conclusion, the oleic acid-modified LAH5 peptide is an effective delivery platform for direct Cas9/RNP delivery, and holds great potential for the development of new CRISPR/Cas9-based therapeutic applications for the treatment of genetic diseases.},
}
@article {pmid40388365,
year = {2025},
author = {Wang, Y and Hu, J and Zhang, J},
title = {Evaluation of Abnormal Growth-related Genes of Hematopoietic Stem and Progenitor Cells by Combining CRISPR/Cas9 Technology with Cell Counting.},
journal = {Journal of visualized experiments : JoVE},
volume = {},
number = {219},
pages = {},
doi = {10.3791/67508},
pmid = {40388365},
issn = {1940-087X},
mesh = {Animals ; *Hematopoietic Stem Cells/cytology/physiology/metabolism ; Mice ; *CRISPR-Cas Systems ; Tumor Suppressor Protein p53/genetics ; Cell Proliferation ; Transfection ; },
abstract = {Hematopoietic stem cells possess the ability for long-term self-renewal and the potential to differentiate into various types of mature blood cells. However, the accumulation of cancerous mutations in hematopoietic stem and progenitor cells (HSPCs) can block normal differentiation, induce aberrant proliferation, and ultimately lead to leukemogenesis. To identify and/or evaluate the cancerous mutations, we integrated CRISPR/Cas9 technology with the Cell Counting Kit-8 (CCK-8) assay to investigate a model gene Trp53, that is essential for abnormal proliferative ability of HSPCs. Specifically, bone marrow cells enriched for HSPCs from Cas9 mice were harvested and then subjected to viral transfection with single-guide RNAs (sgRNAs) targeting one or several candidate genes to introduce genetic alterations in HSPCs. Then, a CCK-8 assay was performed to investigate the proliferative capacity of transfected HSPCs. The sgRNA targeting efficiency was confirmed by a Tracking of Indels by Decomposition assay. These identified genes may play crucial roles in leukemogenesis and could serve as potential therapeutic targets.},
}
@article {pmid40387535,
year = {2025},
author = {Liu, S and Yu, T and Song, L and Kalantar-Zadeh, K and Liu, G},
title = {CRISPR/Cas on Microfluidic Paper-Based Analytical Devices for Point-of-Care Screening of Cervical Cancer.},
journal = {ACS sensors},
volume = {},
number = {},
pages = {},
doi = {10.1021/acssensors.5c00863},
pmid = {40387535},
issn = {2379-3694},
abstract = {Highly sensitive point-of-care early screening for high-risk human papillomavirus (HPV) infections is urgently needed, particularly in resource-limited settings. Nucleic acid amplification methods, especially CRISPR/Cas-based biosensors, have emerged as promising tools for sensitive HPV detection; however, current approaches typically rely on tedious tube-based formats coupled with lateral flow assays for signal readout in point-of-care testing (POCT). Here, we developed customized microfluidic paper-based analytical devices (μPADs) with valves that seamlessly integrated recombinase polymerase amplification (RPA) with CRISPR/Cas12a biosensing (RPA-CRISPR/Cas12a) on the filter paper substrate. This innovation achieved sensitive and cost-effective high-risk HPV detection in POCT. The RPA-CRISPR/Cas12a system with a linear reporter on μPADs, enabled fluorescence detection of the E7 gene, achieving a sensitivity of 1 pM at approximately 1 h. The sensitivity was further enhanced by introducing a circular reporter into the fluorescence-based RPA-CRISPR/Cas12a system on μPADs, enabling detection of the E7 gene with a detection limit of 1 fM and an assay time of 35 min. The system was validated using 50 cervical swab clinical samples, demonstrating 95% sensitivity and 100% specificity when compared to qPCR. This sample-to-answer detection platform holds significant promise for early screening of high-risk HPV infections in point-of-care scenarios.},
}
@article {pmid40387367,
year = {2025},
author = {Li, J and Liu, Y and Jiang, J and Chen, F and Zhang, N and Kang, X and Liu, L and Wang, Y and Xia, Q and Zhu, C and Kuang, D},
title = {Type I-E* CRISPR-Cas of Klebsiella pneumoniae upregulates bacterial virulence by targeting endogenous histidine utilization system.},
journal = {mSphere},
volume = {},
number = {},
pages = {e0021525},
doi = {10.1128/msphere.00215-25},
pmid = {40387367},
issn = {2379-5042},
abstract = {Klebsiella pneumoniae is a globally recognized microbial pathogen with significant clinical impact. The bacterium harbors the clustered regularly interspaced short palindromic repeats (CRISPR)-Cas systems, which provide adaptive immunity against invading foreign nucleic acids. Recent studies suggest that certain CRISPR-Cas systems can regulate endogenous genes, influencing bacterial virulence. However, their role in regulating pathogenicity in K. pneumoniae remains poorly understood. This study investigates the regulatory role of the type I-E* CRISPR-Cas system in a hypervirulent K. pneumoniae strain, focusing on its impact on histidine metabolism and pathogenicity. Transcriptome analyses identified differentially expressed genes (DEGs) between the casABECD-deletion and wild-type strains, including significant upregulation of the histidine utilization (Hut) operon and downregulation of biofilm-related genes. These molecular changes resulted in enhanced histidine metabolic activity, reduced biofilm formation, attenuated virulence in A549 lung epithelial cells, and improved survival of Galleria mellonella, as validated through phenotypic and virulence assays. Our bioinformatic analysis indicated that the CRISPR-Cas system in K. pneumoniae targets the hutT sequence, which is part of the Hut operon. Furthermore, the overexpression of hutT mitigated CRISPR-Cas-mediated repression of the Hut operon, as observed in virulence assays, while simultaneous deletion of hutH and casABECD restored the reduced virulence in the ΔcasABECD strain. Additionally, deletion of casABECD significantly enhances the growth of the strain in medium with histidine as the sole carbon source, highlighting the intricate regulatory role of the CRISPR-Cas system in metabolic adaptation. Collectively, these findings uncover a novel role for the CRISPR-Cas system in regulating metabolic pathways and virulence in hypervirulent K. pneumoniae.IMPORTANCEClustered regularly interspaced short palindromic repeats (CRISPR)-Cas systems are primarily recognized for their roles in adaptive immunity against foreign genetic elements in bacteria. However, emerging evidence indicates that these systems can also regulate endogenous genes, thereby influencing bacterial physiology and virulence. In this study, we demonstrate that the type I-E* CRISPR-Cas system in Klebsiella pneumoniae targets the hutT gene, a critical component of the histidine utilization (Hut) pathway. This targeting potentially impacts hutT transcription and alters the expression of other hut genes, ultimately enhancing bacterial virulence. Our findings reveal a previously unrecognized regulatory mechanism through which CRISPR-Cas systems facilitate metabolic adaptation and pathogenicity in K. pneumoniae. This study broadens our understanding of the multifaceted roles of CRISPR-Cas systems in bacterial physiology and pathobiology, with implications for clinically relevant pathogens.},
}
@article {pmid40387084,
year = {2025},
author = {Belato, HB and Knight, AL and D'Ordine, AM and Pindi, C and Fan, Z and Luo, J and Palermo, G and Jogl, G and Lisi, GP},
title = {Structural and dynamic impacts of single-atom disruptions to guide RNA interactions within the recognition lobe of Geobacillus stearothermophilus Cas9.},
journal = {eLife},
volume = {13},
number = {},
pages = {},
pmid = {40387084},
issn = {2050-084X},
support = {P30 GM133893/GM/NIGMS NIH HHS/United States ; R01GM141329/NH/NIH HHS/United States ; R01GM136815/NH/NIH HHS/United States ; R01 GM136815/GM/NIGMS NIH HHS/United States ; TC-24-063//Camille and Henry Dreyfus Foundation/ ; CHE2144823//National Science Foundation/ ; R01 GM141329/GM/NIGMS NIH HHS/United States ; MCB2143760//National Science Foundation/ ; FG-2023-20431//Alfred P. Sloan Foundation/ ; },
mesh = {*Geobacillus stearothermophilus/enzymology/genetics ; *RNA, Guide, CRISPR-Cas Systems/metabolism/genetics/chemistry ; *CRISPR-Associated Protein 9/metabolism/chemistry/genetics ; *Bacterial Proteins/metabolism/chemistry/genetics ; CRISPR-Cas Systems ; Protein Conformation ; },
abstract = {The intuitive manipulation of specific amino acids to alter the activity or specificity of CRISPR-Cas9 has been a topic of great interest. As a large multi-domain RNA-guided endonuclease, the intricate molecular crosstalk within the Cas9 protein hinges on its conformational dynamics, but a comprehensive understanding of the extent and timescale of the motions that drive its allosteric function and association with nucleic acids remains elusive. Here, we investigated the structure and multi-timescale molecular motions of the recognition (Rec) lobe of GeoCas9, a thermophilic Cas9 from Geobacillus stearothermophilus. Our results provide new atomic details about the GeoRec subdomains (GeoRec1, GeoRec2) and the full-length domain in solution. Two rationally designed mutants, K267E and R332A, enhanced and redistributed micro-millisecond flexibility throughout GeoRec, and NMR studies of the interaction between GeoRec and its guide RNA showed that mutations reduced this affinity and the stability of the ribonucleoprotein complex. Despite measured biophysical differences due to the mutations, DNA cleavage assays reveal no functional differences in on-target activity, and similar specificity. These data suggest that guide RNA interactions can be tuned at the biophysical level in the absence of major functional losses but also raise questions about the underlying mechanism of GeoCas9, since analogous single-point mutations have significantly impacted on- and off-target DNA editing in mesophilic Streptococcus pyogenes Cas9. A K267E/R332A double mutant did also did not enhance GeoCas9 specificity, highlighting the robust tolerance of mutations to the Rec lobe of GeoCas9 and species-dependent complexity of Rec across Cas9 paralogs. Ultimately, this work provides an avenue by which to modulate the structure, motion, and guide RNA interactions at the level of the Rec lobe of GeoCas9, setting the stage for future studies of GeoCas9 variants and their effect on its allosteric mechanism.},
}
@article {pmid40382983,
year = {2025},
author = {Pulito, C and Vaccarella, S and Palcau, AC and Ganci, F and Brandi, R and Frascolla, C and Sacconi, A and Canu, V and Benedetti, A and De Pascale, V and Donzelli, S and Fisch, AS and Manciocco, V and Covello, R and Pimpinelli, F and Morrone, A and Fazi, F and Pellini, R and Muti, P and Meens, J and Karamboulas, C and Nichols, AC and Strano, S and Klinghammer, K and Tinhofer, I and Ailles, L and Fontemaggi, G and Blandino, G},
title = {MicroRNA-mediated PTEN downregulation as a novel non-genetic mechanism of acquired resistance to PI3Kα inhibitors of head &amp; neck squamous cell carcinoma.},
journal = {Drug resistance updates : reviews and commentaries in antimicrobial and anticancer chemotherapy},
volume = {81},
number = {},
pages = {101251},
doi = {10.1016/j.drup.2025.101251},
pmid = {40382983},
issn = {1532-2084},
mesh = {Humans ; *PTEN Phosphohydrolase/genetics/metabolism ; *MicroRNAs/genetics ; *Drug Resistance, Neoplasm/genetics/drug effects ; *Squamous Cell Carcinoma of Head and Neck/drug therapy/genetics/pathology ; Down-Regulation ; *Head and Neck Neoplasms/drug therapy/genetics/pathology ; Cell Line, Tumor ; *Class I Phosphatidylinositol 3-Kinases/antagonists &amp; inhibitors/genetics ; Gene Expression Regulation, Neoplastic/drug effects ; Thiazoles/pharmacology/therapeutic use ; Polo-Like Kinase 1 ; Cell Cycle Proteins/antagonists &amp; inhibitors/metabolism ; *Phosphoinositide-3 Kinase Inhibitors/pharmacology ; Protein Serine-Threonine Kinases/antagonists &amp; inhibitors/metabolism ; Proto-Oncogene Proteins/antagonists &amp; inhibitors/metabolism ; Animals ; Signal Transduction/drug effects ; CRISPR-Cas Systems ; Phosphorylation ; RNA, Long Noncoding ; },
abstract = {AIMS: Head and neck squamous cell carcinomas (HNSCCs) frequently harbor alterations in the PI3K signalling axis and, particularly, in the PIK3CA gene. The promising rationale of using PI3K inhibitors for the treatment of HNSCC has, however, clashed with the spontaneous development of resistance over time.<br><br>METHODS: To identify valuable targets for overcoming acquired resistance to PI3K&#x3b1; inhibitors in HNSCC, we performed microRNA profiling on a cohort of HNSCC PDXs that were treated with alpelisib, including both responsive and resistant tumors. Using CRISPR/Cas9, siRNA, and PTEN-/- isogenic and alpelisib-resistant cell models, we examined the role of PTEN in resistance acquisition. Phospho-proteomic analysis identified PTEN-dependent phosphorylation events, while PI3K&#x3b1; inhibitor-resistant organoids were used to assess PLK1 inhibitor efficacy.<br><br>RESULTS: We identified microRNAs altered in resistant PDXs, including members of the miR-17-92 cluster. Mechanistically, we observed that the hyperactive c-Myc was recruited to MIR17HG regulatory regions in alpelisib-resistant cells, sustaining miR-17-5p, miR-19b-3p, and miR-20a-5p expression, which downregulated PTEN. PTEN knockout or depletion conferred alpelisib resistance in HNSCC cells. We identified PTEN-dependent phosphorylation events, such as p-PLK1-T210, involved in resistance. Interestingly, pharmacological inhibition of PLK1 strongly reduced the viability of PI3K&#x3b1;-resistant organoids derived from HNSCC PDXs and cell line models.<br><br>CONCLUSION: Overall, this study unveils a novel, microRNA-driven, non-genetic mechanism contributing to acquired resistance to PI3K&#x3b1; inhibitors in HNSCC. Indeed, linking hyperactive c-Myc to sustain miR-17-92 expression and consequent PTEN downregulation, we also propose that targeting PTEN-dependent downstream effectors, such as PLK1, may offer a powerful therapeutic strategy for resistant HNSCC.},
}
@article {pmid40380978,
year = {2025},
author = {Pipes, BL and Nishiguchi, MK},
title = {Generation and validation of a versatile inducible multiplex CRISPRi system to examine bacterial regulation in the Euprymna-Vibrio fischeri symbiosis.},
journal = {Archives of microbiology},
volume = {207},
number = {7},
pages = {147},
pmid = {40380978},
issn = {1432-072X},
support = {EXO 80NSSC18K1053//National Aeronautical and Space Adminstration/ ; EXO 80NSSC18K1053//National Aeronautical and Space Adminstration/ ; DBI-2214028//National Science Foundation/ ; DBI-2214028//National Science Foundation/ ; },
mesh = {*Aliivibrio fischeri/genetics/physiology ; *Symbiosis/genetics ; Animals ; *Gene Expression Regulation, Bacterial ; *CRISPR-Cas Systems ; *Decapodiformes/microbiology ; Genetic Vectors ; Bacterial Proteins/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats ; Plasmids/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; },
abstract = {The Vibrio fischeri-Euprymna scolopes symbiosis has become a powerful animal-microbe model system to examine the genetic underpinnings of symbiont development and regulation. Although there has been a number of elegant bacterial genetic technologies developed to examine this symbiosis, there is still a need to develop more sophisticated methodologies to better understand complex regulatory pathways that lie within the association. Therefore, we have developed a suite of CRISPR interference (CRISPRi) vectors for inducible repression of specific V. fischeri genes associated with symbiotic competence. The suite utilizes both Tn7-integrating and shuttle vector plasmids that allow for inducible expression of CRISPRi dCas9 protein along with single-guide RNAs (sgRNA) modules. We validated this CRISPRi tool suite by targeting both exogenous (an introduced mRFP reporter) and endogenous genes (luxC in the bioluminescence producing lux operon, and flrA, the major regulatory gene controlling flagella production). The suite includes shuttle vectors expressing both single and multiple sgRNAs complementary to the non-template strand of multiple targeted genetic loci, which were effective in inducible gene repression, with significant reductions in targeted gene expression levels. V. fischeri cells harboring a version of this system targeting the luxC gene and suppressing the production of luminescence were used to experimentally validate the hypothesis that continuous luminescence must be produced by the symbiont in order to maintain the symbiosis at time points longer than the known 24-h limit. This robust new CRISPRi genetic toolset has broad utility and will enhance the study of V. fischeri genes, bypassing the need for gene disruptions by standard techniques of allelic knockout-complementation-exchange and the ability to visualize symbiotic regulation in vivo.},
}
@article {pmid40380066,
year = {2025},
author = {Garduño-Tamayo, YQ and Acosta-García, G},
title = {Gene Silencing in Plants Through Exogenous Application of miRNAs.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2900},
number = {},
pages = {249-255},
pmid = {40380066},
issn = {1940-6029},
mesh = {*MicroRNAs/genetics ; *Gene Silencing ; *Plants/genetics ; Gene Expression Regulation, Plant ; CRISPR-Cas Systems ; RNA Interference ; Gene Editing/methods ; Plants, Genetically Modified/genetics ; RNA, Plant/genetics ; },
abstract = {Although omics technologies allow us to identify genes involved in various biological processes, we still rely on the analysis of the function of individual genes. Studies of the function of a specific gene include technologies such as gene silencing by RNAi or genome editing by CRISPR-cas9. However, most of them depend on the availability of transformation methods, so they are not established for all plants. In this chapter, we report a protocol that involves the exogenous application of miRNAs for the specific silencing of genes of interest. The advantage of this protocol is that it does not require a transformation event and can be applied to a certain tissue or developmental stage. This novel technology facilitates the analysis of specific gene functions in crops of economic interest.},
}
@article {pmid40380065,
year = {2025},
author = {Rivera-Toro, DM and Alvarez-Venegas, R},
title = {CRISPR-Activation: Boosting Expression of Plant MIRs.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2900},
number = {},
pages = {229-247},
pmid = {40380065},
issn = {1940-6029},
mesh = {*MicroRNAs/genetics ; *CRISPR-Cas Systems ; *Gene Expression Regulation, Plant ; *Gene Editing/methods ; Promoter Regions, Genetic ; *Plants/genetics ; Plants, Genetically Modified/genetics ; Genetic Vectors/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; },
abstract = {In the field of plant genome editing, the CRISPR/Cas system stands out as an exceptionally versatile tool. Originally conceived for gene editing, the catalytically inactive Cas9 variant, known as dead Cas9 (dCas9), takes on a new role when coupled with transcriptional effectors, offering a finely tuned mechanism for controlling gene expression. This innovation has paved the way for the evolution of sophisticated systems such as CRISPR-Act 2.0 and CRISPR-Act 3.0, which significantly enhance transcriptional activation in plants. This chapter serves as an introduction to a protocol that harnesses CRISPR-activation (CRISPRa) to modulate the transcription of plant miRNA genes. Recognizing the imperative for meticulous construct design and promoter identification, the protocol outlines steps encompassing vector construction. It underscores the critical importance of comprehending miRNA promoter regions and cis-acting elements, presenting a holistic approach for effectively employing CRISPRa in manipulating miRNA expression to modulate traits in plants.},
}
@article {pmid40380064,
year = {2025},
author = {Martínez-Estrada, E and de la Mora-Franco, D and de Folter, S},
title = {A Simple and Efficient Protocol to Transform and Regenerate CRISPR-Cas9-Mediated Genome-Edited Tomato Plants.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2900},
number = {},
pages = {213-228},
pmid = {40380064},
issn = {1940-6029},
mesh = {*Solanum lycopersicum/genetics/growth &amp; development ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; *Genome, Plant ; Plants, Genetically Modified/genetics ; Cotyledon/genetics/growth &amp; development ; Transformation, Genetic ; Agrobacterium/genetics ; },
abstract = {CRISPR-Cas9-mediated genome editing has revolutionized functional genomics and crop improvement. However, to maximize the adoption of the CRISPR-Cas9 technology, an efficient method to transform and regenerate genetically edited plants is necessary. In this protocol, we describe a detailed method to generate a CRISPR-Cas9 construct based on the Golden Gate cloning system and a simple and efficient method to transform and regenerate tomato plants from cotyledons co-cultured with Agrobacterium. Our protocol allows the production of at least ten Cas-positive independent lines from one hundred cotyledons. This protocol is routinely used in our laboratory to obtain tomato mutant lines and has been proven effective across several genotypes.},
}
@article {pmid40379664,
year = {2025},
author = {Nam, H and Xie, K and Majumdar, I and Wang, J and Yang, S and Starzyk, J and Lee, D and Shan, R and Li, J and Wu, H},
title = {Engineering tripartite gene editing machinery for highly efficient non-viral targeted genome integration.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {4569},
pmid = {40379664},
issn = {2041-1723},
support = {DP2 GM154019/GM/NIGMS NIH HHS/United States ; },
mesh = {*Gene Editing/methods ; Humans ; CRISPR-Cas Systems/genetics ; Transgenes/genetics ; DNA, Single-Stranded/genetics/metabolism ; HEK293 Cells ; Receptors, Chimeric Antigen/genetics/metabolism ; T-Lymphocytes/metabolism ; DNA, Circular/genetics ; Genetic Engineering/methods ; CRISPR-Associated Protein 9/metabolism/genetics ; Homologous Recombination ; Gene Knock-In Techniques/methods ; },
abstract = {Non-viral DNA donor templates are commonly used for targeted genomic integration via homologous recombination (HR), with efficiency improved by CRISPR/Cas9 technology. Circular single-stranded DNA (cssDNA) has been used as a genome engineering catalyst (GATALYST) for efficient and safe gene knock-in. Here, we introduce enGager, an enhanced GATALYST associated genome editor system that increases transgene integration efficiency by tethering cssDNA donors to nuclear-localized Cas9 fused with single-stranded DNA binding peptide motifs. This approach further improves targeted integration and expression of reporter genes at multiple genomic loci in various cell types, showing up to 6-fold higher efficiency compared to unfused Cas9, especially for large transgenes in primary cells. Notably, enGager enables efficient integration of a chimeric antigen receptor (CAR) transgene in 33% of primary human T cells, enhancing anti-tumor functionality. This 'tripartite editor with ssDNA optimized genome engineering (TESOGENASE) offers a safer, more efficient alternative to viral vectors for therapeutic gene modification.},
}
@article {pmid40378977,
year = {2025},
author = {Qin, B and Shen, S and Chen, H and Wang, Y and Ding, J and Ding, J},
title = {Inactivation of the key ORFs of HBV for antiviral therapy by non-cleavage base editing.},
journal = {Microbial pathogenesis},
volume = {205},
number = {},
pages = {107689},
doi = {10.1016/j.micpath.2025.107689},
pmid = {40378977},
issn = {1096-1208},
mesh = {*Hepatitis B virus/genetics/drug effects ; Humans ; *Gene Editing/methods ; CRISPR-Cas Systems ; *Open Reading Frames/genetics ; Antiviral Agents/pharmacology/therapeutic use ; Virus Replication/genetics ; DNA, Viral/genetics ; Point Mutation ; Hepatitis B/therapy/virology ; },
abstract = {OBJECTIVES: Hepatitis B virus (HBV) infection is the key cause of chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma. Currently available anti-HBV drugs are more or less defective owing to the unremovable covalently closed circular DNA (cccDNA). Thus, CRISPR/Cas9 is a promising therapeutic strategy for anti-HBV therapy. Double-strand breaks (DSBs) and uncontrolled genomic rearrangements occur inevitably. In this study, we aimed to use base editors to control HBV infection.<br><br>METHODS: Base editors precisely instal targeted point mutations without requiring DSBs or donor DNA templates, and without relying on homology-directed repair (HDR) or nonhomologous end joining (NHEJ). Adenine base editors (ABEs) and cytosine base editors (CBEs) catalyse A&#x2022; T to G &#x2022;C and C&#x2022; G to T &#x2022;A conversions, respectively. In this study, to control HBV replication by modifying and inactivating key HBV genes, recently developed CRISPR/Cas-mediated SpRY-ABE8e and CBE4-max were utilised to falsify and invalidate the ATG initiation codons of the S, Pre-S1, PreS2, C, Pre-C, X, and P genes.<br><br>RESULTS: The ATG initiation codons of HBV genes were edited by ABE/CBE. The expected point mutations were successfully introduced, resulting in the simultaneous suppression of HBV antigen expression and replication to varying degrees.<br><br>CONCLUSIONS: Our study focused on clearing HBV using base and provided experimental and theoretical evidence for the treatment of chronic HBV infection. Thus, base editing is a potential strategy for curing CHB by permanently inactivating the integrated DNA and cccDNA without using DSBs.},
}
@article {pmid40378975,
year = {2025},
author = {Kumar, V and Nagano, T and Takasuka, TE},
title = {Genome integration and expression of β-glucosidase in Priestia megaterium enhanced poly(3-hydroxybutyrate) production from cellobiose and cellulose.},
journal = {Bioresource technology},
volume = {432},
number = {},
pages = {132681},
doi = {10.1016/j.biortech.2025.132681},
pmid = {40378975},
issn = {1873-2976},
mesh = {*Cellobiose/metabolism ; *Cellulose/metabolism ; *Hydroxybutyrates/metabolism ; *beta-Glucosidase/genetics/metabolism ; *Polyesters/metabolism ; Promoter Regions, Genetic/genetics ; Polyhydroxyalkanoates ; *Genome, Bacterial/genetics ; Polyhydroxybutyrates ; },
abstract = {Polyhydroxyalkanoates (PHAs) production using cellulosic biomass is a promising way for sustainable manufacturing of bioplastics. Priestia megaterium is an ideal choice as it can use glucose and xylose for PHA production. To further improve the strain for PHA production from cellobiose, we integrate exogenous β-glucosidase (Bgl) from Bacillus sp. GL1 (Bsbgl) in the PHA depolymerase (phaZ1) deletion background (ΔZ1) using CRISPR-Cas. The deletion of phaZ1 in P. megaterium showed a significant improvement in the PHA accumulation whereas BsBgl expression resulted in robust activity and improved growth using cellobiose as a sole carbon source compared to other Bgl targets. To further improve the strain, four native promoters were examined for intracellular BsBgl expression, and the PHA promoter (PphaR) and citrate synthase promoter (Pcitz) showed 2.0- and 4.5-fold higher activities of BsBgl, compared to the xylose promoter (Pxyl). The rate of cellobiose utilization in engineered strains P2 (PphaRBsbgl_ΔZ1) and P4 (PcitzBsbgl_ΔZ1) was improved to 1.6-fold and 2.6-fold, whereas poly(3-hydroxybutyrate) (P3HB) yield for the respective strains was around 3-fold to the wild-type. The strain P2 turned out to be better for cellobiose to PHA production. Further, the strain P2 in a co-culture with cellulolytic Streptomyces sp. SirexAA-E in a consolidated bioprocessing yielded 76 mg of P3HB/ g of carboxymethylcellulose, which is 4.3-times higher than the co-culture with the wild-type. Thus, the present work improved the cellobiose utilization and P3HB accumulation of P. megaterium. The current study paves the way for designing efficient cell factories for cellulosic biomass into bioplastic in the future.},
}
@article {pmid40378589,
year = {2025},
author = {Chen, Q and Wang, H and Xu, H and Peng, Y and Yao, B and Chen, Z and Yang, J and Adeloju, S and Chen, W},
title = {One-pot RPA-CRISPR/Cas12a integrated dual-mode electrochemical lateral flow strip for ultrasensitive and precise detection of Salmonella.},
journal = {Biosensors &amp; bioelectronics},
volume = {285},
number = {},
pages = {117529},
doi = {10.1016/j.bios.2025.117529},
pmid = {40378589},
issn = {1873-4235},
mesh = {*Biosensing Techniques/methods ; *Salmonella/isolation &amp; purification/genetics ; *Electrochemical Techniques/methods ; Limit of Detection ; CRISPR-Cas Systems/genetics ; Food Microbiology ; Equipment Design ; Salmonella Infections/microbiology/diagnosis ; Humans ; },
abstract = {Rapid and accurate screening of pathogenic contamination is essential for timely intervention and infection prevention. In this work, one-pot RPA-CRISPR/Cas12a strategy combined with an innovative electrochemical lateral flow strip (OPRCC-eLFS) was presented for ultrasensitive and precise detection of Salmonella. Highly sensitive dual-mode detection of Salmonella in various samples has been simultaneously achieved with electrochemical detection limit of 3.84 CFU/mL and visual detection limit of 384 CFU/mL, respectively, with improved detection efficiency and prevention of uncapping-related aerosol-contamination. This dual-mode biosensing platform demonstrates exceptional stability, remarkable sensitivity, and robust on-site quantification capability, emphasizing its potential in food safety monitoring and disease prevention.},
}
@article {pmid40377871,
year = {2025},
author = {Rahmati, R and Zarimeidani, F and Ghanbari Boroujeni, MR and Sadighbathi, S and Kashaniasl, Z and Saleh, M and Alipourfard, I},
title = {CRISPR-Assisted Probiotic and In Situ Engineering of Gut Microbiota: A Prospect to Modification of Metabolic Disorders.},
journal = {Probiotics and antimicrobial proteins},
volume = {},
number = {},
pages = {},
pmid = {40377871},
issn = {1867-1314},
abstract = {The gut microbiota, a substantial group of microorganisms residing in the human body, profoundly impacts various physiological and pathological mechanisms. Recent studies have elucidated the association between gut dysbiosis and multiple organ diseases. Gut microbiota plays a crucial role in maintaining gastrointestinal stability, regulating the immune system and metabolic processes not only within the gastrointestinal tract but also in other organs such as the brain, lungs, and skin. Dysbiosis of the gut microbiota can disrupt biological functioning and contribute to the development of metabolic disorders. The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR-associated proteins (Cas) modules are adaptive immune systems in numerous archaea and bacteria. CRISPR/Cas is a versatile gene-editing tool that enables modification of the genome in live cells, including those within the gut microbiota. This technique has revolutionized gene editing due to its simplicity and effectiveness. It finds extensive applications in diverse scientific arenas, facilitating the functional screening of genomes during various biological processes. Additionally, CRISPR has been instrumental in creating model organisms and cell lines for research purposes and holds great potential for developing personalized medical treatments through precise genetic alterations. This review aims to explore and discuss the possibilities of CRISPR/Cas and the current trends in using this technique for editing gut microbiota genes in various metabolic disorders. By uncovering the valuable potential of CRISPR/Cas in modifying metabolic disorders through the human gut microbiota, we shed light on its promising applications.},
}
@article {pmid40377313,
year = {2025},
author = {Scarrone, M and Turner, D and Dion, M and Tremblay, D and Moineau, S},
title = {In silico and in vitro comparative analysis of 79 Acinetobacter baumannii clinical isolates.},
journal = {Microbiology spectrum},
volume = {},
number = {},
pages = {e0284924},
doi = {10.1128/spectrum.02849-24},
pmid = {40377313},
issn = {2165-0497},
abstract = {Acinetobacter baumannii is a significant nosocomial bacterial pathogen that poses a substantial infection risk due to its high resistance to antibiotics and ability to survive in hospital environments. In this study, we performed comprehensive in silico and in vitro analyses on 79 A. baumannii clinical isolates from different geographical locations to uncover their genomic and epidemiological characteristics as well as their antibiotic and phage susceptibilities. Our findings revealed considerable genomic diversity among the isolates, as shown by average nucleotide identity (ANI) heat maps, multilocus sequence typing (MLST), and core genome MLST (cgMLST). We identified several international clones known for their high antibiotic resistance and global prevalence. Surprisingly, we also observed that the number of antimicrobial resistance genes (ARGs) was higher in isolates containing CRISPR-Cas systems. Plaque assays with 13 phages indicated that Acinetobacter phages have a narrow host range, with capsule loci (KL) serving as a good indicator of phage-bacteria interactions. The presence of CRISPR-Cas systems and other antiviral defense mechanisms in A. baumannii genomes also appears to play a key role in providing phage resistance, regardless of the phage receptors. We also found that spacers associated with subtypes I-F1 and I-F2 CRISPR-Cas systems predominantly target prophages, suggesting a role in maintaining genomic stability and contributing to phage-bacteria co-evolution. Overall, this study provides a set of highly characterized A. baumannii clinical isolates for future studies on antibiotic-phage-bacteria interactions.IMPORTANCEAcinetobacter baumannii poses a significant challenge to the healthcare system due to its antibiotic resistance and strong survival mechanisms. This study examines a diverse collection of 79 clinical isolates to deepen our understanding of A. baumannii's genetic characteristics and its defense mechanisms against both antibiotics and phages. Genomic analysis revealed globally prevalent, highly resistant clones and uncovered a complex role for CRISPR-Cas systems. Although CRISPR-Cas systems were not widespread among these isolates, they primarily targeted prophages. Additionally, the study emphasizes the importance of capsule types as indicators of phage susceptibility. Together, these findings provide insights into the pathogen's resilience and evolutionary adaptations, potentially guiding future research on infection control strategies and new therapeutic approaches to combat A. baumannii infections.},
}
@article {pmid40377112,
year = {2025},
author = {Kellari, LM and Dalakouras, A and Tsiouri, O and Vletsos, P and Katsaouni, A and Uslu, VV and Papadopoulou, KK},
title = {Cross-kingdom RNAi induced by a beneficial endophytic fungus to its host requires transitivity and amplification of silencing signals.},
journal = {Plant biology (Stuttgart, Germany)},
volume = {27},
number = {4},
pages = {504-514},
pmid = {40377112},
issn = {1438-8677},
support = {6236//the Hellenic Foundation for Research and Innovation (HFRI)/ ; 7322//Research Committee of UTH/ ; //PRIMA Programme/ ; },
mesh = {*Nicotiana/microbiology/genetics/metabolism ; *Endophytes/physiology ; *Fusarium/physiology/genetics ; *RNA Interference ; Green Fluorescent Proteins/metabolism/genetics ; },
abstract = {Cross-kingdom transfer of small RNA (sRNA) molecules has been identified as a means of communication between plants and interacting microorganisms, but the mechanistic details of this sRNA-based interaction remain elusive. We have previously shown that the beneficial root-colonizing fungus Fusarium solani strain K (FsK) translocates sRNAs to its host, Nicotiana benthamiana (Nb), leading to systemic silencing of a reporter gene. Here, we investigated the mechanistic details of the endophyte-induced systemic silencing using an RNAi sensor system. We inoculated three Nb GFP expressing lines with conidia of an FsK transformant containing a transgene that targets host GFP (FsK-hpGF). The efficiency of silencing mediated by FsK-hpGF was monitored both phenotypically under ultraviolet light as well as quantitatively by RT-qPCR. sRNA sequencing was performed to evaluate the production of sRNAs targeting host GFP. Finally, bisulfite sequencing was used to assess plant GFP methylation levels. We show that the translocated fungal sRNAs induced production of secondary sRNAs, mainly of 22-24-nt in size, with the conspicuous absence of 21-nt sRNAs. Importantly, systemic silencing could not be induced in an RNA-DEPENDENT RNA POLYMERASE 6 (RDR6) CRISPR/Cas knockout background, nor in an intron-containing target gene. Overall, our data show that endophyte-induced silencing in the host requires RDR6-mediated transitivity and amplification of silencing signals. Despite being based on an artificial RNAi sensor system, our observations may reflect a more generalized and so far unexplored facet of cross-kingdom RNAi, with RDR6-based transitivity influencing the way symbionts and pathogens elicit systemic phenotypes in their host plants.},
}
@article {pmid40376763,
year = {2025},
author = {Wang, K and Liu, Z},
title = {Plant synthetic biology-based biofortification, strategies and recent progresses.},
journal = {Journal of integrative plant biology},
volume = {},
number = {},
pages = {},
doi = {10.1111/jipb.13934},
pmid = {40376763},
issn = {1744-7909},
support = {startup fund//Shenzhen University of Advanced Technology/ ; },
abstract = {Hidden hunger, caused by chronic micronutrient deficiencies, affects billions of people worldwide and remains a critical public health issue despite progress in food production. Biofortification offers a promising solution by enhancing nutrient levels within plant tissues through traditional breeding or advanced biotechnologies. Recent advancements in plant synthetic biology have significantly improved biofortification strategies, enabling precise and targeted nutrient enrichment. This mini-review outlines five core strategies in synthetic biology-based biofortification: overexpression of endogenous biosynthetic genes, introduction of heterologous biosynthetic pathways, expression of nutrient-specific transporters, optimization of transcriptional regulation, and protein (directed) evolution. Vitamin B1 biofortification serves as a primary illustrative example due to its historical importance and ongoing relevance. Recent breakthroughs, particularly from Chinese research teams, are also highlighted. Together, these strategies offer transformative potential for addressing global nutritional challenges through precise, sustainable and innovative plant-based approaches.},
}
@article {pmid40376717,
year = {2025},
author = {Marzluf, JP and Daniela, K and Klein, J and Zehe, C and Leroux, AC},
title = {Utilizing Stable Gene-Edited Knockout Pools for Genetic Screening and Engineering in Chinese Hamster Ovary Cells.},
journal = {Biotechnology journal},
volume = {20},
number = {5},
pages = {e70033},
pmid = {40376717},
issn = {1860-7314},
mesh = {CHO Cells ; Animals ; Cricetulus ; *Gene Knockout Techniques/methods ; *Gene Editing/methods ; CRISPR-Cas Systems/genetics ; Cricetinae ; *Genetic Testing/methods ; Fibronectins/genetics ; Genetic Engineering/methods ; },
abstract = {Chinese hamster ovary (CHO) cells are the primary host for biopharmaceutical production. To meet increasing demands for productivity, quality, and complex molecule expression, genetic engineering, particularly clustered regularly interspaced short palindromic repeats (CRISPR)-mediated gene knockout (KO), is widely used to optimize host cell performance. However, systematic screening of KO targets remains challenging due to the labor-intensive process of generating and evaluating individual clones. In this study, we present a robust, high-throughput CRISPR workflow using stable KO pools in CHO cells. These pools maintain genetic stability for over 6 weeks, including in multiplexed configurations targeting up to seven genes simultaneously. Compared to clonal approaches, KO pools reduce variability caused by clonal heterogeneity and better reflect the host cell population phenotype. We demonstrate the utility of this approach by reproducing the beneficial phenotypic effects of fibronectin 1 (FN1) KO, specifically prolonged culture duration and improved late-stage viability in fed-batch processes. This workflow enables efficient identification and evaluation of promising KO targets without the need to generate and test large numbers of clones. Overall, screening throughput is increased 2.5-fold and timelines are compressed from 9 to 5 weeks. This provides a scalable, efficient alternative to traditional clonal screening, accelerating discovery for CHO cell line engineering for biopharmaceutical development.},
}
@article {pmid40376300,
year = {2025},
author = {Feussner, M and Migur, A and Mitrofanov, A and Alkhnbashi, OS and Backofen, R and Beisel, CL and Weinberg, Z},
title = {Disparate mechanisms counteract extraneous CRISPR RNA production in type II-C CRISPR-Cas systems.},
journal = {microLife},
volume = {6},
number = {},
pages = {uqaf007},
pmid = {40376300},
issn = {2633-6693},
abstract = {CRISPR-Cas adaptive immune systems in bacteria and archaea enable precise targeting and elimination of invading genetic elements. An inherent feature of these systems is the 'extraneous' CRISPR RNA (ecrRNA), which is produced via the extra repeat in a CRISPR array lacking a corresponding spacer. As ecrRNAs would interact with the Cas machinery yet not direct acquired immunity, they pose a potential barrier to defence. Type II-A CRISPR-Cas systems resolve this barrier through the leader sequence upstream of a CRISPR array, which forms a hairpin structure with the extra repeat that inhibits ecrRNA production. However, the fate of ecrRNAs in other CRISPR types and subtypes remains to be explored. Here, we report that II-C systems likely employ disparate strategies to resolve the ecrRNA due to their distinct configuration in comparison to II-A. Applying bioinformatics analyses to over 650 II-C systems followed by experimental validation, we identified three strategies applicable to these systems: formation of an upstream Rho-independent terminator, formation of a hairpin that sequesters the ecrRNA guide, and mutations in the repeat expected to disrupt ecrRNA formation. These findings expand the list of mechanisms in CRISPR-Cas systems that could resolve the ecrRNA to optimize immune response.},
}
@article {pmid40376084,
year = {2024},
author = {Shi, H and Al-Sayyad, N and Wasko, KM and Trinidad, MI and Doherty, EE and Vohra, K and Boger, RS and Colognori, D and Cofsky, JC and Skopintsev, P and Bryant, Z and Doudna, JA},
title = {Rapid two-step target capture ensures efficient CRISPR-Cas9-guided genome editing.},
journal = {bioRxiv : the preprint server for biology},
volume = {},
number = {},
pages = {},
doi = {10.1101/2024.10.01.616117},
pmid = {40376084},
issn = {2692-8205},
abstract = {RNA-guided CRISPR-Cas enzymes initiate programmable genome editing by recognizing a 20-base-pair DNA sequence adjacent to a short protospacer-adjacent motif (PAM). To uncover the molecular determinants of high-efficiency editing, we conducted biochemical, biophysical and cell-based assays on S. pyogenes Cas9 (Spy Cas9) variants with wide-ranging genome editing efficiencies that differ in PAM binding specificity. Our results show that reduced PAM specificity causes persistent non-selective DNA binding and recurrent failures to engage the target sequence through stable guide RNA hybridization, leading to reduced genome editing efficiency in cells. These findings reveal a fundamental trade-off between broad PAM recognition and genome editing effectiveness. We propose that high-efficiency RNA-guided genome editing relies on an optimized two-step target capture process, where selective but low-affinity PAM binding precedes rapid DNA unwinding. This model provides a foundation for engineering more effective CRISPR-Cas and related RNA-guided genome editors.},
}
@article {pmid40375877,
year = {2025},
author = {Khamwut, A and Nimnual, J and Chomta, N and Nimsamer, P and Mayuramart, O and Kaewsapsak, P and Pasittungkul, S and Poovorawan, Y and Payungporn, S},
title = {Detection of respiratory syncytial virus based on RT-RPA and CRISPR-Cas12a.},
journal = {Experimental biology and medicine (Maywood, N.J.)},
volume = {250},
number = {},
pages = {10387},
pmid = {40375877},
issn = {1535-3699},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Respiratory Syncytial Virus, Human/isolation &amp; purification/genetics ; *Respiratory Syncytial Virus Infections/diagnosis/virology ; Sensitivity and Specificity ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {Human respiratory syncytial virus (hRSV) is one of the most prevalent viruses infecting children globally. In this study, we employed the RT-RPA with CRISPR/Cas12a detection methodology to detect and differentiate RSV-A and RSV-B, particularly in resource-limited settings. The detection limit for RSV-A and RSV-B was approximately 10[2] and 10[3] copies/reaction, respectively. The assay revealed 100% specificity in detecting both RSV-A and RSV-B. Diagnostic accuracy was 90.32 and 93.55% for RSV-A and RSV-B, respectively, compared to RT-qPCR. These data indicate a proficient strategy for RSV screening, demonstrating promise for prospective applications in detecting diverse viral infections.},
}
@article {pmid40375406,
year = {2025},
author = {Rhode, J and Edwards, S and Tzvetkova, A and Jensen, LR and Hossain, MF and Nowack, B and Hagenau, L and Kuss, AW},
title = {Two iPSC lines with a heterozygous frameshift mutation in the floating-harbour syndrome locus of the SRCAP gene.},
journal = {Stem cell research},
volume = {86},
number = {},
pages = {103730},
doi = {10.1016/j.scr.2025.103730},
pmid = {40375406},
issn = {1876-7753},
mesh = {Humans ; *Frameshift Mutation/genetics ; *Induced Pluripotent Stem Cells/metabolism/cytology ; Heterozygote ; Cell Line ; *Intellectual Disability/genetics/pathology ; CRISPR-Cas Systems ; },
abstract = {We present two CRISPR/Cas9-modified human iPSC lines with a heterozygous frameshift mutation (NM_006662.3:c.7300_7301insA) in the FLHS-locus of the SRCAP gene, which is associated with Floating-Harbor syndrome, a congenital neurodevelopmental disorder with symptoms including short stature and intellectual disability. The iPSCs express the pluripotency markers OCT4, SOX2, NANOG and TRA 1-60. They show differentiation into cells from all 3 germ layers, no chromosomal abnormalities and no off-target mutations in the tested regions. The mutation leads to a stop codon previously found in patients. Thus, either cell line can serve as disease-specific model for studying SRCAP in the context of FLHS.},
}
@article {pmid40374732,
year = {2025},
author = {Hagman, A and Stenström, O and Carlström, G and Akke, M and Grey, C and Carlquist, M},
title = {Biocatalytic reductive amination with CRISPR-Cas9 engineered yeast.},
journal = {Scientific reports},
volume = {15},
number = {1},
pages = {16972},
pmid = {40374732},
issn = {2045-2322},
mesh = {*CRISPR-Cas Systems ; Amination ; *Saccharomyces cerevisiae/genetics/metabolism ; *Metabolic Engineering/methods ; Biocatalysis ; Transaminases/genetics/metabolism ; Chromobacterium/enzymology/genetics ; Alanine/metabolism ; Alanine Transaminase/genetics/metabolism ; Bioreactors ; },
abstract = {Metabolically engineered baker's yeast can be used to produce chiral amines through whole-cell bioconversion of prochiral ketones. This study investigates the modulation of the alanine-pyruvate metabolic node to enhance reductive amination, using the stereoselective conversion of benzylacetone to (S)-1-methyl-3-phenylpropylamine (MPPA) as a model reaction. Chromosomal integration of multiple copies of the promiscuous omega transaminase from Chromobacterium violaceum (cv-ATA) resulted in an active yeast catalyst. Physiological characterization in bioreactors under aerobic batch cultivation revealed that amine production occurred only under post-diauxic growth on ethanol. To reduce native alanine utilization, the endogenous alanine aminotransferase (ALT1) was knocked out and replaced with cv-ATA. To rapidly employ this strategy in other strains, a simple CRISPR/cas9 method for universal gene replacement was developed. The replacement of ALT1 with cv-ATA improved the reaction by 2.6-fold compared to the control strain with intact ALT1. NMR measurements of metabolites originating from [15]N L-alanine and [13]C glucose indicated that pyruvate formation during growth on glucose inhibited amine production. Under optimal conditions, the biocatalytic bioconversion of benzylacetone to MPPA reached a yield of 58%.},
}
@article {pmid40373980,
year = {2025},
author = {Deng, X and Wang, W and Tao, Y and Yao, L and Li, Y and Huang, X and He, J},
title = {Application of a rapid and sensitive RPA-CRISPR/Cas12a assay for BCSP31-based Brucella detection.},
journal = {Journal of microbiological methods},
volume = {235},
number = {},
pages = {107148},
doi = {10.1016/j.mimet.2025.107148},
pmid = {40373980},
issn = {1872-8359},
mesh = {*Brucella/genetics/isolation &amp; purification ; *Brucellosis/diagnosis/microbiology ; *CRISPR-Cas Systems/genetics ; Sensitivity and Specificity ; *Nucleic Acid Amplification Techniques/methods ; Humans ; Recombinases/genetics/metabolism ; DNA, Bacterial/genetics ; *Molecular Diagnostic Techniques/methods ; Animals ; Bacterial Proteins/genetics ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {Brucellosis, a zoonotic disease caused by Brucella species, poses significant health risks to humans and animals. Due to the limitations of current diagnostic methods, such as serological testing and PCR, in terms of sensitivity, specificity, and speed, this study explores the potential of integrating recombinase polymerase amplification (RPA) with the CRISPR-Cas12a system for Brucella detection. This combination leverages the strengths of both technologies for rapid, sensitive, and specific molecular diagnostics. RPA primers and CRISPR RNA (crRNA) targeting the Brucella-specific conserved sequence BCSP31 were designed, followed by optimization of the RPA-CRISPR/Cas12a system. Its performance was evaluated using genomic DNA from Brucella and non-Brucella species. The system's capabilities were assessed on clinical blood samples, demonstrating high sensitivity (detection limit of 10 copies per reaction and 16.6 attomoles for Brucella DNA) and excellent specificity. Testing on clinical samples showed strong agreement with qPCR results and an improvement over the RBT. The RPA-CRISPR/Cas12a platform represents a rapid, ultra-sensitive, and accurate method for Brucella detection and holds promise as a valuable tool for brucellosis control.},
}
@article {pmid40373829,
year = {2025},
author = {Trujillo, E and Angulo, C},
title = {Perspectives on the use of the CRISPR system in plants to improve recombinant therapeutic protein production.},
journal = {Journal of biotechnology},
volume = {405},
number = {},
pages = {111-123},
doi = {10.1016/j.jbiotec.2025.05.010},
pmid = {40373829},
issn = {1873-4863},
abstract = {The plant-based system is a promising platform for producing biotherapeutics due to its scalability, cost-effectiveness, and lower risk of contamination by human pathogens. However, several challenges remain, including optimizing yield, stability, functionality, and the immunogenic properties of recombinant proteins. In this context, this review explores the application of CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) technology to improve the production of recombinant therapeutic proteins in plants. Traditional tools and strategies for plant-based recombinant protein production are discussed, highlighting their limitations and the potential of CRISPR to overcome these boundaries. It delves into the components of the CRISPR-Cas system and its application in optimizing therapeutic protein function and yield. Major strategies include modifying glycosylation patterns to humanize plant-produced proteins, metabolic pathway engineering to increase protein accumulation, and the precise integration of transgenes into specific genomic loci to enhance expression stability and productivity. These advancements demonstrate how CRISPR system can overcome bottlenecks in plant molecular farming and enable the production of high-quality therapeutic proteins. Lastly, future trends and perspectives are examined, emphasizing ongoing innovations and challenges in the field. The review underscores the potential of CRISPR to reshape plant biotechnology and support the growing demand for recombinant therapeutics, offering new avenues for sustainable and efficient protein production systems. KEY MESSAGE: CRISPR technology has the potential to improve plant-based therapeutic protein production by optimizing yield, stability, and humanization, overcoming bottlenecks, and enabling sustainable, efficient systems for recombinant biotherapeutics.},
}
@article {pmid40373688,
year = {2025},
author = {Thakur, RK and Aggarwal, K and Sood, N and Kumar, A and Joshi, S and Jindal, P and Maurya, R and Patel, P and Kurmi, BD},
title = {Harnessing advances in mechanisms, detection, and strategies to combat antimicrobial resistance.},
journal = {The Science of the total environment},
volume = {982},
number = {},
pages = {179641},
doi = {10.1016/j.scitotenv.2025.179641},
pmid = {40373688},
issn = {1879-1026},
mesh = {*Drug Resistance, Microbial ; Humans ; Bacteria/drug effects ; *Anti-Infective Agents ; *Drug Resistance, Bacterial ; Anti-Bacterial Agents ; },
abstract = {Antimicrobial resistance (AMR) is a growing global health crisis, threatening the effectiveness of antibiotics and other antimicrobial agents, leading to increased morbidity, mortality, and economic burdens. This review article provides a comprehensive analysis of AMR, beginning with a timeline of antibiotics discovery and the year of first observed resistance. Main mechanisms of AMR in bacteria, fungi, viruses, and parasites are summarized, and the main mechanisms of bacteria are given in detail. Additionally, we discussed in detail methods for detecting AMR, including phenotypic, genotypic, and advanced methods, which are crucial for identifying and monitoring AMR. In addressing AMR mitigation, we explore innovative interventions such as CRISPR-Cas systems, nanotechnology, antibody therapy, artificial intelligence (AI), and the One Health approach. Moreover, we discussed both finished and ongoing clinical trials for AMR. This review emphasizes the urgent need for global action and highlights promising technologies that could shape the future of AMR surveillance and treatment. By integrating interdisciplinary research and emerging clinical insights, this study aims to guide individuals toward impactful solutions in the battle against AMR.},
}
@article {pmid40373119,
year = {2025},
author = {Witte, IP and Lampe, GD and Eitzinger, S and Miller, SM and Berríos, KN and McElroy, AN and King, RT and Stringham, OG and Gelsinger, DR and Vo, PLH and Chen, AT and Tolar, J and Osborn, MJ and Sternberg, SH and Liu, DR},
title = {Programmable gene insertion in human cells with a laboratory-evolved CRISPR-associated transposase.},
journal = {Science (New York, N.Y.)},
volume = {388},
number = {6748},
pages = {eadt5199},
doi = {10.1126/science.adt5199},
pmid = {40373119},
issn = {1095-9203},
mesh = {Humans ; *Transposases/genetics/metabolism ; *CRISPR-Cas Systems ; *Directed Molecular Evolution/methods ; *Gene Editing/methods ; HEK293 Cells ; RNA, Guide, CRISPR-Cas Systems ; *Mutagenesis, Insertional ; },
abstract = {Programmable gene integration in human cells has the potential to enable mutation-agnostic treatments for loss-of-function genetic diseases and facilitate many applications in the life sciences. CRISPR-associated transposases (CASTs) catalyze RNA-guided DNA integration but thus far demonstrate minimal activity in human cells. Using phage-assisted continuous evolution (PACE), we generated CAST variants with >200-fold average improved integration activity. The evolved CAST system (evoCAST) achieves ~10 to 30% integration efficiencies of kilobase-size DNA cargoes in human cells across 14 tested genomic target sites, including safe harbor loci, sites used for immunotherapy, and genes implicated in loss-of-function diseases, with undetected indels and low levels of off-target integration. Collectively, our findings establish a platform for the laboratory evolution of CASTs and advance a versatile system for programmable gene integration in living systems.},
}
@article {pmid40372912,
year = {2025},
author = {Xiang, W and Lin, X and Yang, Y and Huang, L and Chen, Y and Chen, J and Liu, L},
title = {Cas12h is a crRNA-guided DNA nickase that can be utilized for precise gene editing.},
journal = {Cell reports},
volume = {44},
number = {5},
pages = {115718},
doi = {10.1016/j.celrep.2025.115718},
pmid = {40372912},
issn = {2211-1247},
mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; *CRISPR-Associated Proteins/metabolism/genetics/chemistry ; DNA/metabolism/genetics ; *RNA, Guide, CRISPR-Cas Systems/metabolism/genetics ; Humans ; *Deoxyribonuclease I/metabolism/chemistry/genetics ; },
abstract = {Type V-H CRISPR-Cas system, an important subtype of type V CRISPR-Cas systems, has remained enigmatic in terms of its structure and function despite being discovered several years ago. Here, we comprehensively characterize the type V-H CRISPR-Cas system and elucidate its role as a DNA nicking system. The unique CRISPR RNA (crRNA) employed by Cas12h effector protein enables specific targeting of double-stranded DNA (dsDNA), while its RuvC domain is responsible for cleaving the non-target strand (NTS) of dsDNA. We present the structure of Cas12h bound to crRNA and target DNA. Our structural analysis reveals that the RuvC domain possesses a narrow active pocket that facilitates recognition of NTS but potentially hinders access to the target strand. Furthermore, we demonstrate that Cas12h confers adaptive immunity against invading mobile genetic elements through transcriptional gene inhibition. We have engineered an adenine base editor by fusing Cas12h with an adenine deaminase, achieving effective A-to-G substitution.},
}
@article {pmid40371045,
year = {2025},
author = {Tewari, M and Rana, P and Pande, V},
title = {Nanomaterial-Based Biosensors for the Detection of COVID-19.},
journal = {Indian journal of microbiology},
volume = {65},
number = {1},
pages = {120-136},
pmid = {40371045},
issn = {0046-8991},
abstract = {The COVID-19 outbreak began in December 2019 and has affected people worldwide. It was declared a pandemic in 2020 by the World Health Organization. Developing rapid and reliable diagnostic techniques is crucial for identifying COVID-19 early and preventing the disease from becoming severe. In addition to conventional diagnostic techniques such as RT-PCR, computed tomography, serological assays, and sequencing methods, biosensors have become widely accepted for identifying and screening COVID-19 infection with high accuracy and sensitivity. Their low cost, high sensitivity, specificity, and portability make them ideal for diagnostics. The use of nanomaterials improves the performance of biosensors by increasing their sensitivities and limiting detection by several orders of magnitude. This manuscript briefly reviews the COVID-19 outbreak and its pathogenesis. Furthermore, it comprehensively discusses the currently available biosensors for SARS-CoV-2 detection, with a special emphasis on nanomaterials-based biosensors developed to detect this emerging virus and its variants efficiently.},
}
@article {pmid40370093,
year = {2025},
author = {Qiu, S and Chen, L and Zhuang, D and Cao, Y and Wei, R and Cao, X and Chen, Y and Lai, X and Wang, S and Lin, Y and Lin, Z and Zhang, S},
title = {Fluorescence Aptasensor for sST2 Detection Using In Vitro Selected Aptamers.},
journal = {Analytical chemistry},
volume = {97},
number = {20},
pages = {10910-10918},
doi = {10.1021/acs.analchem.5c01855},
pmid = {40370093},
issn = {1520-6882},
mesh = {*Aptamers, Nucleotide/chemistry ; Humans ; *Interleukin-1 Receptor-Like 1 Protein/blood/analysis ; *Biosensing Techniques/methods ; Limit of Detection ; Fluorescence ; CRISPR-Cas Systems ; },
abstract = {Soluble suppression of tumorigenicity 2 (sST2) is a critical biomarker for heart failure (HF) diagnosis and prognosis, yet conventional antibody-based detection methods suffer from time-consuming protocols and high costs and involve complex detection procedures. To address these challenges, we first screened high-affinity aptamers under clinically relevant conditions and then coupled with the CRISPR/Cas12a system to develop a fluorescence aptasensor for rapid and sensitive sST2 detection. A serum matrix was introduced during aptamer selection to enhance specificity and anti-interference performance in real biological environments. Three sST2-specific aptamers (Apt-1, Apt-2, and Apt-3) were identified with dissociation constants (KD) of 8.42, 46.08, and 25.02 nM, respectively. Among these, Apt-1 demonstrated superior performance, which was utilized to construct a fluorescence biosensor combining aptamer recognition with CRISPR/Cas12a trans-cleavage signal amplification. The sensor achieved a broad linear detection range (5-120 ng/mL) and an ultralow limit of detection (LOD, 0.816 ng/mL) when applied in detecting sST2 in both the buffer and human serum. Notably, the platform exhibited exceptional resistance to interference from HF-related proteins and maintained high accuracy in clinical serum samples, showing a strong correlation (R[2] = 0.9794) with enzyme-linked immunosorbent assay (ELISA) results. By integration of serum-matrix screening and CRISPR-based signal enhancement, this work establishes a robust, cost-effective, and rapid diagnostic tool for sST2 detection.},
}
@article {pmid40369543,
year = {2025},
author = {Zhang, T and Liu, G and Sun, S and Meng, Z and Qiu, Y and Ding, P},
title = {A novel tri-mode detection platform for ampicillin and drug resistance genes by CRISPR-driven luminescent nanozymes.},
journal = {Journal of nanobiotechnology},
volume = {23},
number = {1},
pages = {346},
pmid = {40369543},
issn = {1477-3155},
support = {82373635//the National Natural Science Foundation of China/ ; },
mesh = {*Ampicillin/analysis/pharmacology ; *Biosensing Techniques/methods ; *Anti-Bacterial Agents/analysis/pharmacology ; *CRISPR-Cas Systems ; Colorimetry ; Aptamers, Nucleotide/chemistry ; Smartphone ; Manganese Compounds/chemistry ; Oxides/chemistry ; Polymers/chemistry ; Ferrosoferric Oxide/chemistry ; Limit of Detection ; Clustered Regularly Interspaced Short Palindromic Repeats ; *Drug Resistance, Bacterial/genetics ; beta-Lactamases/genetics ; Indoles ; },
abstract = {The antibiotic residues pose significant risks for bacterial resistance. To address the practical requirements for rapid, accurate, and on-site detection of antibiotic residues and monitoring the abundance of associated resistance genes, we report a smartphone-integrated multi-mode platform. The platform is aimed to simultaneous, accurate, and visual quantitative detection of ampicillin (AMP) and β-lactam antibiotic resistance genes (blaTEM). Specifically, we developed a magnetically controlled fluorescence, colorimetric, and photothermal biosensor based on a magnetic separation unit (aminated modified complementary DNA chain (NH2-cDNA) loading on the surface of Ferrosoferric Oxide@polydopamine (Fe3O4@PDA, FP), FP@cDNA) and a signal unit (the aptamer nucleic acid chain modified by phosphate group linked to Prussian blue@UiO-66@manganese dioxide (PB@UiO-66@MnO2, PUM) through Zr-O-P bond, PUM@Apt), for the integrated detection of AMP and blaTEM. By utilizing complementary base pairing between FP@cDNA and PUM@Apt, along with precise aptamer recognition the AMP, we achieved the fluorescence quantitative detection of AMP by measuring the signal unit in the supernatant. Subsequently, the difference of signal units in colorimetric process leads to a varying conversion rate of oxidized 3,3',5,5'-Tetramethylbenzidine (oxTMB), enabling the output of colorimetric and photothermal signals. The competitive binding of aptamers permitting the determination of AMP in the range of 0-160 pM with a low detection limit (0.34 pM). Additionally, in the presence of blaTEM, the activated CRISPR/Cas12a indiscriminately cleaves the single-stranded portion of the FP@DNA@PUM complex obtained by magnetic separation. A PUM-based three-signal detection scheme was established for the sensitive determination of blaTEM with the limit of detection (LOD) of 1.03 pM. The integration of smartphone-assisted analysis broadens the potential of the platform for visual detection. Notably, the innovative platform, with its excellent stability, exhibits great potential as a simple yet robust approach for the simultaneously visually monitoring antibiotics and drug resistance genes, and holds promise in the field of kit development.},
}
@article {pmid40369382,
year = {2025},
author = {Ye, Y and Li, L and Chen, Y and Li, B and Xu, Z},
title = {Molecular methods for rapid detection and identification of foodborne pathogenic bacteria.},
journal = {World journal of microbiology &amp; biotechnology},
volume = {41},
number = {5},
pages = {175},
pmid = {40369382},
issn = {1573-0972},
mesh = {*Foodborne Diseases/microbiology/diagnosis ; *Bacteria/genetics/isolation &amp; purification/classification/pathogenicity ; *Molecular Diagnostic Techniques/methods ; *Food Microbiology/methods ; Humans ; Nucleic Acid Amplification Techniques/methods ; Polymerase Chain Reaction/methods ; Nucleic Acid Hybridization/methods ; Food Safety ; Clustered Regularly Interspaced Short Palindromic Repeats ; CRISPR-Cas Systems ; },
abstract = {Foodborne pathogenic bacteria are one of the main factors causing food safety issues. The rapid and accurate detection of pathogenic bacteria using molecular techniques is an effective and powerful strategy for preventing and controlling outbreaks of foodborne diseases, thereby ensuring food safety. This article summarizes the rapid and efficient molecular diagnostic techniques for detecting pathogenic bacteria, including polymerase chain reaction and its derivatives, isothermal amplification, DNA hybridization, genomic sequencing, and Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)/CRISPR-associated (CRISPR/Cas)-based detection technique. Through a comparative analysis of the technical principles, advantages, and potential limitations of these diagnostic methods, as well as an outlook on the future development directions for molecular biological detection technology, which will provide a valuable reference for developing more accurate, convenient, and sensitive methods for foodborne pathogens detection, and will help better address the challenges posed by foodborne diseases, thereby ensuring public health and safety.},
}
@article {pmid40369306,
year = {2025},
author = {Nie, Y and Li, X and Yang, W and Fei, S and Wang, Y and Li, Y and Zhang, K and Kang, J and Cheng, Y and Wang, H and Liu, D},
title = {Concanavalin-A-assisted extraction-free one-pot RPA-CRISPR/Cas12a assay for rapid detection of HPV16.},
journal = {Mikrochimica acta},
volume = {192},
number = {6},
pages = {354},
pmid = {40369306},
issn = {1436-5073},
support = {22QNFC098//Youth Independent Innovation Science Foundation of the General Hospital of the Chinese People's Liberation Army/ ; 22QNFC098//Youth Independent Innovation Science Foundation of the General Hospital of the Chinese People's Liberation Army/ ; 24JSZ12//Military Family Planning Program of China/ ; 24JSZ12//Military Family Planning Program of China/ ; },
mesh = {*Human papillomavirus 16/genetics/isolation &amp; purification ; Humans ; *Nucleic Acid Amplification Techniques/methods ; *CRISPR-Cas Systems ; *Concanavalin A/chemistry ; DNA, Viral/genetics/analysis ; Papillomavirus Infections/diagnosis/virology ; Female ; Recombinases/metabolism ; },
abstract = {Human papillomavirus (HPV) infection is a major threat to women's health worldwide. High-risk subtypes, particularly HPV16, require rigorous screening and long-term surveillance to control cervical cancer. However, traditional HPV testing is hampered by the need for nucleic acid extraction, reliance on specialized technicians, and fluorescence detection equipment, limiting its suitability for rapid on-site testing. In this study, we developed a Concanavalin A-assisted extraction-free one-pot recombinase polymerase amplification (RPA) CRISPR/Cas12a assay (ConRCA) for HPV16. Concanavalin A-coated magnetic beads were used for target enrichment and nucleic acid-extraction-free processing. Suboptimal protospacer-adjacent motifs were used to achieve a one-pot RPA-CRISPR/Cas12a assay. The ConRCA assay can be completed in approximately 25 min under isothermal conditions and can detect at least 1.2 copies/μL of HPV16 genomic DNA using a fluorescence reader or test strip, demonstrating comparable sensitivity to qPCR. The feasibility of this detection method was evaluated with 31 unextracted clinical samples. Compared with qPCR, the overall sensitivity was 95% (19/20), and the specificity was 100% (11/11). Our results indicate that the ConRCA assay has great potential utility as a point-of-care testing for the rapid identification of HPV.},
}
@article {pmid40369106,
year = {2025},
author = {Guo, T and Yang, J and Zhou, N and Sun, X and Huan, C and Lin, T and Bao, G and Hu, J and Li, G},
title = {Cas3 of type I-Fa CRISPR-Cas system upregulates bacterial biofilm formation and virulence in Acinetobacter baumannii.},
journal = {Communications biology},
volume = {8},
number = {1},
pages = {750},
pmid = {40369106},
issn = {2399-3642},
support = {82073611//National Natural Science Foundation of China (National Science Foundation of China)/ ; },
mesh = {*Biofilms/growth &amp; development ; *Acinetobacter baumannii/pathogenicity/genetics/physiology ; Animals ; Virulence/genetics ; Mice ; *CRISPR-Cas Systems ; *Acinetobacter Infections/microbiology ; *Bacterial Proteins/genetics/metabolism ; Gene Expression Regulation, Bacterial ; Virulence Factors/genetics/metabolism ; *CRISPR-Associated Proteins/genetics/metabolism ; Female ; Mice, Inbred BALB C ; },
abstract = {Acinetobacter baumannii (A. baumannii) is an important pathogen causing various nosocomial infections. CRISPR-Cas system is the adaptive immune system of bacteria, which is also closely related to the drug resistance and virulence of bacteria. However, the effect and mechanism of cas3 (type I-Fa) in A. baumannii is still unclear. In this study, we successfully constructed a cas3 deletion mutant (19606Δcas3) and complemented strain (19606Δcas3/pcas3) to study the regulatory mechanism of type I-Fa cas3 on bacterial virulence. Our results showed that deletion of cas3(type I-Fa) significantly reduced the biofilm formation, virulence and pathogenicity to mice. The organ bacterial load of mice infected with cas3 deletion strain was significantly reduced, the lung inflammation was slightly changed, and the serum cytokine level was also decreased. All results demonstrated that cas3 enhanced the virulence and pathogenicity of A. baumannii. Mechanism analysis showed that deletion of cas3 can lead to the down-regulation of virulence factors such as biofilm formation related factors and outer membrane protein A(ompA). In addition, cas3 was also involved in the regulation of carbon metabolism and oxidative phosphorylation pathway of A. baumannii. Altogether, our study may provide cas3 as a therapeutic target in the future because of the close link to the virulence of A. baumannii.},
}
@article {pmid40368251,
year = {2025},
author = {Ramadan, NK and Gaber, N and Ali, NM and Amer, OSO and Soliman, H},
title = {SHERLOCK, a novel CRISPR-Cas13a-based assay for detection of infectious bursal disease virus.},
journal = {Journal of virological methods},
volume = {337},
number = {},
pages = {115185},
doi = {10.1016/j.jviromet.2025.115185},
pmid = {40368251},
issn = {1879-0984},
mesh = {*Infectious bursal disease virus/isolation &amp; purification/genetics ; Animals ; Chickens/virology ; *Birnaviridae Infections/diagnosis/veterinary/virology ; *Poultry Diseases/diagnosis/virology ; Sensitivity and Specificity ; *CRISPR-Cas Systems ; *Molecular Diagnostic Techniques/methods ; *Nucleic Acid Amplification Techniques/methods ; RNA, Viral/genetics ; },
abstract = {Infectious bursal disease (IBD) is an extremely contagious viral infection that primarily affects young chicks, leading to significant economic losses in the poultry industry. The disease is caused by a double-stranded RNA virus of the genus Avibirnavirus, family Birnaviridae, namely, the infectious bursal disease virus (IBDV). Unfortunately, current methods for detecting IBDV lack adequate sensitivity. Accordingly, the advantages of the Specific High Sensitivity Enzymatic Reporter UnLOCKing (SHERLOCK) assay were employed to develop an ultrasensitive assay (IBD-SHERLOCK assay) for the detection of IBDV in clinical chicken tissues. The assay comprises two steps: isothermal preamplification of the target RNA through reverse transcription recombinase polymerase amplification (RT-RPA) and a subsequent detection step, which is based on the CRISPR-Cas13a system. The integration of lateral flow (LFD) visual detection of the IBD-SHERLOCK products strengthens the feasibility of the assay for use as a point-of-care test in chicken farms. Compared with RT-qPCR, this method exhibited ultra-analytical and clinical sensitivity. The assay has a lower detection limit of 5 aM, which is equivalent to three IBDV-RNA molecules. The assay demonstrated the ability to detect IBDV-RNA in 70 clinical field samples, 15 of which tested negative by RT-qPCR. This evidence highlights its superior sensitivity and potential for early detection of IBDV in chicken tissues. This study effectively established and verified a CRISPR-based diagnostic test for the early detection of IBDV in clinical chicken tissues, demonstrating remarkable specificity and sensitivity. The IBD-SHERLOCK assay can be used as a highly sensitive point-of-care diagnostic tool in chicken farms.},
}
@article {pmid40366179,
year = {2025},
author = {Zhang, Y and Li, W and Chen, S and Zhang, Y and Zhu, Y and Lan, F and Du, H and Fan, R and Zhu, J and Pan, W and Situ, B and Zheng, L and Luo, S and Yan, X},
title = {Layered-Responsive Multivalent Tetrahedral DNA Framework-Decorated CRISPR-Cas12a Nanocapsule Enables Precise and Enhanced Tumor Chemotherapy.},
journal = {ACS nano},
volume = {19},
number = {20},
pages = {19274-19286},
doi = {10.1021/acsnano.5c01747},
pmid = {40366179},
issn = {1936-086X},
mesh = {*Doxorubicin/pharmacology/chemistry/administration &amp; dosage ; Humans ; *DNA/chemistry ; *CRISPR-Cas Systems ; *Nanocapsules/chemistry ; Animals ; Cell Line, Tumor ; Mice ; *Antineoplastic Agents/pharmacology/chemistry ; *Antibiotics, Antineoplastic/pharmacology/chemistry ; Neoplasms/drug therapy ; },
abstract = {The lack of selective tumor targeting and the high toxicity of conventional chemotherapy treatments remain major challenges in cancer therapy. Here, we develop a self-controlled DNA nanostructure-CRISPR-12a system, a triple-locked cascade tumor therapy nanocapsule (Tatna), for efficient and targeted tumor treatment. Tatna integrates structural DNA tetrahedrons (DTs) with high drug-loading capacity, Cas12a/crRNA ribonucleoprotein (Cas12a RNP), and doxorubicin (DOX) to enable multisite response for precise drug delivery and augmented tumor treatment. By incorporation of a nucleolin-targeting aptamer, Tatna achieves selective targeting and efficient tumor cell internalization. Encapsulation in pH-responsive poly l-lactic-co-glycolic acid (PLGA) nanocapsule ensures stable circulation and controlled release of both DOX and Cas12a until tumor-specific activation in the acidic microenvironment. The Cas12a RNP, triggered by APE1 mRNA overexpression in tumor cells, induces trans-cleavage of DTs, releasing DOX and Cas12a to transport into the nucleus and induce enhanced cell apoptosis. This self-regulating and multifunctional approach enhances the efficacy of chemotherapy while reducing off-target effects. Tatna's programmable, tumor-specific delivery system represents a powerful strategy for advancing precision medicine and personalized cancer treatment.},
}
@article {pmid40363763,
year = {2025},
author = {Wang, L and Liu, Y and Song, H and Zhang, X and Wang, Y},
title = {Conditional Control of CRISPR/Cas9 Function by Chemically Modified Oligonucleotides.},
journal = {Molecules (Basel, Switzerland)},
volume = {30},
number = {9},
pages = {},
pmid = {40363763},
issn = {1420-3049},
support = {22307107//the National Natural Science Foundation of China/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; *Oligonucleotides/chemistry/genetics ; Humans ; *RNA, Guide, CRISPR-Cas Systems/genetics/chemistry ; Animals ; },
abstract = {The CRISPR (clustered regularly interspaced short palindromic repeats) system has emerged as a revolutionary gene-editing tool with immense potential in gene therapy, functional genomics, and beyond. However, achieving precise spatiotemporal control of gene editing in specific cells and tissues while effectively mitigating potential risks, such as off-target effects, remains a key challenge for its clinical translation. To overcome these limitations, researchers have developed innovative strategies based on chemical modifications of oligonucleotides to enhance the precision, efficiency, and controllability of CRISPR/Cas9-mediated gene editing. By introducing conditional responsive elements, such as photosensitive groups, small-molecule responsive units, and supramolecular structures, they have successfully achieved precise spatiotemporal and dose-dependent regulation of CRISPR/Cas9 function. This review provides a comprehensive overview of recent advancements in gRNA regulation strategies based on chemical modifications of oligonucleotides, discussing their applications in improving the efficiency, specificity, and controllability of CRISPR/Cas9 editing. We also highlight the challenges associated with the conditional control of gRNA and offer insights into future directions for the chemical regulation of gRNA to further advance CRISPR/Cas9 technology.},
}
@article {pmid40362657,
year = {2025},
author = {Seijas, A and Cora, D and Novo, M and Al-Soufi, W and Sánchez, L and Arana, &#xc1;J},
title = {CRISPR/Cas9 Delivery Systems to Enhance Gene Editing Efficiency.},
journal = {International journal of molecular sciences},
volume = {26},
number = {9},
pages = {},
pmid = {40362657},
issn = {1422-0067},
support = {ND//Campus Terra, University of Santiago de Compostela/ ; ND//Fundaci&#xf3;n Caixa Rural Galega Tom&#xe1;s Notario Vacas/ ; investment line no.1 of its component number 17//Spain's Recovery and Resilience Plan, Complementary RTDI Plan for Marine Science/ ; },
mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems ; Humans ; Animals ; *Gene Transfer Techniques ; Nanoparticles/chemistry ; Lipids/chemistry ; },
abstract = {CRISPR/Cas9 has revolutionized genome editing by enabling precise and efficient genetic modifications across multiple biological systems. Despite its growing therapeutic potential, key challenges remain in mitigating off-target effects, minimizing immunogenicity, and improving the delivery of CRISPR components into target cells. This review provides an integrated analysis of physical, viral, and non-viral delivery systems, highlighting recent advances in the use of lipid nanoparticles, polymeric carriers, and hybrid platforms. We also examine an often overlooked factor: the aggregation behavior of the Cas9 protein, which may interfere with cellular uptake, the encapsulation efficiency, and nuclear localization. By comparing delivery platforms and their reported editing outcomes, we identify critical physicochemical parameters that influence therapeutic success. Finally, we propose standardized methods to assess Cas9 encapsulation and aggregation and discuss translational barriers such as manufacturing scalability and regulatory requirements. These insights aim to guide the development of safer and more effective CRISPR/Cas9-based therapies.},
}
@article {pmid40362595,
year = {2025},
author = {Luo, X and Weidinger, E and Burghardt, T and Höhn, M and Wagner, E},
title = {CRISPR/Cas9 Ribonucleoprotein Delivery Enhanced by Lipo-Xenopeptide Carriers and Homology-Directed Repair Modulators: Insights from Reporter Cell Lines.},
journal = {International journal of molecular sciences},
volume = {26},
number = {9},
pages = {},
pmid = {40362595},
issn = {1422-0067},
support = {SFB1032 (project-ID 201269156) sub-project B4//Deutsche Forschungsgemeinschaft/ ; No 825825 UPGRADE//European Union/ ; CNATM, number 03ZU1201AA//Federal Ministry of Education and Research/ ; doctoral fellowship to XL//China Scholarship Council/ ; },
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; *Recombinational DNA Repair/drug effects ; *Ribonucleoproteins/genetics/metabolism ; HeLa Cells ; DNA End-Joining Repair ; Peptides ; },
abstract = {CRISPR-Cas9 genome editing is a versatile platform for studying and treating various diseases. Homology-directed repair (HDR) with DNA donor templates serves as the primary pathway for gene correction in therapeutic applications, but its efficiency remains a significant challenge. This study investigates strategies to enhance gene correction efficiency using a T-shaped lipo-xenopeptide (XP)-based Cas9 RNP/ssDNA delivery system combined with various HDR enhancers. Nu7441, a known DNA-PKcs inhibitor, was found to be most effective in enhancing HDR-mediated gene correction. An over 10-fold increase in HDR efficiency was achieved by Nu7441 in HeLa-eGFPd2 cells, with a peak HDR efficiency of 53% at a 5 nM RNP concentration and up to 61% efficiency confirmed by Sanger sequencing. Surprisingly, the total gene editing efficiency including non-homologous end joining (NHEJ) was also improved. For example, Nu7441 boosted exon skipping via NHEJ-mediated splice site destruction by 30-fold in a DMD reporter cell model. Nu7441 modulated the cell cycle by reducing cells in the G1 phase and extending the S and G2/M phases without compromising cellular uptake or endosomal escape. The enhancement in genome editing by Nu7441 was widely applicable across several cell lines, several Cas9 RNP/ssDNA carriers (LAF-XPs), and also Cas9 mRNA/sgRNA/ssDNA polyplexes. These findings highlight a novel and counterintuitive role for Nu7441 as an enhancer of both HDR and total gene editing efficiency, presenting a promising strategy for Cas9 RNP-based gene therapy.},
}
@article {pmid40362588,
year = {2025},
author = {Arana, &#xc1;J and González-Llera, L and Barreiro-Iglesias, A and Sánchez, L},
title = {Emerging Frontiers in Zebrafish Embryonic and Adult-Derived Cell Lines.},
journal = {International journal of molecular sciences},
volume = {26},
number = {9},
pages = {},
pmid = {40362588},
issn = {1422-0067},
support = {PID2020-115121GB-I00//Ministerio de Ciencia, Innovaci&#xf3;n y Universidades/ ; PID2023-147266NB-I00//Ministerio de Ciencia, Innovaci&#xf3;n y Universidades/ ; ED431C 2022/33//Xunta de Galicia/ ; },
mesh = {Animals ; *Zebrafish/embryology/genetics ; Cell Line ; *Embryo, Nonmammalian/cytology ; CRISPR-Cas Systems ; Cell Culture Techniques/methods ; Humans ; },
abstract = {Zebrafish (Danio rerio) has become a pivotal vertebrate model in biomedical research, renowned for its genetic similarity to humans, optical transparency, rapid embryonic development, and amenability to experimental manipulation. In recent years, the derivation of cell lines from zebrafish embryos has unlocked new possibilities for in vitro studies across developmental biology, toxicology, disease modeling, and genetic engineering. These embryo-derived cultures offer scalable, reproducible, and ethically favorable alternatives to in vivo approaches, enabling high-throughput screening and mechanistic exploration under defined conditions. This review provides a comprehensive overview of protocols for establishing and maintaining zebrafish embryonic cell lines, emphasizing culture conditions, pluripotency features, transfection strategies, and recent innovations such as genotype-defined mutant lines generated via CRISPR/Cas9 and feeder-free systems. We also highlight emerging applications in oncology, regenerative medicine, and functional genomics, positioning zebrafish cell lines as versatile platforms bridging animal models and next-generation in vitro systems. Its continued optimization holds promise for improved reproducibility, reduced animal use, and expanded translational impact in biomedical research.},
}
@article {pmid40362571,
year = {2025},
author = {Herreno-Pachón, AM and Leal, AF and Khan, S and Alméciga-Díaz, CJ and Tomatsu, S},
title = {CRISPR/nCas9-Edited CD34+ Cells Rescue Mucopolysaccharidosis IVA Fibroblasts Phenotype.},
journal = {International journal of molecular sciences},
volume = {26},
number = {9},
pages = {},
pmid = {40362571},
issn = {1422-0067},
support = {1R01HD102545-01A1/GF/NIH HHS/United States ; },
mesh = {Humans ; *CRISPR-Cas Systems ; *Fibroblasts/metabolism/pathology ; *Gene Editing/methods ; *Antigens, CD34/metabolism/genetics ; Genetic Therapy/methods ; *Mucopolysaccharidosis IV/therapy/genetics/metabolism/pathology ; Phenotype ; Animals ; Mice ; Chondroitinsulfatases/genetics/metabolism ; },
abstract = {Mucopolysaccharidosis (MPS) IVA is a bone-affecting lysosomal storage disease (LSD) caused by impaired degradation of the glycosaminoglycans (GAGs) keratan sulfate (KS) and chondroitin 6-sulfate (C6S) due to deficient N-acetylgalactosamine-6-sulfatase (GALNS) enzyme activity. Previously, we successfully developed and validated a CRISPR/nCas9-based gene therapy (GT) to insert an expression cassette at the AAVS1 and ROSA26 loci in human MPS IVA fibroblasts and MPS IVA mice, respectively. In this study, we have extended our approach to evaluate the effectiveness of our CRISPR/nCas9-based GT in editing human CD34+ cells to mediate cross-correction of MPS IVA fibroblasts. CD34+ cells were electroporated with the CRISPR/nCas9 system, targeting the AAVS1 locus. The nCas9-mediated on-target donor template insertion, and the stemness of the CRISPR/nCas-edited CD34+ cells was evaluated. Additionally, MPS IVA fibroblasts were co-cultured with CRISPR/nCas-edited CD34+ cells to assess cross-correction. CRISPR/nCas9-based gene editing did not affect the stemness of CD34+ cells but did lead to supraphysiological levels of the GALNS enzyme. Upon co-culture, MPS IVA fibroblasts displayed a significant increase in the GALNS enzyme activity along with lysosomal mass reduction, pro-oxidant profile amelioration, mitochondrial mass recovery, and pro-apoptotic and pro-inflammatory profile improvement. These results show the potential of our CRISPR/nCas9-based GT to edit CD34+ cells to mediate cross-correction.},
}
@article {pmid40362308,
year = {2025},
author = {Haider, S and Mussolino, C},
title = {Fine-Tuning Homology-Directed Repair (HDR) for Precision Genome Editing: Current Strategies and Future Directions.},
journal = {International journal of molecular sciences},
volume = {26},
number = {9},
pages = {},
pmid = {40362308},
issn = {1422-0067},
support = {CA311-41//Deutsche Forschungsgemeinschaft/ ; },
mesh = {*Gene Editing/methods ; Humans ; *Recombinational DNA Repair ; *CRISPR-Cas Systems ; DNA End-Joining Repair ; Animals ; DNA Breaks, Double-Stranded ; },
abstract = {CRISPR-Cas9 is a powerful genome-editing technology that can precisely target and cleave DNA to induce double-strand breaks (DSBs) at almost any genomic locus. While this versatility holds tremendous therapeutic potential, the predominant cellular pathway for DSB repair-non-homologous end-joining (NHEJ)-often introduces small insertions or deletions that disrupt the target site. In contrast, homology-directed repair (HDR) utilizes exogenous donor templates to enable precise gene modifications, including targeted insertions, deletions, and substitutions. However, HDR remains relatively inefficient compared to NHEJ, especially in postmitotic cells where cell cycle constraints further limit HDR. To address this challenge, numerous methodologies have been explored, ranging from inhibiting key NHEJ factors and optimizing donor templates to synchronizing cells in HDR-permissive phases and engineering HDR-enhancing fusion proteins. These strategies collectively aim to boost HDR efficiency and expand the clinical and research utility of CRISPR-Cas9. In this review, we discuss recent advances in manipulating the balance between NHEJ and HDR, examine the trade-offs and practical considerations of these approaches, and highlight promising directions for achieving high-fidelity genome editing in diverse cell types.},
}
@article {pmid40362304,
year = {2025},
author = {Shiryaeva, O and Tolochko, C and Alekseeva, T and Dyachuk, V},
title = {Targets and Gene Therapy of ALS (Part 1).},
journal = {International journal of molecular sciences},
volume = {26},
number = {9},
pages = {},
pmid = {40362304},
issn = {1422-0067},
mesh = {Humans ; *Amyotrophic Lateral Sclerosis/therapy/genetics/pathology ; *Genetic Therapy/methods ; Animals ; Superoxide Dismutase-1/genetics ; C9orf72 Protein/genetics ; Mutation ; Gene Editing ; RNA-Binding Protein FUS/genetics ; Oligonucleotides, Antisense/therapeutic use ; CRISPR-Cas Systems ; DNA-Binding Proteins/genetics ; RNA Interference ; MicroRNAs/genetics ; Disease Models, Animal ; RNA, Small Interfering/genetics ; Motor Neurons/metabolism/pathology ; },
abstract = {Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by the selective death of motor neurons, which causes muscle atrophy. Genetic forms of ALS are recorded only in 10% of cases. However, over the past decade, studies in genetics have substantially contributed to our understanding of the molecular mechanisms underlying ALS. The identification of key mutations such as SOD1, C9orf72, FUS, and TARDBP has led to the development of targeted therapy that is gradually being introduced into clinical trials, opening up a broad range of opportunities for correcting these mutations. In this review, we aimed to present an extensive overview of the currently known mechanisms of motor neuron degeneration associated with mutations in these genes and also the gene therapy methods for inhibiting the expression of their mutant proteins. Among these, antisense oligonucleotides, RNA interference (siRNA and miRNA), and gene-editing (CRISPR/Cas9) methods are of particular interest. Each has shown its efficacy in animal models when targeting mutant genes, whereas some of them have proven to be efficient in human clinical trials.},
}
@article {pmid40361099,
year = {2025},
author = {Xie, H and Bourgade, B and Stensjö, K and Lindblad, P},
title = {dCas12a-mediated CRISPR interference for multiplex gene repression in cyanobacteria for enhanced isobutanol and 3-methyl-1-butanol production.},
journal = {Microbial cell factories},
volume = {24},
number = {1},
pages = {104},
pmid = {40361099},
issn = {1475-2859},
support = {2021-01669//Svenska Forskningsr&#xe5;det Formas/ ; CTS 20:412//Carl Tryggers Stiftelse f&#xf6;r Vetenskaplig Forskning/ ; 2024-00443//Vinnova/ ; },
mesh = {*Butanols/metabolism ; *Metabolic Engineering/methods ; *CRISPR-Cas Systems ; *Synechocystis/genetics/metabolism ; *Pentanols/metabolism ; Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {BACKGROUND: Cyanobacteria of the genera Synechocystis and Synechococcus have emerged as promising platforms for metabolic engineering endeavors aimed at converting carbon dioxide into valuable fuels and chemicals, thus addressing the pressing energy demand and mitigating global climate change. Notably, Synechocystis sp. strain PCC 6803 (Synechocystis) has been engineered to produce isobutanol (IB) and 3-methyl-1-butanol (3M1B) via heterologous expression of α-ketoisovalerate decarboxylase (Kivd). Despite these advances, the achieved IB/3M1B titers remain low. CRISPR interference (CRISPRi), an emerging tool for targeted gene repression, has demonstrated success in various cellular systems to enhance biochemical productivity.<br><br>RESULTS: In this study, we developed a dCas12a-mediated CRISPRi system (CRISPRi-dCas12a) that effectively blocked the transcriptional initiation/elongation of essential gene(s), resulting in up to 60% gene repression in Synechocystis. Subsequently, the CRISPRi-dCas12a system was successfully integrated into an IB/3M1B producer strain, where it exhibited target gene repression under optimal cultivation conditions. To identify gene targets involved in metabolic pathways potentially limiting IB/3M1B biosynthesis, we initially designed a CRISPR RNA (crRNA) library targeting fifteen individual gene(s), where repression of ten genes significantly increased IB/3M1B production per cell. Moreover, a synergetic effect was observed on IB/3M1B production by designing a single crRNA targeting multiple genes for simultaneous repression. A final strain HX106, featuring dual repression of ppc and gltA, both involved in the TCA cycle, resulted in 2.6-fold and 14.8-fold improvement in IB and 3M1B production per cell, respectively.<br><br>CONCLUSIONS: Our findings underscore the effectiveness of the CRISPRi-dCas12a system in Synechocystis for identifying competing pathways and redirecting carbon flux to enhance IB/3M1B production. Furthermore, this study established a solid groundwork for utilizing an expanded CRISPRi-crRNA library to undertake genome-wide exploration of potential competing pathways not only for IB/3M1B biosynthesis but also for other diverse biofuels and biochemical production processes.},
}
@article {pmid40361067,
year = {2025},
author = {Avci, FG and Prasun, T and Wendisch, VF},
title = {Metabolic engineering for microbial production of sugar acids.},
journal = {BMC biotechnology},
volume = {25},
number = {1},
pages = {36},
pmid = {40361067},
issn = {1472-6750},
support = {031B1737C//German Federal Ministry of Education and Research (BMBF) as part of the collaborative research project ForceYield2/ ; online publication fund//Deutsche Forschungsgemeinschaft/ ; open access publication fund//Universit&#xe4;t Bielefeld/ ; },
mesh = {*Metabolic Engineering/methods ; *Sugar Acids/metabolism ; Fermentation ; Synthetic Biology ; Oxidation-Reduction ; *Bacteria/metabolism/genetics ; },
abstract = {Carbohydrates including sugar acids are commonly used as carbon sources in microbial biotechnology. These sugar acids are themselves desirable and often overlooked targets for biobased production since they find applications in a broad range of industries, examples include food, construction, medical, textile, and polymer industries. Different stages of oxidation for natural sugar acids can be distinguished. Oxidation of the aldehyde group yields aldonic acids, oxidation of the primary hydroxy group leads to uronic acids, and both oxidations combined yield aldaric acids. While the chemical oxidation of sugars to their acid forms often is a one-pot reaction under harsh conditions, their biosynthesis is much more delicate. Bio-based production can involve enzymatic conversion, whole-cell biotransformation, and fermentation. Generally, the in vivo approaches are preferred because they are less resource-intensive than enzymatic conversion. Metabolic engineering plays a crucial role in optimizing microbial strains for efficient sugar acid production. Strategies include pathway engineering to overexpress key enzymes involved in sugar oxidation, deletion of competing pathways to enhance the precursor availability and eliminate the product consumption, cofactor balancing for efficient redox reactions, and transporter engineering to facilitate precursor import or sugar acid export. Synthetic biology tools, such as CRISPR-Cas and dynamic regulatory circuits, have further improved strain development by enabling precise genetic modifications and adaptive control of metabolic fluxes. The usage of plant biomass hydrolysates for bio-based production further adds to the environmental friendliness of the in vivo approaches. This review highlights the different approaches for the production of C5 and C6 sugar acids, their applications, and their catabolism in microbes.},
}
@article {pmid40360969,
year = {2025},
author = {Dong, J and Ma, W and Zhou, S and Li, X and Deng, L and Hou, C and Huo, D},
title = {Tri-Mode CRISPR-Based Biosensor for miRNA Detection: Enhancing Clinical Diagnostics with Cross-Validation.},
journal = {Analytical chemistry},
volume = {97},
number = {20},
pages = {10938-10946},
doi = {10.1021/acs.analchem.5c02348},
pmid = {40360969},
issn = {1520-6882},
mesh = {*MicroRNAs/analysis/genetics ; *Biosensing Techniques/methods ; Humans ; *CRISPR-Cas Systems ; Nucleic Acid Amplification Techniques ; Limit of Detection ; Electrochemical Techniques ; Metal-Organic Frameworks/chemistry ; Methylene Blue/chemistry ; },
abstract = {In vitro diagnostics require the accurate detection of disease-associated target biomolecules at ultralow concentrations. A multimode sensing strategy is considered as a potential method for in vitro diagnosis because it allows cross-validation of test results through data complementation and self-calibration, and provides double confirmation. Here, we present a CRISPR/Cas12a-powered trimode biosensor (CPTMB) for ultrasensitive and reliable analysis of miRNA. Briefly, the presence of target miRNA initiates rolling circle extension-driven loop-mediated isothermal amplification (R-LAMP), which subsequently activates the trans-cleavage activity of CRISPR/Cas12a. Then, the hairpin probe (HP) biogate on nucleic acid-functionalized MB@Fe-MOF signal probe was degraded by Cas12a, leading to the release of methylene blue (MB) signal molecules encapsulated within Fe-MOF nanocarriers. Due to the capability of MB to generate output responses across three distinct modes: electrochemical (EC), fluorescence (FL), and ultraviolet-visible spectroscopy (UV-vis), a trimodal sensing system is achieved. Benefiting from the efficient signal amplification capabilities of R-LAMP and CRISPR/Cas12a, this strategy enables rapid detection of target miRNA at femtomolar levels within 70 min. Furthermore, the detection results across the three modes cross-validate one another, thereby enhancing the reliability of the analysis. More importantly, the platform has been successfully applied to miRNA analysis in real samples, and the detection results are in good agreement with those of the standard method RT-qPCR, indicating its great potential in the clinical diagnosis of early-stage cancer.},
}
@article {pmid40360740,
year = {2025},
author = {Tei, C and Hata, S and Mabuchi, A and Okuda, S and Ito, KK and Genova, M and Fukuyama, M and Yamamoto, S and Chinen, T and Toyoda, A and Kitagawa, D},
title = {Comparative analysis of multiple DNA double-strand break repair pathways in CRISPR-mediated endogenous tagging.},
journal = {Communications biology},
volume = {8},
number = {1},
pages = {749},
pmid = {40360740},
issn = {2399-3642},
support = {18K06246, 19H05651, 20K15987, 20K22701, 21H02623, 22H02629, 22K19305, 22K19370, 22K20624, 23K14176//Japan Society for the Promotion of Science London (JSPS London)/ ; JPMJPR21EC//MEXT | JST | Precursory Research for Embryonic Science and Technology (PRESTO)/ ; },
mesh = {*DNA Breaks, Double-Stranded ; *CRISPR-Cas Systems ; Humans ; *DNA End-Joining Repair ; *Gene Editing/methods ; *DNA Repair ; Gene Knock-In Techniques ; Recombinational DNA Repair ; HEK293 Cells ; },
abstract = {CRISPR-mediated endogenous tagging is a powerful tool in biological research. Inhibiting the non-homologous end joining (NHEJ) pathway has been shown to improve the low efficiency of accurate knock-in via homology-directed repair (HDR). However, the influence of alternative double-stranded break (DSB) repair pathways on knock-in remains to be fully explored. In this study, our long-read amplicon sequencing analysis reveals various patterns of imprecise repair in CRISPR-mediated knock-in, even with NHEJ inhibition. Further suppressing either microhomology-mediated end joining (MMEJ) or single-strand annealing (SSA) reduces nucleotide deletions around the cut site, thereby elevating knock-in accuracy. Additionally, imprecise donor integration is reduced by inhibiting SSA, but not MMEJ. Particularly, SSA suppression reduced asymmetric HDR, a specific imprecise integration pattern, which we further confirm using a novel reporter system. These findings demonstrate the complex interplay of multiple DSB repair pathways in CRISPR-mediated knock-in and offer novel strategies, including SSA pathway targeting, to improve precise gene editing efficiency.},
}
@article {pmid40359875,
year = {2025},
author = {Morán Torres, JP and Lyu, J and Chen, X and Klaas, AM and Vonk, PJ and Lugones, LG and de Cock, H and Wösten, HAB},
title = {Single and combinatorial gene inactivation in Aspergillus niger using selected as well as genome-wide gRNA library pools.},
journal = {Microbiological research},
volume = {298},
number = {},
pages = {128204},
doi = {10.1016/j.micres.2025.128204},
pmid = {40359875},
issn = {1618-0623},
mesh = {*Aspergillus niger/genetics ; CRISPR-Cas Systems ; Gene Editing/methods ; *RNA, Guide, CRISPR-Cas Systems/genetics ; Plasmids/genetics ; *Gene Silencing ; Gene Library ; Genome, Fungal ; Genetic Vectors ; Phenotype ; Genes, Fungal ; Transformation, Genetic ; },
abstract = {Aspergillus niger is a saprotroph, a pathogen, an endophyte, a food spoiler and an important cell factory. Only a minor fraction of its genes has been experimentally characterized. We here set up a CRISPR/Cas9 mutagenesis screen for functional gene analysis using co-transformation of a pool of gene editing plasmids that are maintained under selection pressure and that each contain a gRNA. First, a pool of gRNA vectors was introduced in A. niger targeting five genes with easy selectable phenotypes. Transformants were obtained with all possible single, double, triple, quadruple and quintuple gene inactivation phenotypes. Their genotypes were confirmed using the gRNA sequences in the transforming vector as barcodes. Next, a gRNA library was introduced in A. niger targeting > 9600 genes. Gene nsdC was identified as a sporulation gene using co-transformation conditions that favored uptake of one or two gRNA construct(s) from the genome-wide vector pool. Together, CRISPR/Cas9 vectors with a (genome-wide) pool of gRNAs can be used for functional analysis of genes in A. niger with phenotypes that are the result of the inactivation of a single or multiple genes.},
}
@article {pmid40359808,
year = {2025},
author = {Liu, S and Xiao, G and Li, P and Xu, Y and Fan, X and Tian, L and Zhang, S and Zhang, G},
title = {Plasma-based ultrasensitive detection of Mycobacterium tuberculosis ESAT6/CFP10 fusion antigen using a CRISPR-driven aptamer fluorescence testing (CRAFT).},
journal = {Biosensors &amp; bioelectronics},
volume = {284},
number = {},
pages = {117566},
doi = {10.1016/j.bios.2025.117566},
pmid = {40359808},
issn = {1873-4235},
mesh = {*Mycobacterium tuberculosis/isolation &amp; purification/genetics ; Humans ; *Biosensing Techniques/methods ; *Bacterial Proteins/blood/genetics/isolation &amp; purification ; *Aptamers, Nucleotide/chemistry/genetics ; *Antigens, Bacterial/blood/genetics/isolation &amp; purification ; *Tuberculosis/diagnosis/microbiology/blood ; Limit of Detection ; CRISPR-Cas Systems ; Fluorescence ; SELEX Aptamer Technique ; },
abstract = {Tuberculosis (TB) screening in clinical diagnosis is challenging due to issues such as sputum dependence, time-consuming procedures, and high costs. In this study, we introduce a CRAFT (CRISPR-Driven Aptamer Fluorescence Testing), an aptamer-based CRISPR/Cas12a assay designed for the rapid and sensitive detection of Mycobacterium tuberculosis (Mtb) antigens from peripheral blood. Aptamer 3 (Ap3) and the aptamer-mediated probe (Aptamer-blocker 3-7) were selected through the Systematic Evolution of Ligands by Exponential Enrichment (SELEX). Ap3 demonstrated a dissociation constant (Kd) of 8.3E-7 M with the ESAT6/CFP10 fusion proteins (EC proteins), which are produced during the replicative phase of Mtb. Upon labeling the EC proteins with Aptamer-blocker 3-7 (Ap-blocker 3-7) probe, single-stranded DNA (ssDNA) blocker 3-7 was released, thereby completing the process for RPA-based CRISPR/Cas12a fluorescence detection. After optimizing multiple parameters, CRAFT achieved a detection limit of 0.1 ag/mL EC proteins (equivalent to 3 protein particles per mL) within 120 min from plasma sample to result. The method was validated with 86 clinical plasma samples confirmed the method's high diagnostic accuracy for Mtb infection (sensitivity: 97.1 %, 95 % confidence interval (CI) [0.849-0.998]); specificity: 98.0 %, 95 % CI [0.897-0.999]), supporting its utility in early therapeutic evaluation of tuberculosis management.},
}
@article {pmid40359574,
year = {2025},
author = {Zhou, J and Pang, R and Han, Y and Guo, Y and Wang, Y and Yang, H and Wang, W and Fu, X and Zhang, R and Zheng, X and Zhang, T and Zhang, Y and Wang, Q},
title = {CRISPR-Cas9-mediated knockout of OsKCS11 in rice reveals potential crosstalk between very-long-chain fatty acids and cytokinin.},
journal = {The Plant journal : for cell and molecular biology},
volume = {122},
number = {3},
pages = {e70208},
doi = {10.1111/tpj.70208},
pmid = {40359574},
issn = {1365-313X},
support = {2020B1515120086//Guangdong Basic and Applied Basic Research Foundation/ ; SZKF2103//Open Project Program of Panxi Crops Research and Utilization Key Laboratory of Sichuan Province/ ; 2023ZD04074//the Biological Breeding- National Science and Technology Major Projects/ ; 2021B0707010006//Key-Area Research and Development Program of Guangdong Province/ ; CAAS-CSIAF-202303//Innovation Program of CAAS/ ; 31961143015//National Natural Science Foundation of China/ ; 32070202//National Natural Science Foundation of China/ ; 2021YFYZ0016//Sichuan Science and Technology Program/ ; 2024NSFSC0314//Sichuan Science and Technology Program/ ; },
mesh = {*Oryza/genetics/metabolism ; *Cytokinins/metabolism ; CRISPR-Cas Systems/genetics ; *Fatty Acids/metabolism ; *Plant Proteins/genetics/metabolism ; Gene Knockout Techniques ; Gene Expression Regulation, Plant ; Plants, Genetically Modified ; *3-Oxoacyl-(Acyl-Carrier-Protein) Synthase/genetics/metabolism ; },
abstract = {Very-long-chain fatty acids (VLCFAs) play crucial roles in various physiological processes in plants. Through our investigation using a CRISPR-Cas9 knockout mutant library in rice, we identified a semi-dwarf rice mutant named CRISPR-Cas-based dwarf-1 (csd-1). This mutant displayed multiple developmental defects, such as decreased plant height, panicle length, seed size, and seed-setting rate. Whole-genome resequencing analysis revealed that a T-nucleotide insertion in β-ketoacyl-CoA synthase 11 (KCS11), responsible for the initial step in fatty acid elongation, was responsible for the observed defects in csd-1. The identity of csd-1 was confirmed through genetic complementation and CRISPR-Cas9-mediated knockout. Expression analysis indicated that OsKCS11 was present in various tissues, with differential abundance observed through RT-qPCR and promoter GUS staining, and strong localization at the node position by RNA in situ hybridization; furthermore, OsKCS11 protein was confirmed to be in the endoplasmic reticulum. Furthermore, csd-1 exhibited significantly reduced levels of linolenic acid (18:3), C24:0-OH, C28:0-alkanes, C29:0-alkanes, alpha-tocopherol, and C33:0-alkanes, while trans-nonadecenoic acid and behenic acid levels were increased. Cytokinin analysis revealed significant increases in isopentenyladenine (IPA) and cis-zeatin (cZ) levels in csd-1. Molecular investigations indicated upregulation of genes involved in cytokinin biosynthesis or signaling, suggesting a potential link between VLCFAs and cytokinin synthesis through acetyl-CoA. This study not only proposed an alternative gene mapping method based on whole-genome resequencing but also elucidated the mechanism by which VLCFAs influence cytokinin synthesis and signaling.},
}
@article {pmid40358185,
year = {2025},
author = {Boff, MO and Xavier, FAC and Diz, FM and Gonçalves, JB and Ferreira, LM and Zambeli, J and Pazzin, DB and Previato, TTR and Erwig, HS and Gonçalves, JIB and Bruzzo, FTK and Marinowic, D and da Costa, JC and Zanirati, G},
title = {mTORopathies in Epilepsy and Neurodevelopmental Disorders: The Future of Therapeutics and the Role of Gene Editing.},
journal = {Cells},
volume = {14},
number = {9},
pages = {},
pmid = {40358185},
issn = {2073-4409},
support = {Not applicable//Pontifical Catholic University of Rio Grande do Sul (PUCRS) - Brain Institute of Rio Grande do Sul (BraIns)/ ; },
mesh = {Humans ; *Gene Editing/methods ; *Neurodevelopmental Disorders/therapy/genetics ; *TOR Serine-Threonine Kinases/metabolism/genetics ; *Epilepsy/therapy/genetics ; Animals ; Genetic Therapy ; CRISPR-Cas Systems ; },
abstract = {mTORopathies represent a group of neurodevelopmental disorders linked to dysregulated mTOR signaling, resulting in conditions such as tuberous sclerosis complex, focal cortical dysplasia, hemimegalencephaly, and Smith-Kingsmore Syndrome. These disorders often manifest with epilepsy, cognitive impairments, and, in some cases, structural brain anomalies. The mTOR pathway, a central regulator of cell growth and metabolism, plays a crucial role in brain development, where its hyperactivation leads to abnormal neuroplasticity, tumor formation, and heightened neuronal excitability. Current treatments primarily rely on mTOR inhibitors, such as rapamycin, which reduce seizure frequency and tumor size but fail to address underlying genetic causes. Advances in gene editing, particularly via CRISPR/Cas9, offer promising avenues for precision therapies targeting the genetic mutations driving mTORopathies. New delivery systems, including viral and non-viral vectors, aim to enhance the specificity and efficacy of these therapies, potentially transforming the management of these disorders. While gene editing holds curative potential, challenges remain concerning delivery, long-term safety, and ethical considerations. Continued research into mTOR mechanisms and innovative gene therapies may pave the way for transformative, personalized treatments for patients affected by these complex neurodevelopmental conditions.},
}
@article {pmid40356437,
year = {2025},
author = {Kong, G and Li, R and Huang, W and Yang, Y and Guan, T and Liu, J and Li, W and Hsiang, T and Xi, P and Li, M and Jiang, Z},
title = {A RACK1 family protein regulates pathogenicity of Peronophythora litchii by acting as a scaffold for MAPK signal modules.},
journal = {Virulence},
volume = {16},
number = {1},
pages = {2503429},
pmid = {40356437},
issn = {2150-5608},
mesh = {*Plant Diseases/microbiology ; *Receptors for Activated C Kinase/metabolism/genetics ; *MAP Kinase Signaling System ; *Litchi/microbiology ; Virulence ; *Mitogen-Activated Protein Kinases/metabolism/genetics ; *Fungal Proteins/genetics/metabolism ; CRISPR-Cas Systems ; Gene Expression Regulation, Fungal ; },
abstract = {Litchi downy blight caused by Peronophythora litchii is the most destructive disease of litchi (Litchi chinensis). RACK1 (Receptor for activated C kinase 1) is a group of scaffold proteins, mainly involved in the regulation of various signaling pathways by interacting with signal transduction proteins and affecting the activity of these proteins. In this study, a RACK1 homologue identified in P. litchii, and named PlRACK1. The protein was found to interact with the mitogen-activated protein kinases, PlMAPK1 and PlMAPK2. CRISPR/Cas9-mediated genome editing technology was used to knock out PlRACK1, and we found that it was involved in mycelial growth, cell wall integrity, ROS metabolism, laccase activity, and pathogenicity of P. litchii. PlMAPK1 interacted with RACK1, and they jointly regulated sporangiophore branching of P. litchii. Transcriptome analysis showed that P. litchii MAPK Phosphatase 1 (PlMKP1) and beta-glucoside (PlBglX) were regulated by PlRACK1, both of which were also required for the pathogenicity of P. litchii. As well, PlMKP1 also interacted with PlMAPK1 and PlMAPK2. These results provide insights into the direct interactions between RACK1, MAPKs, and MKP, and their functions in growth, development, and pathogenesis in a plant pathogenic oomycete.},
}
@article {pmid40356298,
year = {2025},
author = {Zubair, A and Sujan, A and Ali, M and Hussain, SM},
title = {Current Challenges With Highly Active Antiretroviral Therapy and New Hope and Horizon With CRISPR-CAS9 Technology for HIV Treatment.},
journal = {Chemical biology &amp; drug design},
volume = {105},
number = {5},
pages = {e70121},
doi = {10.1111/cbdd.70121},
pmid = {40356298},
issn = {1747-0285},
mesh = {Humans ; *CRISPR-Cas Systems ; *HIV Infections/drug therapy/genetics ; Gene Editing ; *Antiretroviral Therapy, Highly Active ; },
abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR/Cas system) is now the predominant approach for genome editing. Compared to conventional genetic editing methods, CRISPR/Cas technology offers several advantages that were previously unavailable. Key benefits include the ability to simultaneously modify multiple locations, reduced costs, enhanced efficiency, and a more user-friendly design. By directing Cas-mediated DNA cleavage to specific genomic targets and utilizing intrinsic DNA repair processes, this system can produce site-specific gene modifications. This goal is achieved through an RNA-guided procedure. As the most effective gene editing method currently available, the CRISPR/Cas system has proven to be highly valuable in genomic research across a wide range of species since its discovery as a component of the adaptive immune system in bacteria. Its applicability extends to various organisms, making it increasingly prevalent in the medical field, where it shows great promise in investigating viral infections, cancer, and genetic disorders. Furthermore, it enhances our understanding of fundamental genetics. This article outlines the current antiretroviral therapy and its adverse effects but also CRISPR/Cas technology. This review article also discusses its mechanism of action and potential applications in the treatment of HIV/AIDS.},
}
@article {pmid40356202,
year = {2025},
author = {Butt, H and Mandava, M and Jacobsohn, D},
title = {Advances in Gene Therapy for Sickle Cell Disease: From Preclinical Innovations to Clinical Implementation and Access Challenges.},
journal = {The CRISPR journal},
volume = {8},
number = {3},
pages = {174-188},
doi = {10.1089/crispr.2024.0101},
pmid = {40356202},
issn = {2573-1602},
mesh = {*Anemia, Sickle Cell/therapy/genetics ; *Genetic Therapy/methods/trends ; Humans ; *Gene Editing/methods ; CRISPR-Cas Systems ; Animals ; beta-Globins/genetics ; Mutation ; },
abstract = {Sickle cell disease (SCD) is a hereditary blood disorder caused by a specific mutation in the β-globin gene, leading to the production of hemoglobin S, which deforms red blood cells, causing occlusion in small blood vessels. This results in pain, anemia, organ damage, infections, and increased stroke risk. Treatment options, including disease-modifying therapies and curative hematopoietic stem cell transplants, have limited accessibility. Recently, autologous gene therapy has emerged as a promising curative option, particularly for SCD. Gene editing techniques such as CRISPR, base editing, and prime editing offer potential to correct this mutation. In this review, we discuss recent preclinical studies and clinical trials of gene and cell therapies, focusing on the progress of FDA-approved treatments like Lyfgenia and Casgevy. We also examine the many challenges, including accessibility, safety, and long-term efficacy, which continue to shape the future of SCD gene therapy.},
}
@article {pmid40355755,
year = {2025},
author = {Wolf, G and Leippe, P and Onstein, S and Goldmann, U and Frommelt, F and Teoh, ST and Girardi, E and Wiedmer, T and Superti-Furga, G},
title = {The genetic interaction map of the human solute carrier superfamily.},
journal = {Molecular systems biology},
volume = {21},
number = {6},
pages = {531-559},
pmid = {40355755},
issn = {1744-4292},
support = {777372//Innovative Medicines Initiative 2/ ; },
mesh = {Humans ; CRISPR-Cas Systems ; *Solute Carrier Proteins/genetics/metabolism ; Gene Knockout Techniques ; Mitochondrial Membrane Transport Proteins/genetics/metabolism ; Cation Transport Proteins/genetics/metabolism ; Cell Line, Tumor ; *Epistasis, Genetic ; Gene Regulatory Networks ; Mitochondrial Proteins ; Organic Anion Transporters ; },
abstract = {Solute carriers (SLCs), the largest superfamily of transporter proteins in humans with about 450 members, control the movement of molecules across membranes. A typical human cell expresses over 200 different SLCs, yet their collective influence on cell phenotypes is not well understood due to overlapping substrate specificities and expression patterns. To address this, we performed systematic pairwise gene double knockouts using CRISPR-Cas12a and -Cas9 in human colon carcinoma cells. A total of 1,088,605 guide combinations were used to interrogate 35,421 SLC-SLC and SLC-enzyme double knockout combinations across multiple growth conditions, uncovering 1236 genetic interactions with a growth phenotype. Further exploration of an interaction between the mitochondrial citrate/malate exchanger SLC25A1 and the zinc transporter SLC39A1 revealed an unexpected role for SLC39A1 in metabolic reprogramming and anti-apoptotic signaling. This full-scale genetic interaction map of human SLC transporters is the backbone for understanding the intricate functional network of SLCs in cellular systems and generates hypotheses for pharmacological target exploitation in cancer and other diseases. The results are available at https://re-solute.eu/resources/dashboards/genomics/ .},
}
@article {pmid40355359,
year = {2025},
author = {Gao, YM and Chang, LX and Zhu, XF},
title = {[Advancements in CRISPR-Cas9 for Fanconi anemia].},
journal = {Zhonghua xue ye xue za zhi = Zhonghua xueyexue zazhi},
volume = {46},
number = {3},
pages = {276-280},
pmid = {40355359},
issn = {0253-2727},
mesh = {*Fanconi Anemia/therapy/genetics ; Humans ; *CRISPR-Cas Systems ; *Gene Editing ; *Genetic Therapy ; },
abstract = {Fanconi anemia (FA) is a hereditary bone marrow failure syndrome that is characterized by genomic instability and heightened sensitivity to DNA cross-linking agents. In recent years, the CRISPR-Cas9 technology has exhibited groundbreaking progress in the field of gene therapy for FA. The traditional CRISPR-Cas9 technology has been successfully applied in FA gene editing. Further, single-base editing technology, based on the CRISPR/Cas9 system, performs precise and efficient gene repair for prevalent gene mutations in patients with FA. The prime editing technology provides new possibilities for gene editing; however, its application in FA has not been initiated. Despite significant advancements in FA gene editing technology, several challenges remain, including the collection of sufficient hematopoietic stem cells, the risk of increased tumorigenesis postgene editing, chromosomal instability, and off-target effects. Future research is recommended to focus on optimizing sgRNA and Cas9 nucleases, designing stricter PAM sequences to reduce off-target effects, and devising personalized gene editing strategies. Further, ethical and regulatory issues as well as long-term follow-ups are crucial priorities for future gene editing work. With continuous technological advancements and in-depth clinical trials, we expect more breakthroughs in FA treatment using the CRISPR-Cas9 technology in the future. This article reviews the latest research progress of CRISPR technology in FA treatment and analyzes the advantages and disadvantages of this technology in FA gene therapy.},
}
@article {pmid40354613,
year = {2025},
author = {Zhao, S and Zhang, Q and Sun, J and Li, S and Wang, S and Zhou, D and Gong, X},
title = {Simply Engineered crRNA with CRISPR/Cas12a System Enables Wide-Scope Nucleic Acid Biomarker Analysis.},
journal = {Nano letters},
volume = {25},
number = {20},
pages = {8431-8441},
doi = {10.1021/acs.nanolett.5c01939},
pmid = {40354613},
issn = {1530-6992},
mesh = {*CRISPR-Cas Systems/genetics ; Humans ; *MicroRNAs/genetics/analysis ; Point Mutation ; Animals ; *RNA/genetics/analysis ; Cell Line, Tumor ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {CRISPR/Cas12a systems have emerged as versatile tools for molecular diagnostics, but directly detecting RNA and identifying specific DNA point mutations remain challenging. Herein, we report a simple engineering approach with a split site in the spacer sequence, enabling activation of CRISPR/Cas12a (LbCas12a) for trans-cleavage with similar efficiency to wild-type crRNA. The engineered crRNA facilitated RNA target recognition by replacing the 3'-end with RNA fragments, enhancing point mutation specificity for ssDNA targets. Based on this, we achieved amplification-free detection of microRNAs and DNA point mutations with high sensitivity and specificity. For clinical sample validation, we constructed reverse fluorescence-enhanced lateral flow test strips (rLFTS), which achieved femtomole-level detection. Moreover, the engineered crRNA-based CRISPR/Cas12a system also effectively recognized tumor cells via intracellular and in vivo imaging of miRNA-21. In conclusion, this engineered crRNA platform enhances CRISPR/Cas12a-based nucleic acid detection, promoting its wide application in molecular diagnostics and bioimaging.},
}
@article {pmid40354233,
year = {2025},
author = {Zhang, X and Duan, M and Zhao, Y and Zhang, K and Liu, F and Jie, J and Li, C and Chen, D and Li, D and Hua, S and Wang, C and Guan, Q and Wu, J and Liu, B and Song, L},
title = {Rapid and Specific Detection of Acinetobacter baumannii Infections Using a Recombinase Polymerase Amplification/Cas12a-based System.},
journal = {Journal of visualized experiments : JoVE},
volume = {},
number = {218},
pages = {},
doi = {10.3791/67542},
pmid = {40354233},
issn = {1940-087X},
mesh = {*Acinetobacter baumannii/isolation &amp; purification/genetics/enzymology ; *Nucleic Acid Amplification Techniques/methods ; *Acinetobacter Infections/diagnosis/microbiology ; *Recombinases/chemistry/metabolism/genetics ; CRISPR-Cas Systems ; *CRISPR-Associated Proteins/genetics/metabolism/chemistry ; *Bacterial Proteins/genetics/metabolism ; *Endodeoxyribonucleases/genetics/metabolism/chemistry ; Humans ; DNA, Bacterial/genetics/analysis ; },
abstract = {Acinetobacter baumannii, a gram-negative bacterium, is notorious for causing severe infections with high mortality rates. Rapid and accurate detection of A. baumannii is crucial for prompt treatment, effective infection control, and curbing antibiotic resistance. However, there is no suitable method for rapid and easy on-site detection of A. baumannii. The DNA Endonuclease Targeted CRISPR Trans Reporter (DETECTR) system offers a rapid, precise, and sensitive approach to A. baumannii detection by integrating the target-specific recognition capabilities of Cas12a with the isothermal amplification efficiency of Recombinase Polymerase Amplification (RPA). This protocol details the detection of A. baumannii using RPA combined with LbaCas12a endonuclease. The following steps are described in this article: extraction of DNA, selection of a specific DNA sequence, design of primer and CRISPR RNA (crRNA), construction of positive recombinant plasmid, setup of Cas12a-RPA assay, optimization of the RPA amplification system, visualization of the RPA-CRISPR/Cas12a assay using a fluorescence detection tool such as a real-time PCR instrument, and evaluation of sensitivity and specificity evaluation.},
}
@article {pmid40354126,
year = {2025},
author = {Lv, B and Liang, P and Chang, C and Li, D},
title = {Sensitive aptasensing of tobramycin through a rational design of catalytic hairpin assembly and hybridization chain reaction amplification monomers for CRISPR/Cas12a activation.},
journal = {The Analyst},
volume = {150},
number = {12},
pages = {2580-2590},
doi = {10.1039/d5an00267b},
pmid = {40354126},
issn = {1364-5528},
mesh = {*CRISPR-Cas Systems ; *Biosensing Techniques/methods ; Limit of Detection ; *Aptamers, Nucleotide/chemistry/genetics ; *Nucleic Acid Amplification Techniques/methods ; Milk/chemistry ; *Tobramycin/analysis ; Nucleic Acid Hybridization ; Animals ; *Anti-Bacterial Agents/analysis ; DNA/chemistry/genetics ; Inverted Repeat Sequences ; *CRISPR-Associated Proteins/metabolism/genetics ; Bacterial Proteins ; Endodeoxyribonucleases ; },
abstract = {The catalytic hairpin assembly (CHA) and hybridization chain reaction amplification (HCR) are enzyme-free isothermal DNA amplification methods based on the self-assembly of hairpin monomers. Recently, CRISPR/Cas12a-based biosensors in combination with CHA or HCR signal amplification have shown promising performance. Herein, several design strategies for hairpin monomers in CHA and HCR were evaluated in the context of CRISPR/Cas12a-based biosensor construction. The SL-HCR strategy, in which the CRISPR/Cas12a target strand is blocked in the loop of one hairpin monomer DNA and released in the duplex HCR products, demonstrated superior performance in terms of a low background signal, wide linear detection range, and high signal-to-noise ratio. With the assistance of an aptamer-containing probe, a highly sensitive aptasensor was constructed for tobramycin detection, whereby the SL-HCR served the function of signal amplification, whereas the CRISPR/Cas12a system acted to cleave the FQ probes, thereby resulting in the production of a fluorescent signal. After optimization, the aptasensor enables linear detection of tobramycin concentrations ranging from 125 pM to 2500 nM, with a limit of detection (LOD) of 92.87 pM. Moreover, the aptasensor was utilized to detect tobramycin in beef and milk samples, yielding satisfactory results. The assay is concise and cost-effective due to the absence of nanomaterial DNA labeling and magnetic separation procedures. Furthermore, the entire detection workflow operates under isothermal conditions, which makes it suitable for use in food safety control and environmental monitoring. In addition, the results presented here may shed new light on the design of CRISPR/Cas12a-based biosensors in combination with CHA or HCR.},
}
@article {pmid40353913,
year = {2025},
author = {Wu, G and Yin, C and Zheng, J and Wang, M and Abdalmegeed, D and Zhang, F and Sun, S and Sun, S and Shao, Y and Xin, Z},
title = {Dynamic regulation of iturin production via reconstructing the quorum-sensing system ComQXPA in Bacillus subtilis.},
journal = {World journal of microbiology &amp; biotechnology},
volume = {41},
number = {5},
pages = {173},
pmid = {40353913},
issn = {1573-0972},
mesh = {*Bacillus subtilis/genetics/metabolism ; *Quorum Sensing/genetics ; Gene Expression Regulation, Bacterial ; *Bacterial Proteins/genetics/metabolism ; Promoter Regions, Genetic ; *Peptides, Cyclic/biosynthesis/genetics/metabolism ; *Lipopeptides/biosynthesis/genetics/metabolism ; CRISPR-Cas Systems ; },
abstract = {Pheromone ComX is a critical element of the quorum-sensing (QS) system in Bacillus subtilis. It activates the surfactin promoter (Psrf) to initiate surfactin production in a cell-density-dependent manner. In this study, the natural promoter Pitu of B. subtilis 1A751 WR-itu, a lipopeptide iturin mono-producing parent strain, was replaced by the constitutive promoter P43, QS Psrf, and the mutated QS PM-srf, generating dynamic regulation systems to improve iturin production. HPLC analysis revealed that the PM-srf promoter-harbouring strain significantly enhanced iturin production to 409.33 ± 16.77 mg·L[- 1], 2.15 times higher than the parent strain. Further identification by UPLC-HRESI-MS/MS and GC-MS indicated that the strain could produce four new C10-C13 iturins. Moreover, the ComX degrading enzymes AprE or NprE were down-regulated by the CRISPR interference (CRISPRi) system, increasing iturin production to 526.46 ± 18.43 mg·L[- 1] and 416.99 ± 17.02 mg·L[- 1], respectively. Interestingly, iturin production was further increased to 579.85 ± 19.83 mg·L[- 1] under simultaneous down-regulation of AprE and NprE, 3.05 times higher than the parent strain. The reconstructed ComQXPA QS system in B. subtilis combines the inhibitory of AprE and NprE to dynamically up and down-regulate the expression of iturin operon, providing an effective pipeline for regulating other bioactive molecules.},
}
@article {pmid40353666,
year = {2025},
author = {Lv, L and Luo, H and Yi, J and Zhang, K and Li, Y and Tong, W and Jiang, Y and Zhou, Y and Tong, G and Liu, C},
title = {IFITM proteins are key entry factors for porcine epidemic diarrhea coronavirus.},
journal = {Journal of virology},
volume = {99},
number = {6},
pages = {e0202824},
pmid = {40353666},
issn = {1098-5514},
support = {No. 2022YFD1800800//National Key Research and Development Program of China/ ; No. 21PJ1416300//Shanghai Pujiang Program/ ; No. 2019JB09//Central Public-interest Scientific Institution Basal Research Fund/ ; No. CAAS-ASTIP-2016-SHVRI-2004-1//Agricultural Science and Technology Innovation Program/ ; },
mesh = {Animals ; *Porcine epidemic diarrhea virus/physiology ; Swine ; Humans ; *Virus Internalization ; *Membrane Proteins/metabolism/genetics ; *RNA-Binding Proteins/metabolism/genetics ; *Coronavirus Infections/virology/metabolism/veterinary ; *Antigens, Differentiation/metabolism/genetics ; *Swine Diseases/virology/metabolism ; Cell Line ; Virus Replication ; Host-Pathogen Interactions ; CRISPR-Cas Systems ; },
abstract = {UNLABELLED: Porcine epidemic diarrhea virus (PEDV) is a highly contagious coronavirus that poses a substantial threat to the global swine industry. However, our current understanding of the host factors crucial for PEDV infection remains limited. To identify these host factors, we conducted a genome-wide CRISPR/Cas9 gene knockout screen using a PEDV-permissive cell line. Our results indicate that the endogenous expression of human interferon-inducible transmembrane protein 3 (IFITM3) enhances PEDV entry and replication. Silencing or eliminating endogenous IFITM3 in Huh7 cells significantly suppressed PEDV entry, whereas reintroducing IFITM3 partially restored susceptibility to PEDV. Overexpression of human IFITM3 or IFITM2, but not IFITM1, in Huh7.5 cells substantially increased PEDV entry and replication. Importantly, our results suggest that human IFITM3 influences PEDV entry at a later stage. Furthermore, the overexpression of porcine IFITM1 significantly enhanced PEDV infection in LLC-PK1 cells, whereas the overexpression of porcine IFITM2/3 did not produce similar effects. Notably, removing the C-terminal 15 amino acids of porcine IFITM2/3 resulted in increased PEDV entry. Coimmunoprecipitation analyses showed that all IFITMs interacted with the PEDV S1 protein, indicating a direct role in the viral entry process. Additionally, porcine IFITM1 colocalized with the PEDV S protein at the cell nuclear periphery and enhanced PEDV infection in porcine small intestinal organoids. Overall, our results suggest that IFITMs are critical in facilitating PEDV entry into cells. Targeting IFITMs may provide a promising strategy for controlling PEDV transmission and developing interventions to mitigate the virus's impact on the swine industry.<br><br>IMPORTANCE: Understanding the mechanisms underlying porcine epidemic diarrhea virus (PEDV) infection is vital for addressing its significant impact on the swine industry. This study reveals that interferon-inducible transmembrane (IFITM) proteins, particularly human IFITM3 and porcine IFITM1, play crucial roles in facilitating PEDV entry and replication. By elucidating these molecular interactions, the research highlights the potential of IFITMs as therapeutic targets for managing PEDV infections and paves the way for antiviral strategies. Moreover, this research extends beyond PEDV management, underscoring the critical role of host factors in controlling the spread of pathogenic coronaviruses.},
}
@article {pmid40352342,
year = {2025},
author = {Kaushik, A and Singh, J and Fatima, Z and Hameed, S},
title = {Establishment and evaluation of a naked-eye diagnostic assay for tuberculosis utilizing reverse isothermal amplification-assisted CRISPR-Cas in resource-limited settings.},
journal = {Drug target insights},
volume = {19},
number = {},
pages = {31-40},
pmid = {40352342},
issn = {1177-3928},
abstract = {INTRODUCTION: The current scenario of tuberculosis (TB) caused by Mycobacterium tuberculosis (MTB) has presented an almost insurmountable challenge to hospitals with high patient numbers. Delayed diagnosis of TB is a major hurdle in preventing the employment of efficient therapeutics, leading to the development of drug resistance. Hence, an easily accessible diagnostic method, particularly for resource for resource-limited settings, is pertinent for the rapid identification of MTB-infected patients. In pursuit of developing such an assay, the present study offers a CLAP-TB (CRISPR-Cas coupled RT-LAMP Amplification Protocol for Tuberculosis) assay, which will allow us to diagnose TB rapidly and visually.<br><br>METHODS AND RESULTS: Herein, the visual MTB detection consists of a method utilizing 232 different samples (sputum, urine, serum) from 82 patients for reverse transcription loop-mediated isothermal amplification (RT-LAMP). Additionally, the assay also utilizes the integration of a CRISPR-Cas12-based system using different guide RNAs of IS6110 and an internal control POP7 (human RNase P) genes along with visual detection via lateral flow readout-based dipsticks with the unaided eye (~134 min). Overall, the limit of detection for CLAP-TB assay was up to 1 ag of RNA, while the clinical sensitivity and specificity were 98.27% and 100%, respectively, on the pilot scale.<br><br>CONCLUSION: Together, our CLAP-TB assay offers proof of concept for a rapid, sensitive, and specific method with the minimum technical expertise required for TB diagnosis in developing and resource-limited settings.},
}
@article {pmid40352243,
year = {2025},
author = {Prasetia, IG and Kurniati, NF and Riani, C and Mudhakir, D},
title = {Design of lipid nanoparticle (LNP) containing genetic material CRISPR/Cas9 for familial hypercholesterolemia.},
journal = {Narra J},
volume = {5},
number = {1},
pages = {e2217},
pmid = {40352243},
issn = {2807-2618},
mesh = {*Hyperlipoproteinemia Type II/therapy/genetics ; *Nanoparticles/chemistry ; *CRISPR-Cas Systems ; Humans ; Receptors, LDL/genetics ; *Lipids/chemistry ; *Genetic Therapy/methods ; Particle Size ; Liposomes ; },
abstract = {Familial hypercholesterolemia is a genetic disorder caused by mutations in the low- density lipoprotein receptor gene (LDLR) and the current treatment still focuses on symptom management. The aim of this study was to develop a lipid nanoparticle (LNP)- based delivery system for the CRISPR/Cas9 component in correcting LDLR gene mutations. LNPs were prepared using an ultrasonic-solvent emulsification technique by varying the surfactant: oil ratio (SOR), homogenization speed and time, and sonication time. Next, the LNP surface was modified by adding DSPE-PEG2000-NH2 and polyethyleneimine. The next stage is to design the single guide RNA (sgRNA) and Donor DNA wildtype (Donor DNA wt). This genetic material was complexed with LNP and then transfected into Hepa1-6 LDLR mt cells, an in vitro representation of cells suffering from familial hypercholesterolemia. This optimization process produced LNPs with a particle size of 118.6 ± 0.8 nm and a polydispersity index of 0.34 ± 0.03. The LNP surface modification resulted in a zeta potential of +7.5 mV. A transmission electron microscope (TEM) analysis showed spherical morphology with size distribution following a regular pattern. LNP cell viability tests showed good biocompatibility at concentrations <15 mM with a half-maximal inhibitory concentration (IC50) value of 27.7 mM. The dominant cellular uptake mechanism of LNP was through the clathrin-mediated endocytosis (CME) pathway. The Hepa1-6 LDLR mt cell model was successfully produced with the transfecting agent Lipofectamine 3000 by homology-directed repair (HDR) mechanism. The LNP-genetic material complex with a ratio of sgRNA:Cas9:Donor DNA wt (1:1:0.04) showed an increase in LDLR gene expression of 3.3 ± 0.2 times and LDLR protein levels reached 12.95 ± 0.25 ng/mL on day 4 after transfection. The results of this study indicate that the developed LNP-based delivery system has the potential for gene therapy applications in familial hypercholesterolemia.},
}
@article {pmid40351170,
year = {2025},
author = {Zang, SS and Zhang, R and Zhang, JR and Zhang, X and Li, J},
title = {Progress, Applications and Prospects of CRISPR-Based Genome Editing Technology in Gene Therapy for Cancer and Sickle Cell Disease.},
journal = {Human gene therapy},
volume = {36},
number = {11-12},
pages = {858-869},
doi = {10.1089/hum.2024.262},
pmid = {40351170},
issn = {1557-7422},
mesh = {Humans ; *Anemia, Sickle Cell/therapy/genetics ; *Gene Editing/methods ; *CRISPR-Cas Systems ; *Genetic Therapy/methods ; *Neoplasms/therapy/genetics ; Animals ; Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {The advent of genome-editing technologies, particularly the RNA-guided the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated system (Cas) 9, which originates from prokaryotic CRISPR/Cas adaptive immune mechanisms, has revolutionized molecular biology. Renowned for its simplicity, cost-effectiveness, and capacity for multiplexed gene editing, CRISPR/Cas9 has emerged as the most versatile and widely adopted genome-editing platform. Its applications span fundamental research, biotechnology, medicine, and therapeutics. This review highlights recent advancements in CRISPR-based technologies, focusing on CRISPR/Cas9, CRISPR/Cas12a, and CRISPR/Cas12f. It emphasizes precision editing methods like base editing and prime editing, which enable targeted nucleotide changes without double-strand breaks. The specificity of these tools, including on-target accuracy and off-target risks, is critically evaluated. Additionally, recent preclinical and clinical efforts to treat diseases such as cancer and sickle cell disease using CRISPR are summarized. Finally, the challenges and future directions of CRISPR-mediated gene therapy are discussed, emphasizing its potential to integrate with other molecular approaches to address unmet medical needs.},
}
@article {pmid40350476,
year = {2025},
author = {Kumari, G and Gupta, P and Goswami, SG and Jain, R and Anand, S and Biswas, S and Garg, S and Thakur, P and Saravanakumar, V and Arvinden, VR and Goswami, B and Bhowmick, IP and Mohandas, N and Burrows, J and Ramalingam, S and Singh, S},
title = {CRISPR/Cas9-engineering of Kell null erythrocytes to unveil host targeted irresistible antimalarial.},
journal = {Communications biology},
volume = {8},
number = {1},
pages = {730},
pmid = {40350476},
issn = {2399-3642},
support = {NER/84/2022-ECD-I//Indian Council of Medical Research (ICMR)/ ; },
mesh = {*Erythrocytes/parasitology/metabolism/drug effects ; *Plasmodium falciparum/drug effects/genetics ; *Antimalarials/pharmacology ; *CRISPR-Cas Systems ; Animals ; Humans ; *Malaria, Falciparum/drug therapy/parasitology ; Mice ; },
abstract = {Malaria elimination faces challenges from drug resistance, stemming from mutations within the parasite's genetic makeup. Genetic adaptations in key erythrocyte proteins offer malaria protection in endemic regions. Emulating nature's approach, and implementing methodologies to render indispensable host proteins inactive, holds the potential to reshape antimalarial therapy. This study delves into the functional implication of the single-span membrane protein Kell ectodomain, which shares consensus sequence with the zinc endopeptidase family, possesses extracellular enzyme activity crucial for parasite invasion into host erythrocytes. Through generating Kell-null erythrocytes from an erythroid progenitor, BEL-A, we demonstrate the indispensable nature of Kell activity in P. falciparum invasion. Additionally, thiorphan, a metallo-endopeptidase inhibitor, which specifically inhibits Kell activity, inhibited Plasmodium infection at nanomolar concentrations. Interestingly, individuals in malaria-endemic regions exhibit low Kell expression and activity, indicating a plausible Plasmodium-induced evolutionary pressure. Both thiorphan and its prodrug racecadotril, demonstrated potent antimalarial activity in vivo, highlighting Kell's protease role in invasion and proposing thiorphan as a promising host-oriented antimalarial therapeutic.},
}
@article {pmid40350377,
year = {2025},
author = {Zhang, L and Zhao, D and Wei, Z and Zhu, X and Sha, T and Tang, W and Bi, C and Zhang, X},
title = {The Advancement of Prime Editing Technology.},
journal = {Chembiochem : a European journal of chemical biology},
volume = {26},
number = {12},
pages = {e202500193},
doi = {10.1002/cbic.202500193},
pmid = {40350377},
issn = {1439-7633},
support = {32225031//National Natural Science Foundation of China/ ; 32171449//National Natural Science Foundation of China/ ; 32271483//National Natural Science Foundation of China/ ; TSBICIP-KJGG-017//Tianjin Synthetic Biotechnology Innovation Capacity Improvement/ ; 2022177//the Youth Innovation Promotion Association CAS/ ; },
mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; Humans ; Protein Engineering ; },
abstract = {The advent of CRISPR/Cas genome editing has spurred major breakthroughs across life sciences, offering vast potential across numerous research and application fields. Among the expanding toolkit of CRISPR/Cas-derived methods, prime editing (PE) stands out for its versatility and specificity, enabling precise point mutations and small insertions or deletions without requiring double-stranded DNA breaks. Since its introduction, PE has undergone multiple rounds of optimization to improve performance. In this review, the core components and mechanism of prime editors, followed by four key evolution strategies: protein engineering, pegRNA modifications, accessory protein recruitment, and paired pegRNA approaches are outlined. Further persistent challenges and outline possible refinements are discussed, highlighting how further innovations can expand PE's utility across diverse areas of research, biotechnology, and potential therapeutic interventions.},
}
@article {pmid40349461,
year = {2025},
author = {Li, W and Song, H and Cai, X and Wang, Y and Zhang, Y and Sun, C and Han, B and Zhang, J},
title = {Deletion of a novel upstream promoter of p53 impairs cold tolerance capacity in zebrafish.},
journal = {Biochemical and biophysical research communications},
volume = {769},
number = {},
pages = {151969},
doi = {10.1016/j.bbrc.2025.151969},
pmid = {40349461},
issn = {1090-2104},
mesh = {Animals ; *Zebrafish/genetics/physiology ; *Promoter Regions, Genetic/genetics ; *Tumor Suppressor Protein p53/genetics ; *Zebrafish Proteins/genetics/metabolism ; *Cold-Shock Response/genetics ; Oxidative Stress/genetics ; Cold Temperature ; CRISPR-Cas Systems ; Reactive Oxygen Species/metabolism ; *Sequence Deletion ; },
abstract = {P53 is a tumor suppressor that plays a crucial role in stress responses. We previously identified a novel upstream p53 promoter in cold acclimated zebrafish cells, however the functional significance of this upstream promoter under cold stress in zebrafish remains unclear. We generated novel p53 promoter[-/-] zebrafish using CRISPR/Cas9 and characterized their responses to cold stress. While novel p53 promoter[-/-] zebrafish exhibited normal development, growth, and locomotion at 28 °C, they showed enhanced locomotor capacity at 18 °C and reduced cold tolerance capacity at 8 °C. RNA-seq revealed the expression of genes related to oxidative stress was downregulated at 8 °C in the gill of novel p53 promoter[-/-] zebrafish, compared with WT zebrafish. Further experiments confirmed that the ROS levels increased, and the expression of SOD and GSH-PX reduced under cold stress in novel p53 promoter[-/-] zebrafish, compared with WT zebrafish. The novel p53 promoter plays a critical role in maintaining antioxidant defense and low-temperature tolerance in zebrafish. Our findings provide new theoretical insights into the environmental adaptation mechanisms of fish and offer experimental evidence for the regulatory role of the p53 gene in response to low-temperature stress.},
}
@article {pmid40348775,
year = {2025},
author = {Jalan, M and Brambati, A and Shah, H and McDermott, N and Patel, J and Zhu, Y and Doymaz, A and Wu, J and Anderson, KS and Gazzo, A and Pareja, F and Yamaguchi, TN and Vougiouklakis, T and Ahmed-Seghir, S and Steinberg, P and Neiman-Golden, A and Azeroglu, B and Gomez-Aguilar, J and da Silva, EM and Hussain, S and Higginson, D and Boutros, PC and Riaz, N and Reis-Filho, JS and Powell, SN and Sfeir, A},
title = {RNA transcripts serve as a template for double-strand break repair in human cells.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {4349},
pmid = {40348775},
issn = {2041-1723},
support = {U01CA231019//U.S. Department of Health &amp; Human Services | NIH | National Cancer Institute (NCI)/ ; U2C CA271894/CA/NCI NIH HHS/United States ; P30 CA008748/CA/NCI NIH HHS/United States ; P30 CA016042/CA/NCI NIH HHS/United States ; R01 CA244729/CA/NCI NIH HHS/United States ; P50 CA247749/CA/NCI NIH HHS/United States ; U01 CA231019/CA/NCI NIH HHS/United States ; R01 CA229161/CA/NCI NIH HHS/United States ; },
mesh = {Humans ; *DNA Breaks, Double-Stranded ; *DNA Repair/genetics ; CRISPR-Cas Systems/genetics ; *RNA, Messenger/genetics/metabolism ; *RNA/genetics/metabolism ; DNA-Directed DNA Polymerase/metabolism/genetics ; Templates, Genetic ; Genomic Instability ; },
abstract = {Double-strand breaks (DSBs) are toxic lesions that lead to genome instability. While canonical DSB repair pathways typically operate independently of RNA, growing evidence suggests that RNA:DNA hybrids and nearby transcripts can influence repair outcomes. However, whether transcript RNA can directly serve as a template for DSB repair in human cells remains unclear. In this study, we develop fluorescence and sequencing-based assays to show that RNA-containing oligonucleotides and messenger RNA can serve as templates during DSB repair. We conduct a CRISPR/Cas9-based genetic screen to identify factors that promote RNA-templated DSB repair (RT-DSBR). Of the candidate polymerases, we identify DNA polymerase zeta (Polζ) as a potential reverse transcriptase that facilitates RT-DSBR. Furthermore, analysis of cancer genome sequencing data reveals whole intron deletions - a distinct genomic signature of RT-DSBR that occurs when spliced mRNA guides repair. Altogether, our findings highlight RT-DSBR as an alternative pathway for repairing DSBs in transcribed genes, with potential mutagenic consequences.},
}
@article {pmid40348052,
year = {2025},
author = {Koike, A and Brindley, PJ},
title = {CRISPR/Cas genome editing, functional genomics, and diagnostics for parasitic helminths.},
journal = {International journal for parasitology},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.ijpara.2025.05.001},
pmid = {40348052},
issn = {1879-0135},
abstract = {Functional genomics using CRISPR (Clustered Regulatory Interspaced Short Palindromic Repeats)/Cas (CRISPR-associated endonuclease)-based approaches has revolutionized biomedical sciences. Gene editing is also widespread in parasitology generally and its use is increasing in studies on helminths including flatworm and roundworm parasites. Here, we survey the progress, specifically with experimental CRISPR-facilitated functional genomics to investigate helminth biology and pathogenesis, and also with the burgeoning use of CRISPR-based methods to assist in diagnosis of helminth infections. We also provide an historical timeline of the introduction and uses of CRISPR in helminth species to date.},
}
@article {pmid40347811,
year = {2025},
author = {Wang, R and He, B and Liang, Z and Liu, Y and Yang, J and Jin, H and Wei, M and Ren, W and Suo, Z and Xu, Y},
title = {A dual-mode sensor for rapid detection of procymidone: "Dark box" qualitative analysis and electrochemical quantification mediated by PdHPCN-222/PEI-rGO and CRISPR/Cas12a.},
journal = {Food chemistry},
volume = {486},
number = {},
pages = {144586},
doi = {10.1016/j.foodchem.2025.144586},
pmid = {40347811},
issn = {1873-7072},
mesh = {*Electrochemical Techniques/methods/instrumentation ; *Biosensing Techniques/methods/instrumentation ; CRISPR-Cas Systems ; Graphite/chemistry ; Limit of Detection ; Polyethyleneimine/chemistry ; Fluorescence ; Food Contamination/analysis ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {This study presents an integrated dual-mode sensing strategy, in which qualitative fluorescence screening is followed by quantitative electrochemical detection, improving detection efficiency and enabling rapid PCM analysis. It develops a novel fluorescence electrochemical aptasensor that combines in vitro "Dark-box" applications with CRISPR/Cas12a system electrode surface sensing technology. PCM activates the DNA walker, and the DNAzyme induces cyclic cleavage of DNA strands bearing the Carboxyfluorescein (FMA) group. After magnetic separation, the fluorescence reaction combined with the "Dark box" enables the preliminary qualitative analysis of procymidone (PCM). Following the preliminary qualitative detection, the solution is introduced to the electrochemical aptasensor platform integrated with the CRISPR/Cas12 system. The Cas12a system triggers cyclic cleavage, producing a signal change that enables the electrochemical quantitative detection of PCM. An fluorescence (FL) response occurs when the PCM concentration in spiked samples is at or above 1 pg·mL[-1], allowing for qualitative fluorescence detection. The EC platform's detection limit is 8.51 × 10[-6] ng·mL[-1], with a range of 1 × 10[-2] ng·mL[-1] to 1 × 10[4] ng·mL[-1]. The designed dual-mode sensor provides reliable monitoring of PCM in real samples.},
}
@article {pmid40347696,
year = {2025},
author = {Martin, OL and Lynch, CRH and Fleming, R},
title = {Advancing forensic body fluid identification: A comparative analysis of RT-LAMP+CRISPR-Cas12a and established mRNA-based methods.},
journal = {Forensic science international. Genetics},
volume = {78},
number = {},
pages = {103297},
doi = {10.1016/j.fsigen.2025.103297},
pmid = {40347696},
issn = {1878-0326},
mesh = {Humans ; *RNA, Messenger/genetics ; Female ; *CRISPR-Cas Systems ; *Nucleic Acid Amplification Techniques/methods ; *CRISPR-Associated Proteins/genetics ; Sensitivity and Specificity ; Cervix Mucus/chemistry ; Male ; Forensic Genetics/methods ; Semen/chemistry ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Bacterial Proteins ; Endodeoxyribonucleases ; },
abstract = {In forensic science, the analysis of body fluid evidence determines the cellular origin of a sample, aiding in the reconstruction of a potential crime. Messenger ribonucleic acid (mRNA) based confirmatory tests address limitations of current conventional methods, providing increased specificity and sensitivity, minimal sample consumption, and the detection of a broader range of body fluids. However, they require expensive instrumentation, longer reaction times, and lack portability. Reverse-transcription loop-mediated isothermal amplification (RT-LAMP) coupled with clustered regular interspaced short palindromic repeats (CRISPR) with CRISPR-associated protein 12a (Cas12a) has the potential to overcome these challenges. This approach offers reduced testing time and cost, while potentially providing equivalent sensitivity and specificity, as observed in the field of viral diagnostics. Visual detection capabilities enable the development of rapid, portable screening tests suitable for testing at the crime scene. In the context of a sexual assault investigation, RT-LAMP+CRISPR-Cas12a could potentially increase the efficiency and detection rate. This study compares this novel method to two other mRNA-based methods, endpoint reverse transcription polymerase chain reaction (RT-PCR) multiplex assay CellTyper 2, and a real-time reverse transcription quantitative PCR (RT-qPCR) multiplex assay. The tests' sensitivity and specificity were evaluated on single-source and mixed body fluid samples, including rectal mucosa, a fluid which is minimally explored in forensic literature. The RT-qPCR assay demonstrated the highest sensitivity, specificity, and precision in mixed samples. In addition, RT-qPCR offers a greater linear dynamic range, faster processing time and easier methodology compared to CellTyper 2, only limited by its expensive nature. Notably, rectal mucosa samples exhibited non-specific marker expression of CellTyper 2 markers and expression of CYP2B7P (vaginal fluid) for all methods. This emphasises the need for a dedicated rectal mucosa marker. RT-LAMP+CRISPR-Cas12a exhibited a high specificity, displaying off-target expression of CYP2B7P in two fluid types. However, the method lacked sensitivity and precision for most markers except MMP3 (menstrual blood), demonstrating detection down to 1:10,000 with 100 % specificity. RT-LAMP+CRISPR requires further development, but its quick, inexpensive nature and high specificity suggest it has potential as a confirmatory test that could reduce the limitations of existing methods.},
}
@article {pmid40347545,
year = {2025},
author = {Wang, S and Guo, X and Yan, H and Zhou, T and Liu, J},
title = {Generation of an anti-CD5 CAR knock-in human induced pluripotent stem cell line using CRISPR/Cas9 technology.},
journal = {Stem cell research},
volume = {86},
number = {},
pages = {103731},
doi = {10.1016/j.scr.2025.103731},
pmid = {40347545},
issn = {1876-7753},
mesh = {Humans ; *Induced Pluripotent Stem Cells/metabolism/cytology ; *CRISPR-Cas Systems/genetics ; *Gene Knock-In Techniques ; *CD5 Antigens/immunology/metabolism ; *Receptors, Chimeric Antigen/genetics/metabolism ; Cell Line ; Cell Differentiation ; },
abstract = {Theanti-CD5 chimeric antigen receptor (CAR)isa genetically engineered immune cell therapydevelopedto targetCD5-associatedhematologic malignancies,such asT-cell lymphoma and leukemia.UsingCRISPR/Cas9-mediated gene targeting, wegeneratedananti-CD5 CAR knock-in human induced pluripotent stem cell (iPSC) linethat stably expresses the CAR constructand is detectable via FLAG tag and GFP markers. Thisengineeredcell linemaintainsstem cell morphology,displaysa normal karyotype, andexhibits robustexpression ofpluripotency markers while retaining differentiation potential.},
}
@article {pmid40347261,
year = {2025},
author = {Botelho, FBS and Nandy, S and Srivastava, V},
title = {CRISPR/Cas9-based modulation of V-PPase expression in rice improves grain quality and yield under high nighttime temperature.},
journal = {Plant cell reports},
volume = {44},
number = {6},
pages = {119},
pmid = {40347261},
issn = {1432-203X},
support = {1826836//Directorate for Biological Sciences/ ; 2023-11092//National Institute of Food and Agriculture/ ; },
mesh = {*Oryza/genetics/enzymology/growth &amp; development/physiology ; Gene Expression Regulation, Plant ; *Edible Grain/genetics/growth &amp; development ; *CRISPR-Cas Systems/genetics ; *Inorganic Pyrophosphatase/genetics/metabolism ; Plant Proteins/genetics/metabolism ; Hot Temperature ; Plants, Genetically Modified ; Endosperm/genetics ; },
abstract = {Transcriptional modulation of the vacuolar H[+] translocating pyrophosphatase expressed specifically in the endosperm and reproductive tissue of rice improves its spikelet fertility and reduces grain chalkiness under high nighttime temperature.},
}
@article {pmid40346697,
year = {2025},
author = {Guo, T and Wang, H and Wu, F and Lu, W and Zhu, M and Ma, S and Zhang, Y and Yan, Y and Zhou, M and Talanaite, D and Liu, S and Qi, M and Lan, F and Liu, X},
title = {Functional analysis of JPH2-knockout cardiomyocytes identifies ECCD as a novel indicator in a human cardiac modelJPH2.},
journal = {Stem cell research &amp; therapy},
volume = {16},
number = {1},
pages = {234},
pmid = {40346697},
issn = {1757-6512},
support = {2021-RC310-12, 2022I2M-2-001 and 2023-I2M-1-003//CAMS Innovation Fund for Medical Sciences/ ; 2021YFC2701703, 2023YFA0915002//National Key Research and Devel opment Program of China/ ; 2022-GSP-GG-7//National High-Level Hospital Clinical Research Funding/ ; 2019PT320026//Non-profit Central Research Institute Fund of Chinese Academy of Medical Sciences/ ; ZDSYS20200923172000001//Shenzhen Fundamental Research Program/ ; 2024SKL-TJ001//Open Project Fund of State Key Laboratory of Cardiovascular Diseases&#xff0c;Shanghai East Hospital/ ; 2024T170070//China Postdoctoral Science Foundation/ ; 82400381//National Natural Science Foundation of China/ ; NCRCG-PLAGH-2024008//Open Project Fund of National Clinical Research Center for Geriatric Diseases, Chinese PLA General Hospital/ ; CXPY202304//Chinese PLA General Hospital Sixth medical center Innovation and Culture Grant/ ; },
mesh = {Humans ; *Myocytes, Cardiac/metabolism/cytology ; *Membrane Proteins/genetics/metabolism ; Calcium/metabolism ; Human Embryonic Stem Cells/metabolism/cytology ; *Excitation Contraction Coupling ; Myocardial Contraction ; Animals ; CRISPR-Cas Systems ; Muscle Proteins ; },
abstract = {BACKGROUND: Junctophilin-2 (JPH2) is a vital protein in cardiomyocytes, anchoring T-tubule and sarcoplasmic reticulum membranes to facilitate excitation-contraction coupling, a process essential for cardiac contractile function. Dysfunction of JPH2 is associated with cardiac disorders such as heart failure; however, prior studies using mouse models or primary human cardiomyocytes are limited by interspecies differences or poor cell viability, respectively. This study aimed to investigate JPH2's role in human cardiac function and disease using a novel stem cell-derived model, while introducing a new indicator to evaluate related cardiac impairments.<br><br>METHODS: We generated a JPH2-knockout model using human embryonic stem cell-derived cardiomyocytes (hESC-CMs) with CRISPR/Cas9. Cellular morphology, contractile function, calcium dynamics, and electrophysiological properties were assessed via transmission electron microscopy, the CardioExcyte96 system, calcium imaging with Fluo-4 AM, and multi-electrode array recordings, respectively. Wild-type JPH2 was overexpressed through lentiviral transfection to evaluate rescue effects, and two JPH2 variants-one benign (G505S) and one pathogenic (E85K)-were introduced to study mutation-specific effects.<br><br>RESULTS: JPH2 knockout disrupted excitation-contraction coupling in hESC-CMs by impairing junctional membrane complex structure, leading to heart failure-like phenotypes with reduced contractility, altered calcium dynamics, and electrophysiological irregularities. Overexpression of wild-type JPH2 restored these functions, affirming its critical role in cardiac physiology. We identified excitation-contraction coupling delay (ECCD) as a novel indicator that precisely quantified coupling impairment severity, with its applicability validated across distinct JPH2 variants (G505S and E85K).<br><br>CONCLUSIONS: This study demonstrates JPH2's essential role in sustaining excitation-contraction coupling by stabilizing the junctional membrane complex, with its deficiency driving heart failure-like cardiac dysfunction. ECCD is established as a sensitive, comprehensive indicator for assessing JPH2-related impairment severity. These findings advance our understanding of JPH2 in cardiac pathology and position ECCD as a valuable tool for research and potential clinical evaluation, with JPH2 and calcium regulation emerging as promising therapeutic targets.},
}
@article {pmid40346034,
year = {2025},
author = {Villa, S and Jafri, Q and Lazzari-Dean, JR and Sangha, M and Olsson, N and Lefebvre, AEYT and Fitzgerald, ME and Jackson, K and Chen, Z and Feng, BY and Nile, AH and Stokoe, D and Bersuker, K},
title = {BiDAC-dependent degradation of plasma membrane proteins by the endolysosomal system.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {4345},
pmid = {40346034},
issn = {2041-1723},
mesh = {*Lysosomes/metabolism/drug effects ; Humans ; *Proteolysis/drug effects ; Ubiquitination/drug effects ; ErbB Receptors/metabolism ; *Membrane Proteins/metabolism ; *Cell Membrane/metabolism/drug effects ; Proteasome Endopeptidase Complex/metabolism ; Receptor, ErbB-2/metabolism/genetics ; HEK293 Cells ; Endocytosis/drug effects ; CRISPR-Cas Systems ; *Endosomes/metabolism ; Animals ; Protein Transport/drug effects ; },
abstract = {The discovery of bifunctional degradation activating compounds (BiDACs) has led to the development of a new class of drugs that promote the clearance of their protein targets. BiDAC-induced ubiquitination is generally believed to direct cytosolic and nuclear proteins to proteolytic destruction by proteasomes. However, pathways that govern the degradation of other classes of BiDAC targets, such as integral membrane and intraorganellar proteins, have not been investigated in depth. In this study we use morphological profiling and CRISPR/Cas9 genetic screens to investigate the mechanisms by which BiDACs induce the degradation of plasma membrane receptor tyrosine kinases (RTKs) EGFR and Her2. We find that BiDAC-dependent ubiquitination triggers the trafficking of RTKs from the plasma membrane to lysosomes for degradation. Notably, functional proteasomes are required for endocytosis of RTKs upstream of the lysosome. Additionally, our screen uncovers a non-canonical function of the lysosome-associated arginine/lysine transporter PQLC2 in EGFR degradation. Our data show that BiDACs can target proteins to proteolytic machinery other than the proteasome and motivate further investigation of mechanisms that govern the degradation of diverse classes of BiDAC targets.},
}
@article {pmid40345204,
year = {2025},
author = {Wu, Y and Zhong, A and Evangelisti, A and Sidharta, M and Danwei, H and Studer, L and Zhou, T},
title = {Leveraging CRISPR activation for rapid assessment of gene editing products in human pluripotent stem cells.},
journal = {Stem cell reports},
volume = {20},
number = {6},
pages = {102499},
pmid = {40345204},
issn = {2213-6711},
mesh = {Humans ; *Gene Editing/methods ; *Pluripotent Stem Cells/metabolism/cytology ; *CRISPR-Cas Systems/genetics ; Cell Differentiation/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Transcriptional Activation ; DNA Methylation ; Cell Line ; },
abstract = {Verification of genome editing in human pluripotent stem cells (hPSCs), particularly at silent loci, is desirable but challenging, as it often requires complex and time-intensive differentiation to induce their expression. Here, we establish a rapid and effective workflow for verifying genome-edited hPSC lines targeting unexpressed genes using CRISPR-mediated transcriptional activation (CRISPRa). We systematically compared the efficiency of various CRISPRa systems and identified the synergistic activation mediator (SAM) system as the most potent for activating silent genes in hPSCs. Furthermore, combining SAM with TET1, a demethylation module, enhanced the activation of methylated genes. By inducing targeted gene activation in undifferentiated hPSCs using CRISPRa, we successfully verified single- and dual-reporter lines, functionally tested degradation tag (dTAG) knockins, and validated silent gene knockouts within 48 h. This approach bypasses the need to induce target gene expression through differentiation, providing a rapid and effective assay for verifying silent gene editing at the hPSC stage.},
}
@article {pmid40345045,
year = {2025},
author = {Li, C and Peng, X and Zhang, Z and Liu, Y and Pedro, GC and Fu, C and Yang, Y and Dong, Q and Duan, Y and Sun, X},
title = {Establishment of Agrobacterium-mediated genetic transformation and CRISPR/Cas9-guided gene editing in Elymus nutans.},
journal = {Journal of plant physiology},
volume = {310},
number = {},
pages = {154513},
doi = {10.1016/j.jplph.2025.154513},
pmid = {40345045},
issn = {1618-1328},
mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; *Transformation, Genetic ; *Agrobacterium/genetics ; *Elymus/genetics/physiology ; Plants, Genetically Modified/genetics ; },
abstract = {Elymus nutans, an allohexaploid (2n = 6x = 42) species with a StStHHYY genome, is a native perennial in the alpine grasslands of the Qinghai-Xizang Plateau, and has been widely used for artificial pasture and ecological restoration as a forage grass with highest yield on the plateau. Nevertheless, the lack of a stable transformation system has impeded further efforts to trait improvement of E. nutans. In the present study, we established a reliable Agrobacterium-mediated genetic transformation system for E. nutans, and successfully generated EnTCP4-edited plants using the CRISPR/Cas9 system. The editing efficiency achieved 19.23 % in E. nutans. Knocking out EnTCP4 significantly delayed flowering and enhanced water-deficit stress resistance. This research represents a significant breakthrough in the genetic transformation and gene editing of E. nutans, laying a technological foundation to gain insight into gene functions and molecular breeding in E. nutans.},
}
@article {pmid40344932,
year = {2025},
author = {Talanaite, D and Wang, H and Qi, M and Lan, F},
title = {Generation of a human embryonic stem cell line (WAe009-A-3B) carrying homozygous TNNT2 gene knockout by CRISPR/Cas9 editing.},
journal = {Stem cell research},
volume = {86},
number = {},
pages = {103729},
doi = {10.1016/j.scr.2025.103729},
pmid = {40344932},
issn = {1876-7753},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Human Embryonic Stem Cells/metabolism/cytology ; *Troponin T/genetics/metabolism ; *Gene Knockout Techniques ; *Gene Editing ; Cell Line ; Homozygote ; },
abstract = {The TNNT2 gene encodes cardiac troponin T (cTnT), a critical protein in cardiac muscle contraction. Mutations in TNNT2 are associated with various cardiomyopathies, including hypertrophic cardiomyopathy (HCM) and dilated cardiomyopathy (DCM), which contribute to significant morbidity and mortality. In this study, we established a novel TNNT2 knockout human embryonic stem cell (hESC) line, WAe009-A-3B, utilizing the CRISPR/Cas9 genome editing system. This novel hESC line provides an important tool for investigating the molecular mechanisms underlying TNNT2-related cardiomyopathies and may serve as a promising in vitro model for the development of therapeutic strategies targeting TNNT2 mutations in cardiac diseases.},
}
@article {pmid40342201,
year = {2025},
author = {Chulei, Y and Yiyuan, Z and Hong, T and Yanhua, G and Limin, W and Ping, Z},
title = {Analysis of Oestrus and Hormonal Changes in the Progeny of 2 Genotypes of FecB Gene-Edited Sheep.},
journal = {Reproduction in domestic animals = Zuchthygiene},
volume = {60},
number = {5},
pages = {e70038},
doi = {10.1111/rda.70038},
pmid = {40342201},
issn = {1439-0531},
support = {2023TSYCTD0021//the foundation Bingtuan Science and Technology Project/ ; NYHXGG2023AA101//Bingtuan Science and Technology Project/ ; NCG202221//Bingtuan Science and Technology Project/ ; },
mesh = {Animals ; Female ; *Estrus/genetics/physiology ; Genotype ; *Gene Editing/veterinary ; Ovarian Follicle ; Luteinizing Hormone/blood ; *Sheep, Domestic/genetics/physiology ; Follicle Stimulating Hormone/blood ; Estradiol/blood ; Sheep/genetics/physiology ; CRISPR-Cas Systems ; },
abstract = {The FecB gene, the first gene identified for multiple births, can improve reproductive performance in sheep. We used the CRISPR/Cas9 editing system to obtain the FecB gene in Kazakh sheep and mated with unedited Kazakh sheep. After the genotypic screening of the progeny, four B+ and ++ Kazakh ewes, each 2 weeks old and healthy, and close to the same body weight, were selected to study oestrus and hormonal changes in the progeny population of FecB gene-edited Kazakh sheep. Our results showed that the FecB gene was moderately polymorphic (0.25 < PIC < 0.5) in the progeny population, was able to be stably inherited, and showed increased lambing numbers. The number of follicles and tiny follicles differed significantly (p < 0.05) between the 2 genotypes of ewes in the progeny population. Physiologically, FecB gene-edited sheep did not differ from wild-type sheep. Throughout the oestrous cycle, FSH, LH, and E2 in serum hormones of both sheep genotypes showed variability (p < 0.05) between 72-360 h, 360-528 h, and 12-72 h, respectively, compared to other periods. The concentration of FSH and PROG in follicular fluid was much higher than that of LH and E2 (p < 0.05). Overall, it is possible to make Kazakh sheep carry the FecB gene by CRISPR/Cas9 editing and stably inherit it into the progeny population. The enhancement of reproductive hormones by the FecB gene is beneficial to follicular development and ovulation and improves the reproductive efficiency of sheep.},
}
@article {pmid40342053,
year = {2025},
author = {Allemailem, KS and Rahmani, AH and Almansour, NM and Aldakheel, FM and Albalawi, GM and Albalawi, GM and Khan, AA},
title = {Current updates on the structural and functional aspects of the CRISPR/Cas13 system for RNA targeting and editing: A next‑generation tool for cancer management (Review).},
journal = {International journal of oncology},
volume = {66},
number = {5},
pages = {},
pmid = {40342053},
issn = {1791-2423},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Neoplasms/genetics/therapy ; *Gene Editing/methods ; Animals ; Genetic Therapy/methods ; },
abstract = {For centuries, a competitive evolutionary race between prokaryotes and related phages or other mobile genetic elements has led to the diversification of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR‑associated sequence (Cas) genome‑editing systems. Among the different CRISPR/Cas systems, the CRISPR/Cas9 system has been widely studied for its precise DNA manipulation; however, due to certain limitations of direct DNA targeting, off‑target effects and delivery challenges, researchers are looking to perform transient knockdown of gene expression by targeting RNA. In this context, the more recently discovered type VI CRISPR/Cas13 system, a programmable single‑subunit RNA‑guided endonuclease system that has the capacity to target and edit any RNA sequence of interest, has emerged as a powerful platform to modulate gene expression outcomes. All the Cas13 effectors known so far possess two distinct ribonuclease activities. Pre‑CRISPR RNA processing is performed by one RNase activity, whereas the two higher eukaryotes and prokaryotes nucleotide‑binding domains provide the other RNase activity required for target RNA degradation. Recent innovative applications of the type VI CRISPR/Cas13 system in nucleic acid detection, viral interference, transcriptome engineering and RNA imaging hold great promise for disease management. This genome editing system can also be employed by the Specific High Sensitivity Enzymatic Reporter Unlocking platform to identify any tumor DNA. The discovery of this system has added a new dimension to targeting, tracking and editing circulating microRNA/RNA/DNA/cancer proteins for the management of cancer. However, there is still a lack of thorough understanding of the mechanisms underlying some of their functions. The present review summarizes the recent updates on the type VI CRISPR/Cas system in terms of its structural and mechanistic properties and some novel applications of this genome‑editing tool in cancer management. However, some issues, such as collateral degradation of bystander RNA, impose major limitations on its in vivo application. Furthermore, additional challenges and future prospects for this genome editing system are described in the present review.},
}
@article {pmid40341582,
year = {2025},
author = {Dacquay, LC and Antoniou, P and Mentani, A and Selfjord, N and Mårtensson, H and Hsieh, PP and Mustfa, S and Thom, G and Wimberger, S and Firth, M and Akrap, N and Maresca, M and Peterka, M},
title = {Dual inhibition of DNA-PK and Polϴ boosts precision of diverse prime editing systems.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {4290},
pmid = {40341582},
issn = {2041-1723},
mesh = {*Gene Editing/methods ; *DNA-Activated Protein Kinase/antagonists &amp; inhibitors/metabolism/genetics ; Humans ; DNA Polymerase theta ; *DNA-Directed DNA Polymerase/metabolism/genetics ; CRISPR-Cas Systems ; RNA, Guide, CRISPR-Cas Systems/genetics ; DNA Repair ; HEK293 Cells ; Animals ; },
abstract = {Prime editing is a genome engineering tool that allows installation of various small edits with high precision. However, prime editing efficiency and purity can vary widely across different edits, genomic targets, and cell types. Prime editing typically offers more precise editing outcomes compared to other genome editing methods such as homology-directed repair. However, it can still result in significant rates of unintended editing outcomes, such as indels or imprecise prime edits. This issue is particularly notable in systems utilizing a second nicking gRNA, such as PE3 and PE5, as well as in dual pegRNA systems and fully active nuclease systems such as PEn, which increase efficiency but compromise precision. In this work, we show that pharmacologica