@article {pmid40332425,
year = {2025},
author = {Guo, X and Wang, X and Wang, J and Ma, M and Ren, Q},
title = {Current Development of iPSC-Based Modeling in Neurodegenerative Diseases.},
journal = {International journal of molecular sciences},
volume = {26},
number = {8},
pages = {},
doi = {10.3390/ijms26083774},
pmid = {40332425},
issn = {1422-0067},
support = {81901088//National Natural Science Foundation of China/ ; E2019050019//Hundred Talents Program of Hebei Province/ ; H2022206562//Hebei Provincial Natural Science Foundation Precision Medicine Joint Fund Cultivation Project/ ; NV20230011//Key laboratory of Neural and Vascular Biology, Ministry of Education of China/ ; },
mesh = {Humans ; *Induced Pluripotent Stem Cells/metabolism/cytology ; *Neurodegenerative Diseases/pathology/therapy/metabolism/genetics ; Animals ; Gene Editing ; Organoids/metabolism ; CRISPR-Cas Systems ; Cell Differentiation ; *Models, Biological ; Neurons/metabolism ; Huntington Disease/pathology ; },
abstract = {Over the past two decades, significant advancements have been made in the induced pluripotent stem cell (iPSC) technology. These developments have enabled the broader application of iPSCs in neuroscience, improved our understanding of disease pathogenesis, and advanced the investigation of therapeutic targets and methods. Specifically, optimizations in reprogramming protocols, coupled with improved neuronal differentiation and maturation techniques, have greatly facilitated the generation of iPSC-derived neural cells. The integration of the cerebral organoid technology and CRISPR/Cas9 genome editing has further propelled the application of iPSCs in neurodegenerative diseases to a new stage. Patient-derived or CRISPR-edited cerebral neurons and organoids now serve as ideal disease models, contributing to our understanding of disease pathophysiology and identifying novel therapeutic targets and candidates. In this review, we examine the development of iPSC-based models in neurodegenerative diseases, including Alzheimer's disease, Parkinson's disease, and Huntington's disease.},
}

Humans
*Induced Pluripotent Stem Cells/metabolism/cytology
*Neurodegenerative Diseases/pathology/therapy/metabolism/genetics
Animals
Gene Editing
Organoids/metabolism
CRISPR-Cas Systems
Cell Differentiation
*Models, Biological
Neurons/metabolism
Huntington Disease/pathology

@article {pmid40332149,
year = {2025},
author = {Navarro, C and Díaz, MP and Duran, P and Castro, A and Díaz, A and Cano, C and Carbonell-Zabaleta, AK and Solano-Jimenez, DS and Rivera-Porras, D and Contreras-Velásquez, JC and Bermúdez, V},
title = {CRISPR-Cas Systems: A Functional Perspective and Innovations.},
journal = {International journal of molecular sciences},
volume = {26},
number = {8},
pages = {},
doi = {10.3390/ijms26083645},
pmid = {40332149},
issn = {1422-0067},
mesh = {*CRISPR-Cas Systems/genetics ; Gene Editing/methods ; Bacteria/genetics/virology ; Archaea/genetics ; Humans ; Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {Adaptation is a fundamental tenet of evolutionary biology and is essential for the survival of all organisms, including prokaryotes. The evolution of clustered regularity exemplifies this principle of interspaced short palindromic repeats (CRISPR) and associated proteins (Cas), an adaptive immune system that confers resistance to viral infections. By integrating short segments of viral genomes into their own, bacteria and archaea develop a molecular memory that enables them to mount a rapid and targeted response upon subsequent viral challenges. The fortuitous discovery of this immune mechanism prompted many studies and introduced researchers to novel tools that could potentially be developed from CRISPR-Cas and become clinically relevant as biotechnology rapidly advances in this area. Thus, a deeper understanding of the underpinnings of CRISPR-Cas and its possible therapeutic applications is required. This review analyses the mechanism of action of the CRISPR-Cas systems in detail and summarises the advances in developing biotechnological tools based on CRISPR, opening the field for further research.},
}

*CRISPR-Cas Systems/genetics
Gene Editing/methods
Bacteria/genetics/virology
Archaea/genetics
Humans
Clustered Regularly Interspaced Short Palindromic Repeats

@article {pmid40331919,
year = {2025},
author = {Xu, C and Zhang, Y and Zhu, X and Hua, D and Yang, L and Huang, X and Gao, H and Luo, A and Deng, R and Xia, X},
title = {Preamplification-Free Detection of Viable Microorganisms in Fermentation Using Tandem CRISPR Nuclease Probe.},
journal = {Journal of agricultural and food chemistry},
volume = {},
number = {},
pages = {},
doi = {10.1021/acs.jafc.5c01068},
pmid = {40331919},
issn = {1520-5118},
abstract = {Accurate detection of viable bacteria is crucial for evaluating and monitoring the fermentation process. However, the complexity of fermentation samples presents challenges to developing precise and rapid detection tools. Here, we present a Cas13a-Csm6 tandem nuclease probe capable of the one-pot detection of viable microorganisms during fermentation, eliminating the need for nucleic acid preamplification. The RNA-activated CRISPR-Cas13a generates cleavage substrates that serve as activators for the CRISPR/Cas III-A Csm6 system. Leveraging the high specificity and efficient amplification capacity of the CRISPR cascade, this nuclease probe can detect 1% of viable Lactobacillus and Bacillus, facilitating the monitoring of bacterial populations throughout fermentation. This approach completes detection within 30 min and improves sensitivity for bacterial profiling by 16-fold compared with using Cas13 alone. The Cas13a-Csm6 tandem nuclease probe offers a precise and rapid analytical tool for the on-site quality monitoring of fermented foods.},
}

@article {pmid40331862,
year = {2025},
author = {Anglada, T and Rodriguez-Muñoz, M and Pulido-Artola, N and Genescà, A},
title = {Engineering Chromosome Bridges Through CRISPR/Cas9 to Decipher the Impact of Intercentromeric Distance on Resolution Dynamics.},
journal = {FASEB journal : official publication of the Federation of American Societies for Experimental Biology},
volume = {39},
number = {9},
pages = {e70599},
doi = {10.1096/fj.202402258RR},
pmid = {40331862},
issn = {1530-6860},
support = {//Fundació la Marató de TV3 (Fundació la Marató)/ ; //Generalitat de Catalunya (Government of Catalonia)/ ; //Consejo de Seguridad Nuclear (CSN)/ ; },
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Mitosis/genetics ; *Chromatin/genetics/metabolism ; Cell Line ; Kinetochores/metabolism ; *Centromere/genetics ; },
abstract = {Resolution of chromosome bridges during mitosis is a critical yet incompletely understood process with implications for genomic stability and cancer development. In this study, we investigated the impact of the bridging chromatin length on the timing and mechanism of chromosome bridge resolution. Using CRISPR/Cas9 technology, we engineered chromosome bridges with precisely defined intercentromeric distances in human RPE-1 cells. Our study revealed a decline in the frequency of chromosome bridges as cells progressed from early anaphase to late telophase, indicating resolution during mitosis. Moreover, the longer the bridging chromatin length, the higher the frequency of chromosome bridges observed at the mitotic exit, demonstrating that the size of the bridge influences its resolution during mitosis. Additionally, the separation between the bridge kinetochores needed for bridge breakage was strongly dependent on the megabase length of the bridging chromatin, with longer chromosome bridges requiring greater separation for their resolution. Given that chromosome bridge resolution occurs in a concerted manner with spindle elongation and is influenced by the length of the bridging chromatin, we posit that the traction forces generated by microtubules attaching to dicentric chromosomes play a significant role in resolving chromosome bridges during mitosis. Our study underscores the intricate interplay between chromosome bridge geometry and mechanical forces in mitotic chromosome bridge resolution. Our model offers a valuable framework for future investigations into the molecular mechanisms underlying chromosome bridge resolution, with potential implications for cancer biology and genomic stability maintenance.},
}

Humans
*CRISPR-Cas Systems/genetics
*Mitosis/genetics
*Chromatin/genetics/metabolism
Cell Line
Kinetochores/metabolism
*Centromere/genetics

@article {pmid40330464,
year = {2025},
author = {Li, TF and Rothhaar, P and Lang, A and Grünvogel, O and Colasanti, O and Ugarte, SMO and Traut, J and Piras, A and Acosta-Rivero, N and Gonçalves Magalhães, V and Springer, E and Betz, A and Huang, HE and Park, J and Qiu, R and Gnouamozi, GE and Mehnert, AK and Thi, VLD and Urban, S and Muckenthaler, M and Schlesner, M and Wohlleber, D and Binder, M and Bartenschlager, R and Pichlmair, A and Lohmann, V},
title = {RBM39 shapes innate immunity by controlling the expression of key factors of the interferon response.},
journal = {Frontiers in immunology},
volume = {16},
number = {},
pages = {1568056},
pmid = {40330464},
issn = {1664-3224},
mesh = {*RNA-Binding Proteins/genetics/metabolism/immunology ; *Immunity, Innate/genetics ; Humans ; Toll-Like Receptor 3/metabolism/genetics/immunology ; Hepatocytes/immunology/metabolism ; Signal Transduction ; *Interferons/immunology/metabolism ; *Gene Expression Regulation ; CRISPR-Cas Systems ; Cell Line ; },
abstract = {BACKGROUND AND AIMS: The contribution of innate immunity to clearance of viral infections of the liver, in particular sensing via Toll-like receptor 3 (TLR3), is incompletely understood. We aimed to identify the factors contributing to the TLR3 response in hepatocytes via CRISPR/Cas9 screening.

METHODS: A genome-wide CRISPR/Cas9 screen on the TLR3 pathway was performed in two liver-derived cell lines, followed by siRNA knockdown validation. SiRNA knockdown and indisulam treatment were used to study the role of RNA-binding motif protein 39 (RBM39) in innate immunity upon poly(I:C) or cytokine treatment and viral infections. Transcriptome, proteome, and alternative splicing were studied via RNA sequencing and mass spectrometry upon depletion of RBM39.

RESULTS: Our CRISPR/Cas9 screen identified RBM39, which is highly expressed in hepatocytes, as an important regulator of the TLR3 pathway. Knockdown of RBM39 or treatment with indisulam, an aryl sulfonamide drug targeting RBM39 for proteasomal degradation, strongly reduced the induction of interferon-stimulated genes (ISGs) in response to double-stranded RNA (dsRNA) or viral infections. RNA sequencing (seq) and mass spectrometry identified that transcription and/or splicing of the key pathway components IRF3, RIG-I, and MDA5 were affected by RBM39 depletion, along with multiple other cellular processes identified previously. RBM39 knockdown further restrained type I and type III IFN pathways by reducing the expression of individual receptor subunits and STAT1/2. The function of RBM39 was furthermore not restricted to hepatocytes.

CONCLUSION: We identified RBM39 as a regulatory factor of cell intrinsic innate immune signaling. Depletion of RBM39 impaired TLR3, RIG-I/MDA5, and IFN responses by affecting the basal expression of key pathway components.},
}

*RNA-Binding Proteins/genetics/metabolism/immunology
*Immunity, Innate/genetics
Humans
Toll-Like Receptor 3/metabolism/genetics/immunology
Hepatocytes/immunology/metabolism
Signal Transduction
*Interferons/immunology/metabolism
*Gene Expression Regulation
CRISPR-Cas Systems
Cell Line

@article {pmid40329691,
year = {2025},
author = {Su, R and Shen, G and Xiao, X and Zheng, Y and Liu, F and Chen, D},
title = {Generation of a Novel Inducible and Dermal Papilla-Specific Wif1-CreER Knock-In Mouse Line for Hair Follicle Research.},
journal = {Experimental dermatology},
volume = {34},
number = {5},
pages = {e70109},
doi = {10.1111/exd.70109},
pmid = {40329691},
issn = {1600-0625},
support = {//National Natural Science Foundation of China/ ; },
mesh = {Animals ; *Hair Follicle/cytology/metabolism ; Mice ; Gene Knock-In Techniques ; Integrases/genetics/metabolism ; *Adaptor Proteins, Signal Transducing/genetics ; Mice, Transgenic ; *Dermis/cytology/metabolism ; CRISPR-Cas Systems ; Tamoxifen/pharmacology ; },
abstract = {Dermal papilla (DP) cells are essential niche cells that regulate hair follicle development, cycling and regeneration. Despite the establishment of several DP cell mouse lines in prior research, these tools are limited by incomplete specificity and spatiotemporal control. The Wnt inhibitory factor 1 (Wif1) has been identified as a DP signature gene. To address the need for precise labelling and manipulation of DP cells, we developed a novel genetic tool-Wif1-CreER knock-in mice. Using CRISPR/Cas9-mediated homologous recombination, the CreERT2 sequences were inserted into the endogenous Wif1 locus, under the control of the native promoter. PCR and sequencing analysis confirmed the accurate insertion of the CreERT2 sequence. Crossing Wif1-CreER mice with a reporter line demonstrated efficient and specific Cre recombinase activity in DP cells during anagen, catagen and telogen upon tamoxifen treatment across hair types. Importantly, DP-restricted labelling was confirmed by immunofluorescence and colocalised with Crabp1 and alkaline phosphatase (AP)-staining activity, exhibiting minimal to negligible expression in other tissues. This innovative mouse model overcomes the limitations of current tools and provides a valuable resource for advancing our understanding of hair biology and developing targeted therapies for hair-related disorders, offering unprecedented precision in the manipulation of dermal papilla cells.},
}

Animals
*Hair Follicle/cytology/metabolism
Mice
Gene Knock-In Techniques
Integrases/genetics/metabolism
*Adaptor Proteins, Signal Transducing/genetics
Mice, Transgenic
*Dermis/cytology/metabolism
CRISPR-Cas Systems
Tamoxifen/pharmacology

@article {pmid40329173,
year = {2025},
author = {Piché, LC and Bories, S and Liato, V and Paquet, VE and Saucier, L and Létourneau-Montminy, MP and Charette, SJ and Dubar, R and Labrie, SJ and Lagüe, P and Vincent, AT},
title = {Evolutionary responses of Escherichia coli to phage pressure: insights into mucoidy and colanic acid overexpression.},
journal = {BMC genomics},
volume = {26},
number = {1},
pages = {448},
pmid = {40329173},
issn = {1471-2164},
support = {IA120638//MAPAQ (Innov'Action)/ ; IA120638//MAPAQ (Innov'Action)/ ; RGPIN-2022-03321//Natural Sciences and Engineering Research Council of Canada/ ; },
mesh = {*Escherichia coli/virology/genetics/metabolism ; *Evolution, Molecular ; *Bacteriophages/physiology ; Escherichia coli Proteins/genetics/metabolism/chemistry ; *Coliphages/physiology ; Mutation ; Polysaccharides ; },
abstract = {BACKGROUND: Antibiotic resistance is a major issue affecting all spheres of human activity, including agriculture. One significant example is the Avian Pathogenic Escherichia coli (APEC), a bacterium that infects poultry and leads to substantial economic losses in the farming industry. As antibiotics lose efficacity, bacteriophages (phages) -viruses that specifically target bacteria-are emerging as a promising alternative to antibiotics for treating and preventing bacterial infections. However, bacteria can develop resistance to phages through various mechanisms. Studying the coevolution between a phage and its host bacterium is important to gain insight into the phage's potential as a therapeutic agent. This study investigates the evolutionary responses of an APEC strain and a laboratory E. coli strain to a commercial phage originally isolated from APEC.

RESULTS: In most cases, phage resistance resulted in a significant increase in mucoidy. Genomic analysis revealed that this resistance consistently correlated with amino acid changes, particularly in proteins involved in colanic acid production, such as YrfF. Further investigation of a mutation found in the YrfF protein demonstrated that this mutation altered the protein's structure and its interaction with the membrane. Transcriptomic analysis confirmed that the genes involved in colanic acid production were significantly overexpressed. Although the strains possessed a CRISPR-Cas system, it did not contribute to phage resistance.

CONCLUSIONS: This study suggests that specific amino acid changes in key proteins may be a mechanism employed by E. coli, including APEC, to defend against phage infections.},
}

*Escherichia coli/virology/genetics/metabolism
*Evolution, Molecular
*Bacteriophages/physiology
Escherichia coli Proteins/genetics/metabolism/chemistry
*Coliphages/physiology
Mutation
Polysaccharides

@article {pmid40328743,
year = {2025},
author = {Huang, F and Wang, Y and Zhang, X and Gao, W and Li, J and Yang, Y and Mo, H and Prince, E and Long, Y and Hu, J and Jiang, C and Kang, Y and Chen, Z and Hu, YC and Zeng, C and Yang, L and Chen, CW and Chen, J and Huang, H and Weng, H},
title = {m[6]A/IGF2BP3-driven serine biosynthesis fuels AML stemness and metabolic vulnerability.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {4214},
pmid = {40328743},
issn = {2041-1723},
support = {82270168//National Natural Science Foundation of China (National Science Foundation of China)/ ; },
mesh = {*Leukemia, Myeloid, Acute/metabolism/genetics/pathology ; Humans ; *Serine/biosynthesis/metabolism ; *RNA-Binding Proteins/metabolism/genetics ; Animals ; *Neoplastic Stem Cells/metabolism/pathology ; *Adenosine/analogs & derivatives/metabolism ; Mice ; Cell Line, Tumor ; Glycine/metabolism ; Methyltransferases/metabolism/genetics ; CRISPR-Cas Systems ; RNA, Messenger/metabolism/genetics ; Gene Expression Regulation, Leukemic ; },
abstract = {Metabolic reprogramming of amino acids represents a vulnerability in cancer cells, yet the mechanisms underlying serine metabolism in acute myeloid leukemia (AML) and leukemia stem/initiating cells (LSCs/LICs) remain unclear. Here, we identify RNA N[6]-methyladenosine (m[6]A) modification as a key regulator of serine biosynthesis in AML. Using a CRISPR/Cas9 screen, we find that depletion of m[6]A regulators IGF2BP3 or METTL14 sensitizes AML cells to serine and glycine (SG) deprivation. IGF2BP3 recognizies m[6]A on mRNAs of key serine synthesis pathway (SSP) genes (e.g., ATF4, PHGDH, PSAT1), stabilizing these transcripts and sustaining serine production to meet the high metabolic demand of AML cells and LSCs/LICs. IGF2BP3 silencing combined with dietary SG restriction potently inhibits AML in vitro and in vivo, while its deletion spares normal hematopoiesis. Our findings reveal the critical role of m[6]A modification in the serine metabolic vulnerability of AML and highlight the IGF2BP3/m[6]A/SSP axis as a promising therapeutic target.},
}

*Leukemia, Myeloid, Acute/metabolism/genetics/pathology
Humans
*Serine/biosynthesis/metabolism
*RNA-Binding Proteins/metabolism/genetics
Animals
*Neoplastic Stem Cells/metabolism/pathology
*Adenosine/analogs & derivatives/metabolism
Mice
Cell Line, Tumor
Glycine/metabolism
Methyltransferases/metabolism/genetics
CRISPR-Cas Systems
RNA, Messenger/metabolism/genetics
Gene Expression Regulation, Leukemic

@article {pmid40328723,
year = {2025},
author = {Yang, X and Shi, J and Wang, H and Wang, L and Su, H and Chen, C and Zhao, C},
title = {[Construction of mouse podocyte clone-5 cell lines with Smad3 knockout by CRISPR/Cas9].},
journal = {Sheng wu gong cheng xue bao = Chinese journal of biotechnology},
volume = {41},
number = {4},
pages = {1658-1670},
doi = {10.13345/j.cjb.240803},
pmid = {40328723},
issn = {1872-2075},
mesh = {Animals ; *Smad3 Protein/genetics ; *CRISPR-Cas Systems/genetics ; Mice ; *Podocytes/cytology/metabolism ; Transforming Growth Factor beta1/pharmacology ; Cell Line ; *Gene Knockout Techniques ; RNA, Guide, CRISPR-Cas Systems/genetics ; },
abstract = {This study established the mouse podocyte clone-5 (MPC5) with Smad3 knockout and studied the effect of transforming growth factor-beta 1 (TGF-β1) on the dedifferentiation of the MPC5 cells with Smad3 knockout, aiming to provide a cell tool for studying the role of Smad3 in mouse podocytes. The single-guide RNA (sgRNA) sequence targeting Smad3 was designed according to the principles of CRISPR/Cas9 design. The pX458-Smad3 vector was constructed and introduced into competent cells, and then the vector was extracted and used to transfect MPC5 cells. The successfully transfected cells were sorted by a flow cytometer. After single-cell clone expansion, PCR amplification of sequences adjacent to the edition site of Smad3 and sequencing were performed to identify potential cells with gene knockout. Western blotting was employed to verify the knockout efficiency of Smad3. Finally, the effect of Smad3 knockout on TGF-β1-induced dedifferentiation of MPC5 cells was analyzed by reverse transcription-polymerase chain reacting (RT-PCR), Western blotting, and the immunofluorescence method. The sgRNA was designed to target the fifth exon of Smad3. EGFP expression was observed 24 h after transfection of the pX458-Smad3 plasmid into MPC5 cells, with the transfection efficiency of 0.1% as determined by flow cytometry. From the transfected cells, 21 cell clones were obtained through flow cytometric sorting and single-cell clone expansion. PCR amplification and sequencing of the region around the sgRNA target site in Smad3 identified two cell clones with biallelic frameshift mutations. Western blotting results confirmed the absence of Smad3 expression in these clones, indicating successful establishment of the MPC5 cell line with Smad3 knockout. In normal MPC5 cells, TGF-β1 stimulation promoted the expression of fibrosis-related genes fibronectin and Col1a1 (collagen I) and inhibited the expression of the podocyte marker proteins synaptopodin and podocin, which suggested epithelial-mesenchymal transition and podocyte injury. However, in the two MPC5 cell lines with Smad3 knockout, TGF-β1-induced expression of epithelial-mesenchymal transition markers was significantly suppressed. The MPC5 cell lines with Smad3 knockout that were constructed by CRISPR/Cas9 provide a valuable cell model for functional studies of Smad3 protein and highlight the critical role of Smad3 in cell dedifferentiation.},
}

Animals
*Smad3 Protein/genetics
*CRISPR-Cas Systems/genetics
Mice
*Podocytes/cytology/metabolism
Transforming Growth Factor beta1/pharmacology
Cell Line
*Gene Knockout Techniques
RNA, Guide, CRISPR-Cas Systems/genetics

@article {pmid40328722,
year = {2025},
author = {Cai, Q and Wang, M and Zhu, J and Wu, J},
title = {[A universal counter-selection strategy based on replacement of sgRNA expression cassettes targeting multi-copy genes].},
journal = {Sheng wu gong cheng xue bao = Chinese journal of biotechnology},
volume = {41},
number = {4},
pages = {1649-1657},
doi = {10.13345/j.cjb.240505},
pmid = {40328722},
issn = {1872-2075},
mesh = {*CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; *RNA, Guide, CRISPR-Cas Systems/genetics ; Plasmids/genetics ; },
abstract = {Selection markers are essential tools in gene editing, the utility of such systems is inherently constrained by species-specific limitations, governed by divergent host genetic backgrounds and metabolic compatibility. To address this limitation, we leveraged the CRISPR/Cas9 system to develop a universal counter-selection tool. We designed and introduced an sgRNA expression cassettes as counter-selection markers, which directs the Cas9 protein to target and cleave genomic DNA, allowing for the selection of the strains where the sgRNA expression cassette has been replaced. Optimized to target multiple copy sites with sgRNA, this system significantly enhances cell lethality, boosting counter-selection efficiency to over 85.00%. This counter-selection tool is not limited to single strains and is suitable for various scenarios, including multi-copy plasmid assembly and plasmid editing, demonstrating broad application potential.},
}

*CRISPR-Cas Systems/genetics
*Gene Editing/methods
*RNA, Guide, CRISPR-Cas Systems/genetics
Plasmids/genetics

@article {pmid40327602,
year = {2025},
author = {Sharma, I and Hall, K and Moonah, S},
title = {CRISPR genome editing using a combined positive and negative selection system.},
journal = {PloS one},
volume = {20},
number = {5},
pages = {e0321881},
pmid = {40327602},
issn = {1932-6203},
mesh = {*Gene Editing/methods ; Humans ; *CRISPR-Cas Systems/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Polymorphism, Single Nucleotide ; *Selection, Genetic ; beta-Globins/genetics ; },
abstract = {The clustered regularly interspaced short palindromic repeats (CRISPR)/Cas system is a powerful genome editing tool that has revolutionized research. Single nucleotide polymorphisms (SNPs) are the most common form of genetic variation in humans. Only a subset of these SNPs has been shown to be linked to genetic diseases, while the biological relevance of the majority remains unclear. Investigating these variants of unknown significance could provide valuable insights into their roles in biological processes, disease susceptibility, and treatment responses. While CRISPR/Cas has emerged as a transformative technology, its ability to make single nucleotide substitutions remains a significant limitation. Other techniques in single nucleotide editing, such as base editing and prime editing, offer promising possibilities to complement CRISPR/Cas systems, though they also have their own limitations. Hence, alternative approaches are necessary to overcome the limitations of CRISPR. Here, to improve the feasibility of generating single base edits in the genome, we provide a protocol that introduces a multiple expression and dual selection (MEDS) system, which, alongside CRISPR, utilizes the opposing roles of cytosine deaminase/uracil phosphoribosyltransferase (CD/UPRT) for negative selection and neomycin phosphotransferase II (NPT II) for positive selection. As a proof of concept and to demonstrate feasibility of the method, we used MEDS, along with traditional CRISPR-Cas9, to generate sickle hemoglobin by introducing a point mutation (A → T) in the sixth codon of the hemoglobin beta gene.},
}

*Gene Editing/methods
Humans
*CRISPR-Cas Systems/genetics
*Clustered Regularly Interspaced Short Palindromic Repeats
Polymorphism, Single Nucleotide
*Selection, Genetic
beta-Globins/genetics

@article {pmid40327171,
year = {2025},
author = {Yang, J and Li, W and Hu, Y and Han, Y and Lei, C and Wang, H},
title = {Establishment of a rapid RAA-CRISPR/Cas12a system targeting the recN gene for on-site detection of Streptococcus suis in livestock and fresh pork meat.},
journal = {Functional & integrative genomics},
volume = {25},
number = {1},
pages = {99},
pmid = {40327171},
issn = {1438-7948},
support = {2025ZNSFSC0209//Natural Science Foundation of Sichuan Province/ ; U21A20257//National Natural Science Foundation of China/ ; 2021ZDZX0010//Sichuan Science and Technology Programs/ ; },
mesh = {*Streptococcus suis/genetics/isolation & purification ; Animals ; *CRISPR-Cas Systems ; Swine ; *Livestock/microbiology ; *Bacterial Proteins/genetics ; *Pork Meat/microbiology ; *Nucleic Acid Amplification Techniques/methods ; },
abstract = {Streptococcus suis is a major bacterial pathogen in the swine industry, causing meningitis, arthritis, and other diseases in infected pigs. It also poses significant public health risks due to its zoonotic potential, particularly in individuals with skin lesions. Current detection methods, including traditional culture-based techniques and PCR assays, are time-consuming, labor-intensive, and lack sufficient accuracy. To address these limitations, this study aimed to develop a rapid and precise detection method for S. suis. By leveraging whole-genome sequencing (WGS) and multiple sequence alignment, the recN gene was identified as a highly specific molecular target. A novel isothermal detection method, integrating recombinase-aided amplification (RAA) with CRISPR/Cas12a, was subsequently established. This RAA-CRISPR/Cas12a-based system demonstrated superior sensitivity compared to conventional PCR (targeting the gdh gene), achieving detection within 30 min without requiring specialized equipment. This method achieves 2.44 × 10[1] copies/µL and 2.1 × 10[1] CFU sensitivity and 100% specificity within 30 min, outperforming conventional PCR in speed and reliability while eliminating dependency on specialized equipment. Designed for field applications, it offers a cost-effective (US$1/test), user-friendly solution for on-site S. suis detection in swine farms and fresh pork meat, enhancing outbreak control and preventive healthcare in the livestock industry.},
}

*Streptococcus suis/genetics/isolation & purification
Animals
*CRISPR-Cas Systems
Swine
*Livestock/microbiology
*Bacterial Proteins/genetics
*Pork Meat/microbiology
*Nucleic Acid Amplification Techniques/methods

@article {pmid40326084,
year = {2025},
author = {Barman, K and Goswami, P},
title = {Recent Advances in Diagnostics and Therapeutic Interventions for Drug-Resistant Malaria.},
journal = {ACS infectious diseases},
volume = {},
number = {},
pages = {},
doi = {10.1021/acsinfecdis.4c00962},
pmid = {40326084},
issn = {2373-8227},
abstract = {The emergence of drug-resistant malarial parasites has been a growing challenge to medical science to safeguard public health in the malaria-endemic regions of the globe. With time, the parasite develops newer resistance mechanisms to defunct the drug's action one after another. Genetic mutation is the prime weapon parasites rely upon to initiate the resistance mechanism in a case-specific manner, following various strategies such as structural changes in the target protein, metabolic alterations, and tweaking the drug-transported channels. In order to combat these resistances, different approaches have evolved among these developing inhibitors against critical parasite enzymes and metabolic pathways, combinatorial/hybrid drug therapies, exploring new drug targets and analogues of existing drugs, use of resistance-reversal agents, drug-repurposing, gene blocking/altering using RNA interference and CRISPR/Cas systems are prominent. However, the effectiveness of these approaches needs to be earnestly monitored for better management of the disease, which demands the development of a reliable diagnosis technique. Several methodologies have been investigated in search of a suitable diagnosis technique, such as in vivo, in vitro, ex vivo drug efficacy studies, and molecular techniques. A parallel effort to transform the efficient method into an inexpensive and portable diagnosis tool for rapid screening of drug resistance malaria among masses in the societal landscape is advocated. This review gives an insight into the historical perspectives of drug-resistant malaria and the recent developments in malaria diagnosis and antimalarial drug discovery. Efforts have been made to update recent strategies formulated to combat and diagnose drug-resistant malaria. Finally, a concluding remark with a future perspective on the subject has been forwarded.},
}

@article {pmid40294287,
year = {2025},
author = {Huang, P and Liu, Y and Zhao, C and Wang, C and Wang, L and Luo, M and Wang, W and Shan, W and Liu, X and Li, B and Wang, Z and Deng, H and Chen, X},
title = {Permanent Efferocytosis Prevention by Terminating MerTK Recycle on Tumor-Associated Macrophages for Cancer Immunotherapy.},
journal = {Journal of the American Chemical Society},
volume = {147},
number = {18},
pages = {15901-15914},
doi = {10.1021/jacs.5c05640},
pmid = {40294287},
issn = {1520-5126},
mesh = {*c-Mer Tyrosine Kinase/metabolism/genetics/immunology/antagonists & inhibitors ; Animals ; Proto-Oncogene Mas ; Mice ; *Immunotherapy ; *Tumor-Associated Macrophages/metabolism/immunology ; Humans ; Phagocytosis ; Mice, Inbred C57BL ; Cell Line, Tumor ; Phosphatidylserines/metabolism ; *Neoplasms/therapy/immunology ; CRISPR-Cas Systems ; Efferocytosis ; },
abstract = {Efferocytosis of apoptotic tumor cells by tumor-associated macrophages mediated through the phosphatidylserine (PtdSer)/MER proto-oncogene tyrosine kinase (MerTK) axis can exacerbate tumor immunosuppression, and conversely, prevention of efferocytosis via blocking PtdSer-MerTK association using prevalent antibodies represents a promising strategy for reversing tumor immunosuppression and boosting antitumor immunity. However, it remains unclear whether the antibody blockade can induce durable efferocytosis prevention and achieve sustained tumor growth inhibition. Here, we have shown that utilizing PtdSer and MerTK antibodies induced only a transient rather than a persistent efferocytosis prevention effect, and little enhancement was observed even after improving antibody enrichment in tumor sites. Further mechanistic studies suggested that degradation of anti-MerTK antibody and recycling of the MerTK receptor to the cell membrane would compromise the therapeutic benefits of antibody blockade. Based on these findings, we developed a CRISPR/Cas9 gene editing system deployed using Cas9 mRNA and MerTK sgRNA to permanently knock out MerTK, which achieved durable efferocytosis prevention, elicited persistent in situ vaccination immune responses via enhancing X-ray irradiation-induced immunogenic cell death, and led to sustained tumor suppression effects together with anti-PtdSer antibody and X-ray irradiation treatment in multiple B16 melanoma tumor models. Our findings provide a reliable gene-editing-mediated strategy for long-term modulating MerTK homeostasis and overcoming MerTK-dependent cancer immune evasion, generating adaptive antitumor immune responses for sustained cancer immunotherapy.},
}

*c-Mer Tyrosine Kinase/metabolism/genetics/immunology/antagonists & inhibitors
Animals
Proto-Oncogene Mas
Mice
*Immunotherapy
*Tumor-Associated Macrophages/metabolism/immunology
Humans
Phagocytosis
Mice, Inbred C57BL
Cell Line, Tumor
Phosphatidylserines/metabolism
*Neoplasms/therapy/immunology
CRISPR-Cas Systems
Efferocytosis

@article {pmid39951551,
year = {2025},
author = {Zhang, B and Zhang, P and Wang, H and Wang, X and Hu, Z and Wang, F and Li, Z},
title = {Dual Protein Corona-Mediated Target Recognition System for Visual Detection and Single-Molecule Counting of Nucleic Acids.},
journal = {ACS nano},
volume = {19},
number = {7},
pages = {6929-6941},
doi = {10.1021/acsnano.4c13924},
pmid = {39951551},
issn = {1936-086X},
mesh = {Gold/chemistry ; Metal Nanoparticles/chemistry ; Surface Plasmon Resonance/methods ; *Protein Corona/chemistry ; Humans ; *Nucleic Acids/analysis ; CRISPR-Cas Systems ; *Single Molecule Imaging/methods ; Biosensing Techniques/methods ; Point-of-Care Systems ; },
abstract = {Rapid, highly sensitive, and specific nucleic acid detection plays a crucial role in advancing point-of-care (POC) diagnostics for pathogens and viruses, cancer monitoring, and optimizing clinical treatments. Herein, leveraging the precise recognition ability of CRISPR/dCas9 and the powerful localized surface plasmon resonance (LSPR) of gold nanoparticles (AuNPs), we report the design of a dual protein corona-mediated detection platform to simultaneously fulfill rapid POC testing and single-molecule counting of nucleic acids in a one-pot and one-step manner. This system uses guide RNA as a molecular bridge to anchor dCas9 protein onto AuNPs, forming artificial protein coronas. Upon recognizing a target, the interaction between the two protein coronas on the same nucleic acid molecule triggers cross-linked aggregation of AuNPs. Then, a target as low as 100 aM can be visually detected within 30 min, making the platform particularly well-suited for rapid POC application and the screening of emerging epidemics. Additionally, the superior LSPR properties of AuNPs increase the light-scattering signal generated during target-induced aggregation, enabling the visualization of the aggregated AuNPs as diffraction-limited spots under confocal microscopy. By counting these spots, the platform achieves unprecedented detection sensitivity, identifying a target as low as 1 aM, which is equivalent to just 6 molecules in a 10 μL system, demonstrating single-molecule detection capability. This dual protein corona-mediated detection system offers exceptional promise for large-scale screening of pathogenic viruses and the early detection of cancer, particularly in applications requiring ultrahigh sensitivity at the single-molecule level.},
}

Gold/chemistry
Metal Nanoparticles/chemistry
Surface Plasmon Resonance/methods
*Protein Corona/chemistry
Humans
*Nucleic Acids/analysis
CRISPR-Cas Systems
*Single Molecule Imaging/methods
Biosensing Techniques/methods
Point-of-Care Systems

@article {pmid39941065,
year = {2025},
author = {Xia, X and Song, W and Zhang, F and Fan, Y and Zhang, B and Chen, X},
title = {ctdsp2 Knockout Induces Zebrafish Craniofacial Dysplasia via p53 Signaling Activation.},
journal = {International journal of molecular sciences},
volume = {26},
number = {3},
pages = {},
pmid = {39941065},
issn = {1422-0067},
support = {82271186 and 81974143//the General Programs of the National Natural Science Foundation of China/ ; 2022-PUMCH-C-029//the National High Level Hospital Clinical Research Funding/ ; 7232122//the Natural Science Foundation of Beijing, China/ ; },
mesh = {Animals ; *Zebrafish/genetics/metabolism/embryology ; *Tumor Suppressor Protein p53/metabolism/genetics ; *Zebrafish Proteins/genetics/metabolism ; *Signal Transduction ; *Craniofacial Abnormalities/genetics/metabolism/pathology ; Apoptosis/genetics ; Chondrocytes/metabolism/pathology ; Gene Knockout Techniques ; Neural Crest/metabolism ; Cell Proliferation ; Gene Expression Regulation, Developmental ; Cell Differentiation ; Cartilage/metabolism ; CRISPR-Cas Systems ; Disease Models, Animal ; },
abstract = {Hemifacial microsomia (HFM) is a rare congenital craniofacial deformity that significantly impacts the appearance and hearing. The genetic etiology of HFM remains largely unknown, although genetic factors are considered to be primary contributors. We previously identified CTDSP2 as a potential causative gene in HFM cases. Utilizing CRISPR/Cas9, we knocked out ctdsp2 in zebrafish and analyzed the spatiotemporal expression of ctdsp2 and neural crest cell (NCC) markers through in situ hybridization (ISH). Craniofacial cartilage and chondrocyte phenotypes were visualized using Alcian blue and wheat germ agglutinin (WGA) staining. Cell proliferation and apoptosis were assessed via immunofluorescence with PH3 and TUNEL. RNA sequencing was performed on ctdsp2[-/-] embryos and control siblings, followed by rescue experiments. Knockout of ctdsp2 in zebrafish resulted in craniofacial defects characteristic of HFM. We observed abnormalities in NCC apoptosis and proliferation in the pharyngeal arches, as well as impaired differentiation of chondrocytes in ctdsp2[-/-] embryos. RNA-Seq analysis revealed significantly higher expression of genes in the p53 signaling pathway in mutants. Furthermore, ctdsp2 mRNA injection and tp53 knockout significantly rescued pharyngeal arch cartilage dysplasia. Our findings suggest that ctdsp2 knockout induces zebrafish craniofacial dysplasia, primarily by disrupting pharyngeal chondrocyte differentiation and inhibiting NCC proliferation through p53 signaling pathway activation.},
}

Animals
*Zebrafish/genetics/metabolism/embryology
*Tumor Suppressor Protein p53/metabolism/genetics
*Zebrafish Proteins/genetics/metabolism
*Signal Transduction
*Craniofacial Abnormalities/genetics/metabolism/pathology
Apoptosis/genetics
Chondrocytes/metabolism/pathology
Gene Knockout Techniques
Neural Crest/metabolism
Cell Proliferation
Gene Expression Regulation, Developmental
Cell Differentiation
Cartilage/metabolism
CRISPR-Cas Systems
Disease Models, Animal

@article {pmid39939726,
year = {2025},
author = {Stewart, J and Krastev, DB and Brough, R and Zatreanu, D and Song, F and Baxter, JS and Sridhar, S and Frankum, J and Konde, A and Yang, W and Haider, S and Alexander, J and Betteridge, K and Gulati, A and Attygalle, AD and Vroobel, K and Natrajan, R and Khalique, S and Roumeliotis, TI and Choudhary, JS and Yeung, J and Wicks, AJ and Marlow, R and Banerjee, S and Pettitt, SJ and Tutt, ANJ and Lord, CJ},
title = {PPP2R1A mutations cause ATR inhibitor sensitivity in ovarian clear cell carcinoma.},
journal = {Oncogene},
volume = {44},
number = {9},
pages = {618-629},
pmid = {39939726},
issn = {1476-5594},
support = {DRCRPG-Nov21\100001//Cancer Research UK (CRUK)/ ; },
mesh = {Female ; Humans ; *Protein Phosphatase 2/genetics ; *Adenocarcinoma, Clear Cell/genetics/drug therapy/pathology ; *Ovarian Neoplasms/genetics/drug therapy/pathology ; Animals ; *Mutation ; Mice ; Cell Line, Tumor ; *Ataxia Telangiectasia Mutated Proteins/antagonists & inhibitors/genetics ; Transcription Factors/genetics ; DNA-Binding Proteins/genetics ; Drug Resistance, Neoplasm/genetics ; Xenograft Model Antitumor Assays ; CRISPR-Cas Systems ; },
abstract = {Identification of ARID1A/ATR synthetic lethality led to ATR inhibitor phase II trials in ovarian clear cell carcinoma (OCCC), a cancer of unmet need. Using multiple CRISPR-Cas9 mutagenesis and interference screens, we show that inactivation of protein phosphatase 2A (PP2A) subunits, including PPP2R1A, enhance ATRi sensitivity in ARID1A mutant OCCC. Analysis of a new OCCC cohort indicates that 52% possess oncogenic PPP2R1A p.R183 mutations and of these, one half possessed both ARID1A as well as PPP2R1A mutations. Using CRISPR-prime editing to generate new isogenic models of PPP2R1A mutant OCCC, we found that PPP2R1A p.R183W and p.R183P mutations cause ATRi-induced S phase stress, premature mitotic entry, genomic instability and ATRi sensitivity in OCCC tumour cells. p.R183 mutation also enhanced both in vitro and in vivo ATRi sensitivity in preclinical models of ARID1A mutant OCCC. These results argue for the assessment of PPP2R1A mutations as a biomarker of ATRi sensitivity.},
}

Female
Humans
*Protein Phosphatase 2/genetics
*Adenocarcinoma, Clear Cell/genetics/drug therapy/pathology
*Ovarian Neoplasms/genetics/drug therapy/pathology
Animals
*Mutation
Mice
Cell Line, Tumor
*Ataxia Telangiectasia Mutated Proteins/antagonists & inhibitors/genetics
Transcription Factors/genetics
DNA-Binding Proteins/genetics
Drug Resistance, Neoplasm/genetics
Xenograft Model Antitumor Assays
CRISPR-Cas Systems

@article {pmid39933924,
year = {2025},
author = {Lebon, S and Bruneel, A and Drunat, S and Albert, A and Csaba, Z and Elmaleh, M and Ntorkou, A and Ténier, Y and Fenaille, F and Gressens, P and Passemard, S and Boespflug-Tanguy, O and Dorboz, I and El Ghouzzi, V},
title = {A biallelic variant in GORASP1 causes a novel Golgipathy with glycosylation and mitotic defects.},
journal = {Life science alliance},
volume = {8},
number = {4},
pages = {},
pmid = {39933924},
issn = {2575-1077},
mesh = {Humans ; Glycosylation ; *Golgi Apparatus/metabolism/pathology/genetics ; *Golgi Matrix Proteins/genetics/metabolism ; *Mitosis/genetics ; Male ; Female ; Mutation ; Alleles ; Phenotype ; CRISPR-Cas Systems ; *Membrane Proteins/genetics ; },
abstract = {GRASP65 is a Golgi-associated peripheral protein encoded by the GORASP1 gene and required for Golgi cisternal stacking in vitro. A key role of GRASP65 in the regulation of cell division has also been suggested. However, depletion of GRASP65 in mice has little effect on the Golgi structure and the gene has not been associated with any human phenotype to date. Here, we report the identification of the first human pathogenic variant of GORASP1 (c.1170_1171del; p.Asp390Glufs*18) in a patient combining a neurodevelopmental disorder with neurosensory, neuromuscular, and skeletal abnormalities. Functional analysis revealed that the variant leads to a total absence of GRASP65. The structure of the Golgi apparatus did not show fragmentation, but glycosylation anomalies such as hyposialylation were detected. Mitosis analyses revealed an excess of prometaphases and metaphases with polar chromosomes, suggesting a delay in the cell cycle. These phenotypes were recapitulated in RPE cells in which a similar mutation was introduced by CRISPR/Cas9. These results indicate that loss of GRASP65 in humans causes a novel Golgipathy associated with defects in glycosylation and mitotic progression.},
}

Humans
Glycosylation
*Golgi Apparatus/metabolism/pathology/genetics
*Golgi Matrix Proteins/genetics/metabolism
*Mitosis/genetics
Male
Female
Mutation
Alleles
Phenotype
CRISPR-Cas Systems
*Membrane Proteins/genetics

@article {pmid39930358,
year = {2025},
author = {Zhang, Y and Newstead, S and Sarkies, P},
title = {Predicting substrates for orphan solute carrier proteins using multi-omics datasets.},
journal = {BMC genomics},
volume = {26},
number = {1},
pages = {130},
pmid = {39930358},
issn = {1471-2164},
mesh = {Humans ; *Solute Carrier Proteins/metabolism/genetics ; Algorithms ; *Computational Biology/methods ; *Genomics/methods ; Substrate Specificity ; CRISPR-Cas Systems ; Multiomics ; },
abstract = {Solute carriers (SLC) are integral membrane proteins responsible for transporting a wide variety of metabolites, signaling molecules and drugs across cellular membranes. Despite key roles in metabolism, signaling and pharmacology, around one third of SLC proteins are 'orphans' whose substrates are unknown. Experimental determination of SLC substrates is technically challenging, given the wide range of possible physiological candidates. Here, we develop a predictive algorithm to identify correlations between SLC expression levels and intracellular metabolite concentrations by leveraging existing cancer multi-omics datasets. Our predictions recovered known SLC-substrate pairs with high sensitivity and specificity compared to simulated random pairs. CRISPR-Cas9 dependency screen data and metabolic pathway adjacency data further improved the performance of our algorithm. In parallel, we combined drug sensitivity data with SLC expression profiles to predict new SLC-drug interactions. Together, we provide a novel bioinformatic pipeline to predict new substrate predictions for SLCs, offering new opportunities to de-orphanise SLCs with important implications for understanding their roles in health and disease.},
}

Humans
*Solute Carrier Proteins/metabolism/genetics
Algorithms
*Computational Biology/methods
*Genomics/methods
Substrate Specificity
CRISPR-Cas Systems
Multiomics

@article {pmid40325455,
year = {2025},
author = {Gao, Y and Zou, Y and Wu, C and Tao, J and Nie, Z and Yan, J and Wang, P and Huang, X},
title = {Comparative evaluation of immunomodulatory cytokines for oncolytic therapy based on a high-efficient platform for oHSV1 reconstruction.},
journal = {Virology journal},
volume = {22},
number = {1},
pages = {133},
pmid = {40325455},
issn = {1743-422X},
support = {Kunming Medical University graduate Student Innovation Fund(2024S086)//Yufang Zou/ ; National Natural Science Foundation of China (82260486)//Xinwei Huang/ ; },
mesh = {Animals ; *Oncolytic Virotherapy/methods ; *Oncolytic Viruses/genetics/immunology ; *Cytokines/genetics/immunology ; Mice ; *Herpesvirus 1, Human/genetics/immunology ; Humans ; Female ; Cell Line, Tumor ; CRISPR-Cas Systems ; *Triple Negative Breast Neoplasms/therapy/immunology ; Mice, Inbred BALB C ; Tumor Microenvironment ; *Immunologic Factors/genetics ; },
abstract = {BACKGROUND: Triple-negative breast cancer (TNBC) presents significant therapeutic challenges due to its immunosuppressive tumor microenvironment (TME). Oncolytic herpes simplex virus type 1 (oHSV1) offers dual mechanisms of tumor lysis and immune activation, yet the optimal cytokine payloads for TNBC remain undefined.

METHODS: We developed a CRISPR/Cas9-mediated platform for high-efficiency oHSV1 engineering, replacing the ICP47 locus with murine IFN-γ, GM-CSF, or IL-15Rα/IL-15 fusion protein (IL15Fu). Constructs were validated for cytokine secretion, MHC modulation, and cytotoxicity in 4T1 TNBC and a panel of human cancer cell lines. Antitumor efficacy and immune remodeling were evaluated in a syngeneic 4T1 model using RNA sequencing and flow cytometry.

RESULTS: The CRISPR platform achieved 62.5-71.4% homologous recombination efficiency, enabling rapid virus construction. In vitro, OV-IFNG exhibited upregulated MHC I/II expression and potent cytotoxicity, while OV-GMCSF attenuated oncolysis in subsets of breast cancer cell lines. In the 4T1 model, OV-IL15Fu modestly improved tumor control and extended survival without apparent toxicity, while OV-IFNG induced early mortality associated with systemic toxicity. Transcriptomic profiling revealed divergent immune modulation: OV-IL15Fu enriched T cell/NK cytotoxicity pathways, OV-IFNG amplified cytokine/chemokine signaling, and OV-GMCSF paradoxically enhanced myeloid recruitment while inhibiting MHC-II pathways. Flow cytometry confirmed functional differences in immune activation: OV-IL15Fu expanding cytotoxic lymphocytes (CD8⁺ T/NK cells), OV-IFNG preferentially promote Th1 polarization and innate immune activation, and OV-GMCSF failed to activate T cells despite myeloid infiltration.

CONCLUSIONS: Our findings underscore the need for rational cytokine selection in oHSV1-based immunotherapy. While IFN-γ increased immunogenic markers, its systemic toxicity and myeloid effects may limit benefit. GM-CSF exacerbated immune suppression in this context, whereas IL15Fu showed favorable immunostimulatory properties without detectable toxicity. These data support IL15Fu as a contextually promising payload for further evaluation in TNBC-targeted oncolytic virotherapy.},
}

Animals
*Oncolytic Virotherapy/methods
*Oncolytic Viruses/genetics/immunology
*Cytokines/genetics/immunology
Mice
*Herpesvirus 1, Human/genetics/immunology
Humans
Female
Cell Line, Tumor
CRISPR-Cas Systems
*Triple Negative Breast Neoplasms/therapy/immunology
Mice, Inbred BALB C
Tumor Microenvironment
*Immunologic Factors/genetics

@article {pmid40324959,
year = {2025},
author = {Namata, MJ and Xu, J and Habyarimana, E and Palakolanu, SR and Wang, L and Li, J},
title = {Genome editing in maize and sorghum: A comprehensive review of CRISPR/Cas9 and emerging technologies.},
journal = {The plant genome},
volume = {18},
number = {2},
pages = {e70038},
doi = {10.1002/tpg2.70038},
pmid = {40324959},
issn = {1940-3372},
support = {32372134//National Natural Science Foundation of China/ ; S202310879188//Student Innovation and Entrepreneurship Project of Anhui Science and Technology University/ ; No.XK-XJGF001//Key Discipline Construction Funds of Anhui Sciences and Technology University/ ; 2023cxcysj179//Chuzhou "Star of Innovation and Entrepreneurship" Industrial Innovation Team, Postgraduate Innovation and Entrepreneurship Practice Project of Anhui Province/ ; },
mesh = {*Sorghum/genetics ; *Zea mays/genetics ; *Gene Editing/methods ; *CRISPR-Cas Systems ; Plant Breeding/methods ; *Genome, Plant ; Plants, Genetically Modified ; Crops, Agricultural/genetics ; },
abstract = {The increasing changes in the climate patterns across the globe have deeply affected food systems where unparalleled and unmatched challenges are created. This jeopardizes food security due to an ever-increasing population. The extreme efficiency of C4 crops as compared to C3 crops makes them incredibly significant in securing food safety. C4 crops, maize (Zea mays L.) and sorghum (Sorghum bicolor L. Moench) in particular, have the ability to withstand osmotic stress induced by oxidative stress. Osmotic stress causes a series of physical changes in a plant thus facilitating reduced water uptake and photosynthesis inhibition, such as membrane tension, cell wall stiffness, and turgor changes. There has been a great advancement in plant breeding brought by introduction of clustered regularly interspaced short palindromic repeats (CRISPR) gene editing technology. This technology offers precise alterations to an organism's DNA through targeting specific genes for desired traits in a wide number of crop species. Despite its immense opportunities in plant breeding, it faces limitations such as effective delivery systems, editing efficiency, regulatory concerns, and off-target effects. Future prospects lie in optimizing next-generation techniques, such as prime editing, and developing novel genotype-independent delivery methods. Overall, the transformative role of CRISPR/Cas9 in sorghum and maize breeding underscores the need for responsible and sustainable utilization to address global food security challenges.},
}

*Sorghum/genetics
*Zea mays/genetics
*Gene Editing/methods
*CRISPR-Cas Systems
Plant Breeding/methods
*Genome, Plant
Plants, Genetically Modified
Crops, Agricultural/genetics

@article {pmid40324075,
year = {2025},
author = {Engel, NW and Steinfeld, I and Ryan, D and Anupindi, K and Kim, S and Wellhausen, N and Chen, L and Wilkins, K and Baker, DJ and Rommel, PC and Jarocha, D and Gohil, M and Zhang, Q and Milone, MC and Fraietta, JA and Davis, M and Young, RM and June, CH},
title = {Quadruple adenine base-edited allogeneic CAR T cells outperform CRISPR/Cas9 nuclease-engineered T cells.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {122},
number = {20},
pages = {e2427216122},
doi = {10.1073/pnas.2427216122},
pmid = {40324075},
issn = {1091-6490},
support = {1-U54 CA244711//US NIH, National Cancer Institute/ ; Thought Leader Award 2020//Agilent Foundation (Agilent Technologies Foundation)/ ; Sponsored Research//Gilead | Kite Pharma/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; *T-Lymphocytes/immunology/metabolism ; Animals ; Humans ; Mice ; *Receptors, Chimeric Antigen/genetics/immunology/metabolism ; *Adenine/metabolism ; Immunotherapy, Adoptive/methods ; Graft vs Host Disease ; },
abstract = {Genome-editing technologies have enabled the clinical development of allogeneic cellular therapies, yet the optimal gene-editing modality for multiplex editing of therapeutic T cell product manufacturing remains elusive. In this study, we conducted a comprehensive comparison of CRISPR/Cas9 nuclease and adenine base editor (ABE) technologies in generating allogeneic chimeric antigen receptor (CAR) T cells, utilizing extensive in vitro and in vivo analyses. Both methods achieved high editing efficiencies across four target genes, critical for mitigating graft-versus-host disease and allograft rejection: TRAC or CD3E, B2M, CIITA, and PVR. Notably, ABE demonstrated higher manufacturing yields and distinct off-target profiles compared to Cas9, with translocations observed exclusively in Cas9-edited products. Functionally, ABE-edited CAR T cells exhibited superior in vitro effector functions under continuous antigen stimulation, including enhanced proliferative capacity and increased surface CAR expression. Transcriptomic analysis revealed that ABE editing resulted in reduced activation of p53 and DNA damage response pathways at baseline, along with sustained activation of metabolic pathways during antigen stress. Consistently, Assay for Transposase-Accessible Chromatin using sequencing data indicated that Cas9-edited, but not ABE-edited, CAR T cells showed enrichment of chromatin accessibility peaks associated with double-strand break repair and DNA damage response pathways. In a preclinical leukemia model, ABE-edited CAR T cells demonstrated improved tumor control and extended overall survival compared to their Cas9-edited counterparts. Collectively, these findings position ABE as superior to Cas9 nucleases for multiplex gene editing of therapeutic T cells.},
}

*CRISPR-Cas Systems/genetics
*Gene Editing/methods
*T-Lymphocytes/immunology/metabolism
Animals
Humans
Mice
*Receptors, Chimeric Antigen/genetics/immunology/metabolism
*Adenine/metabolism
Immunotherapy, Adoptive/methods
Graft vs Host Disease

@article {pmid40270429,
year = {2025},
author = {Shi, K and Luo, W and Cheng, Y and Li, H and Peng, L and Luo, X and Hu, Y and Zhang, J and Chen, J},
title = {Phosphorothioate-Modified Hairpin G-Triplex Reporter-Assisted Split CRISPR/Cas12a-Powered Biosensor for "Turn-On" Fluorescent Detection of Nucleic Acid and Non-Nucleic Acid Targets.},
journal = {Analytical chemistry},
volume = {97},
number = {17},
pages = {9361-9366},
doi = {10.1021/acs.analchem.5c00140},
pmid = {40270429},
issn = {1520-6882},
mesh = {*Biosensing Techniques/methods ; G-Quadruplexes ; *CRISPR-Cas Systems ; *MicroRNAs/analysis ; *DNA/analysis/chemistry ; Benzothiazoles/chemistry ; *Phosphorothioate Oligonucleotides/chemistry ; Fluorescent Dyes/chemistry ; Limit of Detection ; Humans ; Spectrometry, Fluorescence ; },
abstract = {CRISPR/Cas12a-powered biosensors with guanine (G)-rich sequence reporters (e.g., G-quadruplex and G-triplex) are widely used in detection applications due to their simplicity and sensitivity. However, when these biosensors are employed for molecular detection in complex samples, they may encounter difficulties such as high background signal and susceptibility to interference because of the "turn-off" signal output. Herein, we explore, for the first time, a set of phosphorothioate (ps)-modified G-quadruplex (G4) and G-triplex (G3) sequences that can bind with thioflavin T (ThT) in an active split CRISPR/Cas12a system (SCas12a) to generate a "turn-on" fluorescent signal. To apply this new phenomenon, we develop a universal SCas12a-powered biosensor for "turn-on" fluorescent detection of nucleic acid (miRNA-21) and non-nucleic acid (kanamycin) targets by using ps-modified hairpin G3 as a reporter (SCas12a/psHG3). Target recognition activates SCas12a's trans-cleavage activity, leading to cleavage at the loop region of the psHG3 reporter. The released prelocked psG3 DNA binds ThT to produce a strong fluorescence signal. Without preamplification, this strategy can detect miRNA-21 with a detection limit of 100 fM. Moreover, the SCas12a/psHG3 system was further utilized for detecting kanamycin by incorporating its aptamers, enabling the detection of kanamycin at concentrations as low as 100 pM. This work is the first to develop a "turn-on" SCas12a/psHG3 system, showcasing its improved performance and wide range of applications in synthetic biology-based sensing technology.},
}

*Biosensing Techniques/methods
G-Quadruplexes
*CRISPR-Cas Systems
*MicroRNAs/analysis
*DNA/analysis/chemistry
Benzothiazoles/chemistry
*Phosphorothioate Oligonucleotides/chemistry
Fluorescent Dyes/chemistry
Limit of Detection
Humans
Spectrometry, Fluorescence

@article {pmid40263956,
year = {2025},
author = {Yan, H and Qi, M and Li, H},
title = {Characterization and Full Sequencing of 100 Nt sgRNA and Large RNA Using Site-Directed Cleavage and Liquid Chromatography Tandem Mass Spectrometry.},
journal = {Analytical chemistry},
volume = {97},
number = {17},
pages = {9228-9238},
doi = {10.1021/acs.analchem.4c06606},
pmid = {40263956},
issn = {1520-6882},
mesh = {Tandem Mass Spectrometry/methods ; Ribonuclease H/metabolism ; Chromatography, Liquid ; DNA, Catalytic/metabolism/chemistry ; *RNA, Guide, CRISPR-Cas Systems/genetics/metabolism/chemistry ; *RNA/genetics/chemistry ; *Sequence Analysis, RNA/methods ; CRISPR-Cas Systems ; },
abstract = {CRISPR/Cas9 is widely recognized as the most effective, efficient, and precise genome editing tool, inspiring numerous applications in basic science, medicine, and biotechnology. In the CRISPR/Cas9 system, single guide RNA (sgRNA) and Cas9 enzyme form a ribonucleoprotein complex that specifically and effectively cleaves target DNA. Accurate sequencing of sgRNA, particularly identifying the target sequence within the first 20 nucleotides (nt) at the 5'-end, is crucial for quality assurance and regulatory compliance. In this study, we used site-directed cleavage using ribonuclease H (RNase H) and DNAzyme for the first time to digest 100 nt sgRNA, achieving full sequencing with 100% coverage by analyzing the two cleaved fragments separately via LC MS/MS. We evaluated four different DNA-RNA chimeras as capture probes for the RNase H site-directed cleavage approach, finding that the chimera with four deoxynucleotides provided the most specific cleavage. Compared to RNase H, the DNAzyme demonstrated higher specificity and stability for 100 nt sgRNA digestion, successfully identifying up to 200 nucleotides of large RNA with 100% sequence coverage by fully sequencing the four short cleaved fragments. Due to the high specificity of DNAzyme cleavage, we used this method to study the designed 5'-end N-X truncated impurities of 100 nt sgRNA, demonstrating accurate identification and relative quantification. For 100 nt sgRNA, the limited available cleavage site was set on the scaffold sequence for both site-directed cleavage approaches, and the captured probes designed for RNase H and DNAzyme can be universally applied to sequence all 100 nt sgRNAs because of the conserved scaffold sequence.},
}

Tandem Mass Spectrometry/methods
Ribonuclease H/metabolism
Chromatography, Liquid
DNA, Catalytic/metabolism/chemistry
*RNA, Guide, CRISPR-Cas Systems/genetics/metabolism/chemistry
*RNA/genetics/chemistry
*Sequence Analysis, RNA/methods
CRISPR-Cas Systems

@article {pmid40215333,
year = {2025},
author = {Macias, LA and Lowther, J and Tillotson, EL and Rohde, E and Madsen, JA},
title = {Ion Mobility Gas-Phase Separation Enhances Top-Down Mass Spectrometry of Heavily Modified Guide RNA.},
journal = {Analytical chemistry},
volume = {97},
number = {17},
pages = {9430-9437},
doi = {10.1021/acs.analchem.5c00705},
pmid = {40215333},
issn = {1520-6882},
mesh = {*Mass Spectrometry/methods ; *RNA, Guide, CRISPR-Cas Systems/chemistry/genetics/analysis ; Ion Mobility Spectrometry ; Gases/chemistry ; Phase Separation ; },
abstract = {As gene editing technologies enter the clinic, state-of-the-art characterization methods have been developed in parallel to assess the components of these paradigm-shifting medicines. One such component, the guide RNA (gRNA) element of CRISPR-based drugs, is a large synthetic heavily modified oligonucleotide that programs for the desired gene edit. Conventional oligonucleotide sequencing technologies can inform gRNA composition, but these methods may not completely capture the chemical modifications that are introduced during synthesis. Circumventing these challenges, mass spectrometry has demonstrated use in oligonucleotide analyses and has been combined here with ion mobility to deepen its characterization power. The use of ion mobility enabled us to perform gas-phase separation of the fragment ions produced by top-down mass spectrometry, yielding a significant increase in fragment identifications for a highly modified 100-mer gRNA by uncovering high-confidence assignments for heavily modified regions and for the important spacer region. Furthermore, the high-confidence fragment assignments empowered simultaneous de novo sequencing and chemical modification localization for the 5'-end spacer region as well as for 15 nucleotides on the heavily modified 3'-end. Overall, a total sequence coverage of 95% was achieved for the heavily modified 100-mer, ushering near complete sequence and chemical modification confirmation by top-down mass spectrometry.},
}

*Mass Spectrometry/methods
*RNA, Guide, CRISPR-Cas Systems/chemistry/genetics/analysis
Ion Mobility Spectrometry
Gases/chemistry
Phase Separation

@article {pmid40020602,
year = {2025},
author = {Patra, N and Barker, GC and Maiti, MK},
title = {Knockout of fatty acid elongase1 homeoalleles in amphidiploid Brassica juncea leads to undetectable erucic acid in seed oil.},
journal = {Plant physiology and biochemistry : PPB},
volume = {222},
number = {},
pages = {109679},
doi = {10.1016/j.plaphy.2025.109679},
pmid = {40020602},
issn = {1873-2690},
mesh = {*Mustard Plant/genetics/metabolism/enzymology ; *Erucic Acids/metabolism ; *Fatty Acid Elongases/genetics/metabolism ; *Seeds/metabolism/genetics ; *Plant Oils/metabolism ; Gene Knockout Techniques ; *Plant Proteins/genetics/metabolism ; CRISPR-Cas Systems ; Gene Editing ; },
abstract = {Indian mustard (Brassica juncea L.) is a major oilseed crop with considerable economic and nutritional importance globally. While its seed oil offers valuable dietary benefits due to a balanced ratio of human essential fatty acids, the traditional high oil-yielding varieties contain an elevated level of erucic acid (EA, C22:1) associated with adverse health effects. Therefore, developing low erucic acid (LEA) mustard cultivars is crucial for broader utilization and consumer safety. In this study, CRISPR/Cas9 genome editing tool was employed to disrupt the fatty acid elongase1 (FAE1) gene that encodes a key enzyme in EA biosynthesis in two high erucic acid (HEA) B. juncea cultivars, PCR7 (∼39% EA) and JD6 (∼45% EA). Targeted knockout (KO) of BjFAE1 homeoalleles (BjFAE1.1 and BjFAE1.2) in this amphidiploid plant species using CRISPR/Cas9 constructs, each carrying two guide RNAs led to generation of single (either fae1.1 or fae1.2) and double (fae1.1fae1.2) mutants. Best performing homozygous fae1.1fae1.2 KO lines showed a near-complete elimination of EA in both the cultivars (<0.5% in PCR7, undetectable in JD6) with a marked increase in nutritionally beneficial oleic acid (from ∼18% to ∼32% in PCR7, from ∼9% to ∼38% in JD6). Moreover, the content of essential fatty acids also increased substantially [linoleic acid (C18:2) 1.9-fold in PCR7 and 2.1-fold in JD6; linolenic acid (C18:3) 2.5-fold in PCR7 and 1.4-fold in JD6], suggesting rerouting of carbon flux from EA biosynthesis. Importantly, these LEA lines retained key agronomic traits like plant seed yield and oil content, matching the productivity of the unedited control elite cultivars. Our findings underscore the effectiveness of CRISPR/Cas9 technology for editing B. juncea genome, developing plant lines producing LEA seed oil with improved nutritional quality and broadening the utility of this important oilseed crop for food and non-food applications.},
}

*Mustard Plant/genetics/metabolism/enzymology
*Erucic Acids/metabolism
*Fatty Acid Elongases/genetics/metabolism
*Seeds/metabolism/genetics
*Plant Oils/metabolism
Gene Knockout Techniques
*Plant Proteins/genetics/metabolism
CRISPR-Cas Systems
Gene Editing

@article {pmid39916610,
year = {2025},
author = {Uusi-Mäkelä, M and Harjula, SE and Junno, M and Sillanpää, A and Nätkin, R and Niskanen, MT and Saralahti, AK and Nykter, M and Rämet, M},
title = {The inflammasome adaptor pycard is essential for immunity against Mycobacterium marinum infection in adult zebrafish.},
journal = {Disease models & mechanisms},
volume = {18},
number = {9},
pages = {},
pmid = {39916610},
issn = {1754-8411},
support = {//Tampereen Tuberkuloosisaatio/ ; //Maud Kuistilan Muistosaatio/ ; //Tampere University Doctoral School/ ; //Suomen Kulttuurirahasto/ ; //Finnish Society for Study of Infectious Diseases/ ; 277495//The Academy of Finland/ ; //The Competitive State Research Financing of the Expert Responsibility Area of Oulu University Hospital/ ; //Tampereen Tuberkuloosisäätiö/ ; //Maud Kuistilan Muistosäätiö/ ; //Väinö ja Laina Kiven Säätiö/ ; //Orion Research Foundation/ ; //National Doctoral Education Pilot Based on the Immune System/ ; //Instrumentariumin Tiedesäätiö/ ; //Sigrid Juséliuksen Säätiö/ ; 277495//Research Council of Finland/ ; //Competitive State Research Financing of the Expert Responsibility Area of Oulun Yliopistollinen Sairaala/ ; //Tampereen Yliopisto/ ; },
mesh = {Animals ; *Zebrafish/microbiology/immunology/genetics ; *Mycobacterium marinum/physiology/immunology ; *Mycobacterium Infections, Nontuberculous/immunology/microbiology/genetics ; *Zebrafish Proteins/metabolism/genetics ; *Inflammasomes/metabolism ; Neutrophils/metabolism/immunology ; *CARD Signaling Adaptor Proteins/metabolism ; Granuloma/pathology ; *Immunity ; CRISPR-Cas Systems/genetics ; Kidney/pathology ; Gene Knockout Techniques ; Larva/microbiology ; Gene Expression Profiling ; Transcriptome/genetics ; },
abstract = {Inflammasomes regulate the host response to intracellular pathogens including mycobacteria. We have previously shown that the course of Mycobacterium marinum infection in adult zebrafish (Danio rerio) mimics the course of tuberculosis in human. To investigate the role of the inflammasome adaptor pycard in zebrafish M. marinum infection, we produced two zebrafish knockout mutant lines for the pycard gene with CRISPR/Cas9 mutagenesis. Although the zebrafish larvae lacking pycard developed normally and had unaltered resistance against M. marinum, the loss of pycard led to impaired survival and increased bacterial burden in the adult zebrafish. Based on histology, immune cell aggregates, granulomas, were larger in pycard-deficient fish than in wild-type controls. Transcriptome analysis with RNA sequencing of a zebrafish haematopoietic tissue, kidney, suggested a role for pycard in neutrophil-mediated defence, haematopoiesis and myelopoiesis during infection. Transcriptome analysis of fluorescently labelled, pycard-deficient kidney neutrophils identified genes that are associated with compromised resistance, supporting the importance of pycard for neutrophil-mediated immunity against M. marinum. Our results indicate that pycard is essential for resistance against mycobacteria in adult zebrafish.},
}

Animals
*Zebrafish/microbiology/immunology/genetics
*Mycobacterium marinum/physiology/immunology
*Mycobacterium Infections, Nontuberculous/immunology/microbiology/genetics
*Zebrafish Proteins/metabolism/genetics
*Inflammasomes/metabolism
Neutrophils/metabolism/immunology
*CARD Signaling Adaptor Proteins/metabolism
Granuloma/pathology
*Immunity
CRISPR-Cas Systems/genetics
Kidney/pathology
Gene Knockout Techniques
Larva/microbiology
Gene Expression Profiling
Transcriptome/genetics

@article {pmid39909615,
year = {2025},
author = {Chignon, A and Lettre, G},
title = {Using omics data and genome editing methods to decipher GWAS loci associated with coronary artery disease.},
journal = {Atherosclerosis},
volume = {401},
number = {},
pages = {118621},
doi = {10.1016/j.atherosclerosis.2024.118621},
pmid = {39909615},
issn = {1879-1484},
mesh = {Humans ; *Genome-Wide Association Study ; *Coronary Artery Disease/genetics ; Genetic Predisposition to Disease ; *Gene Editing/methods ; Mendelian Randomization Analysis ; Quantitative Trait Loci ; Phenotype ; *Genomics/methods ; Animals ; CRISPR-Cas Systems ; Polymorphism, Single Nucleotide ; },
abstract = {Coronary artery disease (CAD) is due to atherosclerosis, a pathophysiological process that involves several cell-types and results in the accumulation of lipid-rich plaque that disrupt the normal blood flow through the coronary arteries to the heart. Genome-wide association studies have identified 1000s of genetic variants robustly associated with CAD or its traditional risk factors (e.g. blood pressure, blood lipids, type 2 diabetes, smoking). However, gaining biological insights from these genetic discoveries remain challenging because of linkage disequilibrium and the difficulty to interpret the functions of non-coding regulatory elements in the human genome. In this review, we present different statistical methods (e.g. Mendelian randomization) and molecular datasets (e.g. expression or protein quantitative trait loci) that have helped connect CAD-associated variants with genes, biological pathways, and cell-types or tissues. We emphasize that these various strategies make predictions, which need to be validated in orthologous systems. We discuss specific examples where the integration of omics data with GWAS results has prioritized causal CAD variants and genes. Finally, we review how targeted and genome-wide genome editing experiments using the CRISPR/Cas9 toolbox have been used to characterize new CAD genes in human cells. Researchers now have the statistical and bioinformatic methods, the molecular datasets, and the experimental tools to dissect comprehensively the loci that contribute to CAD risk in humans.},
}

Humans
*Genome-Wide Association Study
*Coronary Artery Disease/genetics
Genetic Predisposition to Disease
*Gene Editing/methods
Mendelian Randomization Analysis
Quantitative Trait Loci
Phenotype
*Genomics/methods
Animals
CRISPR-Cas Systems
Polymorphism, Single Nucleotide

@article {pmid38772706,
year = {2025},
author = {Abate-Shen, C and Politi, K},
title = {The Evolution of Mouse Models of Cancer: Past, Present, and Future.},
journal = {Cold Spring Harbor perspectives in medicine},
volume = {15},
number = {5},
pages = {},
pmid = {38772706},
issn = {2157-1422},
mesh = {Animals ; Mice ; *Disease Models, Animal ; *Neoplasms/genetics ; Humans ; Gene Editing ; CRISPR-Cas Systems ; History, 21st Century ; History, 20th Century ; },
abstract = {In the nearly 50 years since the original models of cancer first hit the stage, mouse models have become a major contributor to virtually all aspects of cancer research, and these have evolved well beyond simple transgenic or xenograft models to encompass a wide range of more complex models. As the sophistication of mouse models has increased, an explosion of new technologies has expanded the potential to both further develop and apply these models to address major challenges in cancer research. In the current era, cancer modeling has expanded to include nongermline genetically engineered mouse models (GEMMs), patient-derived models, organoids, and adaptations of the models better suited for cancer immunology research. New technologies that have transformed the field include the application of CRISPR-Cas9-mediated genome editing, in vivo imaging, and single-cell analysis to cancer modeling. Here, we provide a historical perspective on the evolution of mouse models of cancer, focusing on how far we have come in a relatively short time and how new technologies will shape the future development of mouse models of cancer.},
}

Animals
Mice
*Disease Models, Animal
*Neoplasms/genetics
Humans
Gene Editing
CRISPR-Cas Systems
History, 21st Century
History, 20th Century

@article {pmid40324726,
year = {2025},
author = {Kumbhakar, R and Mondal, M and Thakro, V and Tripathi, S and Parida, SK},
title = {Shaping the future: Unravelling regulators modulating plant architecture for next-generation crops.},
journal = {Plant science : an international journal of experimental plant biology},
volume = {},
number = {},
pages = {112534},
doi = {10.1016/j.plantsci.2025.112534},
pmid = {40324726},
issn = {1873-2259},
abstract = {Plant architecture traits in crops are modulated through intricate interactions of various genetic pathways, which helps them to adapt to diverse environmental conditions. Key developmental pathways involved in forming plant architecture include the LAZY-TAC (Tiller Angle Control) module regulating branch and tiller angle, the CLAVATA-WUSCHEL pathway controlling shoot apical meristem fate and the GID1-DELLA pathway governing plant height and tillering in major food crops. These pathways function in concert to shape the overall architecture of plants, which is essential for optimizing light capture, resource allocation, reproductive success and eventual crop yield enhancement. Presently, plant architecture of modern crops has been shaped especially by artificial selection of natural alleles that target yield traits. Recent advances in CRISPR-Cas-based genome editing and genomics-assisted breeding strategies have enabled precise genetic manipulation of natural alleles in the functionally relevant genes regulating plant architecture traits in crops. This will assist researchers to select and introgress superior natural alleles in popular cultivars strategically for restructuring their desirable plant-types suitable for mechanical harvesting as well as enhancing the crop yield potential.},
}

@article {pmid40324585,
year = {2025},
author = {Jiang, Y and Zheng, H and Bai, L and Wang, Y and Zhong, J and Zhang, W and Xu, T and Pan, Y and Tang, J and Lu, J and Zhang, B and Wu, Y},
title = {Genomic analysis and mobile genetic elements carriage of Clostridium perfringens type A.},
journal = {Anaerobe},
volume = {},
number = {},
pages = {102970},
doi = {10.1016/j.anaerobe.2025.102970},
pmid = {40324585},
issn = {1095-8274},
abstract = {OBJECTIVES: To explore a comprehensive genomic analysis of Clostridium perfringens type A strains from diverse regions in China, investigating their virulence genes, antibiotic resistance genes, and mobile genetic elements (MGEs) to inform strategies for infection control and resistance gene surveillance.

METHODS: We conducted whole-genome sequencing on 168 C. perfringens type A strains from nine provinces in China (2016-2021). Previously described alpha-toxin (PLC) sequence typing for C. perfringens was used for comparisons with core genome multilocus sequence typing. Virulence genes, antibiotic resistance genes, and MGEs, including CRISPR/Cas, prophages, and plasmids of C. perfringens type A were investigated by molecular and bioinformatic methods.

RESULTS: PLC type II contained the largest number of isolates (n=44). The same type strains were largely clustered in the same branches. Tetracycline resistance genes tetA(P) and tetB(P) had high prevalence in type A isolates. 395 prophages were predicted including 265 "incomplete," 55 "questionable," and 75 "intact" prophages. CRISPR/Cas systems were more common in isolates from humans (63%) than in those from animals and food (52% and 46%, respectively). Fifty-seven percent of strains likely had the tcp conjugation locus (tcpC to tcpH), and 12 isolates likely carried the conjugative pCW3 plasmid. Type A strains exhibited fewer plasmid-encoded toxins.

CONCLUSIONS: cgMLST analysis demonstrated some micro-evolution and regional transmission trends within type A, which exhibited partial correlated with PLC typing. This study highlights the need for enhanced surveillance of antimicrobial resistance and pathogenicity-associated MGEs in C. perfringens type A.},
}

@article {pmid40319267,
year = {2025},
author = {García-Calvo, L and Kummen, C and Rustad, S and Rønning, SB and Fagerlund, A},
title = {A toolkit for facilitating markerless integration of expression cassettes in Komagataella phaffii via CRISPR/Cas9.},
journal = {Microbial cell factories},
volume = {24},
number = {1},
pages = {97},
pmid = {40319267},
issn = {1475-2859},
support = {314743//Norwegian Fund for Research Fees for Agricultural Products (FFL)/ ; 314743//Norwegian Fund for Research Fees for Agricultural Products (FFL)/ ; 314743//Norwegian Fund for Research Fees for Agricultural Products (FFL)/ ; 314743//Norwegian Fund for Research Fees for Agricultural Products (FFL)/ ; 336136//The Research Council of Norway/ ; 336136//The Research Council of Norway/ ; 336136//The Research Council of Norway/ ; 336136//The Research Council of Norway/ ; 336136//The Research Council of Norway/ ; },
mesh = {*CRISPR-Cas Systems ; *Saccharomycetales/genetics/metabolism ; *Gene Editing/methods ; Plasmids/genetics ; Recombinant Proteins/genetics/biosynthesis ; Green Fluorescent Proteins/genetics/metabolism ; },
abstract = {BACKGROUND: The yeast Komagataella phaffii (formerly known as Pichia pastoris) has been widely used for functional expression of recombinant proteins, including plant and animal food proteins. CRISPR/Cas9 genome editing systems can be used for insertion of heterologous genes without the use of selection markers. The study aimed to create a convenient markerless knock-in method for integrating expression cassettes into the chromosome of K. phaffii using CRISPR/Cas9 technology. The approach was based on the hierarchical, modular, Golden Gate assembly employing the GoldenPiCS toolkit. Furthermore, the aim was to evaluate the system's efficiency and suitability for producing secreted recombinant food proteins.

RESULTS: Three Cas9/sgRNA plasmids were constructed, along with corresponding donor helper plasmids containing homology regions for chromosomal integration via homology-directed repair. The integration efficiency of an enhanced green fluorescent protein (eGFP) expression cassette was assessed at three genomic loci (04576, PFK1, and ROX1). The 04576 locus showed the highest integration efficiency, while ROX1 had the highest transformation efficiency. Whole genome sequencing revealed variable copy numbers of eGFP expression cassettes among clones, corresponding with increasing levels of fluorescence. Furthermore, the system's applicability for producing recombinant food proteins was validated by successfully expressing and secreting chicken ovalbumin. This constitutes the first report of CRISPR/Cas9 applied to produce recombinant chicken ovalbumin.

CONCLUSIONS: The adapted GoldenPiCS toolkit combined with CRISPR/Cas9 technology enabled efficient and precise genome integration in K. phaffii. This approach holds promise for expanding the production of high-value recombinant proteins. Future research should focus on optimizing integration sites and improving cloning procedures to enhance the system's efficiency and versatility.},
}

*CRISPR-Cas Systems
*Saccharomycetales/genetics/metabolism
*Gene Editing/methods
Plasmids/genetics
Recombinant Proteins/genetics/biosynthesis
Green Fluorescent Proteins/genetics/metabolism

@article {pmid40319033,
year = {2025},
author = {Cheng, B and Peng, SI and Jia, YY and Tong, E and Atwood, SX and Sun, BK},
title = {Comprehensive secretome profiling and CRISPR screen identifies SFRP1 as a key inhibitor of epidermal progenitor proliferation.},
journal = {Cell death & disease},
volume = {16},
number = {1},
pages = {360},
pmid = {40319033},
issn = {2041-4889},
support = {R03AR075844//U.S. Department of Health & Human Services | NIH | National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS)/ ; K08AR067853//U.S. Department of Health & Human Services | NIH | National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS)/ ; },
mesh = {Humans ; Cell Proliferation ; *Keratinocytes/metabolism/cytology ; *Membrane Proteins/metabolism/genetics ; *Stem Cells/metabolism/cytology ; *Intercellular Signaling Peptides and Proteins/metabolism/genetics ; *Secretome/metabolism ; *Epidermis/metabolism ; Wnt Signaling Pathway ; CRISPR-Cas Systems/genetics ; *Epidermal Cells/metabolism ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; },
abstract = {Secreted proteins are crucial for the structure and functions of the human epidermis, but the full repertoire of the keratinocyte secretome has not been experimentally defined. In this study, we performed mass spectrometry on conditioned media from primary human keratinocytes, identifying 406 proteins with diverse roles in adhesion, migration, proliferation, proteolysis, signal transduction, and innate immunity. To leverage this new dataset, we developed a novel colony formation assay-based CRISPR screen to investigate the functions of uncharacterized secreted proteins on epidermal stem cells. The screen identified six candidate proteins that promoted proliferation of epidermal progenitors and two proteins that inhibited it. Secreted frizzled-related protein-1 (SFRP1) was the most potent inhibitor. We discovered that SFRP1 restrained clonogenic keratinocyte proliferation by inhibiting Wnt signaling as well as blocking ectopic expression of leukemia inhibitory factor (LIF). Collectively, our study expands our knowledge of the keratinocyte secretome, establishes a novel CRISPR screen to assess the function of non-cell autonomous factors, and highlights SFRP1's role in regulating epidermal balance.},
}

Humans
Cell Proliferation
*Keratinocytes/metabolism/cytology
*Membrane Proteins/metabolism/genetics
*Stem Cells/metabolism/cytology
*Intercellular Signaling Peptides and Proteins/metabolism/genetics
*Secretome/metabolism
*Epidermis/metabolism
Wnt Signaling Pathway
CRISPR-Cas Systems/genetics
*Epidermal Cells/metabolism
*Clustered Regularly Interspaced Short Palindromic Repeats/genetics

@article {pmid40318042,
year = {2025},
author = {Kang, J and Park, C and Lee, G and Koo, J and Oh, H and Kim, EH and Bae, E and Suh, JY},
title = {Structural Investigation of the Anti-CRISPR Protein AcrIE7.},
journal = {Proteins},
volume = {},
number = {},
pages = {},
doi = {10.1002/prot.26832},
pmid = {40318042},
issn = {1097-0134},
support = {NRF-2022R1A2C1009804//National Research Foundation of Korea/ ; NRF-2021R1A2C1003917//National Research Foundation of Korea/ ; RS-2023-00207820//National Research Foundation of Korea/ ; A412550//Korea Basic Science Institute/ ; },
abstract = {The CRISPR-Cas system is an adaptive immune system in prokaryotes that provides protection against bacteriophages. As a countermeasure, bacteriophages have evolved various anti-CRISPR proteins that neutralize CRISPR-Cas immunity. Here, we report the structural and functional investigation of AcrIE7, which inhibits the type I-E CRISPR-Cas system in Pseudomonas aeruginosa. We determined both crystal and solution structures of AcrIE7, which revealed a novel helical fold. In binding assays using various biochemical methods, AcrIE7 did not tightly interact with a single Cas component in the type I-E Cascade complex or the CRISPR adaptation machinery. In contrast, AlphaFold modeling with our experimentally determined AcrIE7 structure predicted that AcrIE7 interacts with Cas3 in the type I-E CRISPR-Cas system in P. aeruginosa. Our findings are consistent with a model where AcrIE7 inhibits Cas3 and also highlight the effectiveness and limitations of AlphaFold modeling.},
}

@article {pmid40317571,
year = {2025},
author = {da Silva, GC and Rossi, CC},
title = {The Arms Race Between Actinobacillus pleuropneumoniae and Its Genetic Environment: A Comprehensive Analysis of Its Defensome and Mobile Genetic Elements.},
journal = {Molecular microbiology},
volume = {},
number = {},
pages = {},
doi = {10.1111/mmi.15374},
pmid = {40317571},
issn = {1365-2958},
support = {APQ-03498-22//Fundação de Amparo à Pesquisa do Estado de Minas Gerais/ ; APQ-01339-25//Fundação de Amparo à Pesquisa do Estado de Minas Gerais/ ; 408564/2023-7//Conselho Nacional de Desenvolvimento Científico e Tecnológico/ ; },
abstract = {Actinobacillus pleuropneumoniae is the causative agent of pleuropneumonia in swine, a highly contagious and economically significant disease. The genetic variability of A. pleuropneumoniae complicates disease control efforts, as it enables rapid adaptation to various stressors, including antimicrobial treatments. To better understand the molecular mechanisms underlying this adaptability, we investigated the role of the bacterial defensome and its relationship with mobile genetic elements (MGEs), such as prophages, plasmids, and integrative conjugative elements (ICEs). Using bioinformatic tools, we identified a diverse and rich defensome in A. pleuropneumoniae, with an average of 16 different defense systems per strain. We found that CRISPR-Cas systems, along with other defense mechanisms, are actively involved in restricting the entry of foreign genetic material, playing a crucial role in bacterial adaptation. Additionally, we characterized several novel prophages and examined their distribution across different strains, revealing their potential contribution to the bacterium's evolutionary success. Our findings underscore the complex interplay between the bacterium's defense systems and MGEs, shedding light on how A. pleuropneumoniae maintains genetic diversity while also safeguarding itself against external threats. These insights provide a better understanding of the genetic factors that influence the pathogen's adaptability and highlight potential avenues for more effective disease control strategies.},
}

@article {pmid40317300,
year = {2025},
author = {Rathore, RS and Jiang, W and Sedeek, K and Mahfouz, M},
title = {Harnessing neo-domestication of wild pigmented rice for enhanced nutrition and sustainable agriculture.},
journal = {TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik},
volume = {138},
number = {5},
pages = {108},
pmid = {40317300},
issn = {1432-2242},
mesh = {*Oryza/genetics ; Gene Editing ; *Domestication ; *Plant Breeding ; *Agriculture ; Nutritive Value ; Genetic Variation ; CRISPR-Cas Systems ; Crops, Agricultural/genetics ; },
abstract = {Advances in precision gene editing have enabled the rapid domestication of wild crop relatives, a process known as neo-domestication. During domestication, breeding rice for maximum productivity under optimal growth conditions reduced genetic diversity, eliminating variants for stress tolerance and grain nutrients. Wild rice varieties have rich genetic diversity, including variants for disease resistance, stress tolerance, and grain nutritional quality. For example, the grain of pigmented wild rice has abundant antioxidants (anthocyanins, proanthocyanidins, and flavonoids), but low yield, poor plant architecture, and long life cycle limit its cultivation. In this review, we address the neo-domestication of wild pigmented rice, focusing on recent progress, CRISPR-Cas editing toolboxes, selection of key candidate genes for domestication, identifying species with superior potential via generating genomic and multi-omics resources, efficient crop transformation methods and highlight strategies for the promotion and application pigmented rice. We also address critical outstanding questions and potential solutions to enable efficient neo-domestication of wild pigmented rice and thus enhance food security and nutrition.},
}

*Oryza/genetics
Gene Editing
*Domestication
*Plant Breeding
*Agriculture
Nutritive Value
Genetic Variation
CRISPR-Cas Systems
Crops, Agricultural/genetics

@article {pmid40316660,
year = {2025},
author = {Chen, Y and Ren, J and Yang, X and Li, Z and Wu, W and Luo, G and Xu, Y and Yang, H and Wang, Q and Zhang, Q},
title = {The rapid detection of human HLA-B*27 gene based on BASIC isothermal detection.},
journal = {Scientific reports},
volume = {15},
number = {1},
pages = {15427},
pmid = {40316660},
issn = {2045-2322},
support = {82472386//National Natural Science Foundation of China/ ; 23LCYJ038//Sichuan Health Commission/ ; 2023HT073//Industry-University-Research Innovation Fund for Chinese Universities/ ; S21077//Sichuan Medical Association/ ; 23JCYJPT0032, 20SXZRKX0010, 20SXQT0109 and 202001207//Nanchong Science and Technology Fund/ ; 2024CX001, 2023PTZK016 and ZX-51130001-2021-023//Research Fund of Affiliated Hospital of North Sichuan Medical College/ ; 22SXQT0307//Special Fund of the Strategic Science and Technology Cooperation between Nanchong Municipal Science and Technology Bureau and Universities/ ; 2024NSFSC1546//National Natural Science Foundation of Sichuan Province/ ; },
mesh = {Humans ; *HLA-B27 Antigen/genetics ; *Spondylitis, Ankylosing/genetics/diagnosis ; CRISPR-Cas Systems ; *Nucleic Acid Amplification Techniques/methods ; *Molecular Diagnostic Techniques/methods ; Sensitivity and Specificity ; Genotype ; },
abstract = {Ankylosing spondylitis (AS) is a chronic, inflammatory arthritis of the spine and peripheral joints which is known to have a strong association with the human leukocyte antigen B27 (HLA-B27). Quantitative real-time PCR and flow cytometry are the predominant methods for HLA-B27 gene and antigen, respectively, which are too time-consuming and labor-intensive to realize rapid analysis. Therefore, a rapid diagnostic tool is highly required. In this study, we developed a rapid HLA-B*27 detection platform (namely BASIC) by combining our previously invented BASIS isothermal amplification method with the widely used CRISPR/Cas12a signal output tool. The BASIS can efficiently amplify all HLA-B*27 genotypes by using a set of universal primers, which target the conserved regions. The amplicons are subsequently applied to CRISPR/Cas12a analysis. The CRISPR/Cas12a recognizes the pathogenic HLA-B*27 amplicons specifically by using a well-designed gRNA, thereby achieving fluorescence signal output. Our results showed that the BASIC can be completed in 1 h with analytical sensitivity up to 100 aM. It could resist interference of homologous genes, hemoglobin, bilirubin, and triglyceride. For clinical sample detection, the BASIC offered completely consistent results with qPCR. Given the advantages of sensitivity, specificity, simplicity and rapidity, the BASIC was demonstrated a promising HLA-B*27 gene rapid detection tool for the early screening and diagnosis of AS.},
}

Humans
*HLA-B27 Antigen/genetics
*Spondylitis, Ankylosing/genetics/diagnosis
CRISPR-Cas Systems
*Nucleic Acid Amplification Techniques/methods
*Molecular Diagnostic Techniques/methods
Sensitivity and Specificity
Genotype

@article {pmid40316524,
year = {2025},
author = {Berman, A and Su, N and Li, Z and Landau, U and Chakraborty, J and Gerbi, N and Liu, J and Qin, Y and Yuan, B and Wei, W and Yanai, O and Mayrose, I and Zhang, Y and Shani, E},
title = {Construction of multi-targeted CRISPR libraries in tomato to overcome functional redundancy at genome-scale level.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {4111},
pmid = {40316524},
issn = {2041-1723},
support = {757683//EC | EU Framework Programme for Research and Innovation H2020 | H2020 Priority Excellent Science | H2020 European Research Council (H2020 Excellent Science - European Research Council)/ ; 101118769//EC | EU Framework Programme for Research and Innovation H2020 | H2020 Euratom (H2020 Euratom Research and Training Programme 2014-2018)/ ; 101113412//EC | EU Framework Programme for Research and Innovation H2020 | H2020 Euratom (H2020 Euratom Research and Training Programme 2014-2018)/ ; 3419/20//Israel Science Foundation (ISF)/ ; 1462/24//Israel Science Foundation (ISF)/ ; },
mesh = {*Solanum lycopersicum/genetics ; *Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; *Genome, Plant ; RNA, Guide, CRISPR-Cas Systems/genetics ; Gene Library ; Plants, Genetically Modified ; Phenotype ; Plant Breeding ; Clustered Regularly Interspaced Short Palindromic Repeats ; Mutation ; },
abstract = {Genetic variance is vital for breeding programs and mutant screening, yet traditional mutagenesis methods wrestle with genetic redundancy and a lack of specificity in gene targeting. CRISPR-Cas9 offers precise, site-specific gene editing, but its application in crop improvement has been limited by scalability challenges. In this study, we develop genome-wide multi-targeted CRISPR libraries in tomato, enhancing the scalability of CRISPR gene editing in crops and addressing the challenges of redundancy while maintaining its precision. We design 15,804 unique single guide RNAs (sgRNAs), each targeting multiple genes within the same gene families. These sgRNAs are classified into 10 sub-libraries based on gene function. We generate approximately 1300 independent CRISPR lines and successfully identify mutants with distinct phenotypes related to fruit development, fruit flavor, nutrient uptake, and pathogen response. Additionally, we develop CRISPR-GuideMap, a double-barcode tagging system to enable large-scale sgRNA tracking in generated plants. Our results demonstrate that multi-targeted CRISPR libraries are scalable and effective for large-scale gene editing and offer an approach to overcome gene functional redundancy in basic plant research and crop breeding.},
}

*Solanum lycopersicum/genetics
*Gene Editing/methods
*CRISPR-Cas Systems/genetics
*Genome, Plant
RNA, Guide, CRISPR-Cas Systems/genetics
Gene Library
Plants, Genetically Modified
Phenotype
Plant Breeding
Clustered Regularly Interspaced Short Palindromic Repeats
Mutation

@article {pmid40316390,
year = {2025},
author = {Shixing, X and Shengjun, B and He, S and Xinyue, Z and Xingdong, Z and Xiaoying, Z and Leng, H and Enyong, D and Wan, J},
title = {A fluorescence biosensor for detecting LncRNA MALAT1 based on isothermal amplification by cyclic extension.},
journal = {Analytica chimica acta},
volume = {1357},
number = {},
pages = {344076},
doi = {10.1016/j.aca.2025.344076},
pmid = {40316390},
issn = {1873-4324},
mesh = {*RNA, Long Noncoding/analysis/genetics ; Humans ; *Nucleic Acid Amplification Techniques/methods ; *Biosensing Techniques/methods ; Fluorescence ; Limit of Detection ; DNA Probes/chemistry/genetics ; },
abstract = {BACKGROUND: Long non-coding RNA (lncRNA) Metastasis-Associated Lung Adenocarcinoma Transcript 1 (MALAT1), a crucial regulator of gene expression, has emerged as a highly promising biomarker in the progression of various cancers. The clinical detection of lncRNA MALAT1 primarily relies on Reverse Transcription-Polymerase Chain Reaction (RT-PCR), which requires skilled operators and large, expensive thermal cycling equipment. These limitations have restricted the application of RT-PCR, particularly in resource-constrained settings.

RESULTS: In this study, we developed a novel signal amplification method, termed Isothermal Amplification by Cyclic Extension (IACE), based on the linear extension of a single-stranded DNA probe. IACE operates through the continuous extension of Probe 1 (a) into long single-stranded DNA with multiple repetitive sequences, facilitated by Probe 2 (a∗a∗) and Bst DNA polymerase. We found that the single-stranded DNA product of IACE could directly activate the CRISPR-Cas12a system without requiring a protospacer adjacent motif (PAM). By integrating IACE with a three-way junction structure and a nicking enzyme, we established a one-step signal amplification strategy for the detection of lncRNA MALAT1, achieving a detection limit as low as 37.5 fM using the CRISPR-Cas system.

SIGNIFICANCE: The biosensor developed in the present study simplifies workflows, minimizes contamination risks, and demonstrates exceptional detection performance in tumor patient samples, highlighting its potential to advance clinical tumor diagnostic approaches.},
}

*RNA, Long Noncoding/analysis/genetics
Humans
*Nucleic Acid Amplification Techniques/methods
*Biosensing Techniques/methods
Fluorescence
Limit of Detection
DNA Probes/chemistry/genetics

@article {pmid40316384,
year = {2025},
author = {Chang, Z and Zhou, J and Li, D and Wang, H and Chen, M and Liu, L and Ding, J and Wang, Y and Gao, Z and Sai, N},
title = {Selection and identification of the ssDNA aptamer against polymyxin B sulfate.},
journal = {Analytica chimica acta},
volume = {1357},
number = {},
pages = {344067},
doi = {10.1016/j.aca.2025.344067},
pmid = {40316384},
issn = {1873-4324},
mesh = {*Aptamers, Nucleotide/chemistry ; *DNA, Single-Stranded/chemistry ; SELEX Aptamer Technique ; *Polymyxin B/analysis ; Biosensing Techniques ; CRISPR-Cas Systems ; *Anti-Bacterial Agents/analysis ; Animals ; },
abstract = {BACKGROUND: Polymyxin B (PMB) is a potent antibiotic, and its sulfate form, Polymyxin B Sulfate (PMBS), is widely used. When PMBS is used as a veterinary drug, excessive use can lead to drug residues in animal bodies, which is a significant food safety issue and raises serious concerns. Therefore, there is an urgent need to strengthen research in this area. In this study, we obtained a ssDNA aptamer against PMBS using the Capture-SELEX method, which has notable advantages in small molecule selection. Then, we integrated the obtained aptamer with a biosensor based on the CRISPR/Cas14a system for a series of validations.

RESULTS: In this study, specific aptamers against PMBS were discovered via the Capture-SELEX process. The selection process consisted of ten rounds. The affinity of the candidate sequences was determined by Isothermal Titration Calorimetry (ITC), and the PMBS24 aptamer with the highest affinity was ultimately identified, with a dissociation constant (Kd) of 3.89 ± 0.46 μM. To further obtain high-affinity aptamers, we attempted to truncate the PMBS24 aptamer. However, the results demonstrated that the aptamer with the full-length sequence exhibited superior affinity. Subsequently, it was applied in the CRISPR/Cas14a-based sensor for the detection of PMBS. This method had a LOD of 0.99 ng mL[-1], with good specificity, and was successfully used for the detection in milk.

SIGNIFICANCE: An important recognition element of PMBS has been successfully obtained, filling a part of the gap in the detection of PMBS. Moreover, the aptamer has been applied to the CRISPR/Cas14a-based sensor for amplification-free PMBS detection, yielding favorable detection results. This also opens up the possibility of applying the aptamer to other similar sensors.},
}

*Aptamers, Nucleotide/chemistry
*DNA, Single-Stranded/chemistry
SELEX Aptamer Technique
*Polymyxin B/analysis
Biosensing Techniques
CRISPR-Cas Systems
*Anti-Bacterial Agents/analysis
Animals

@article {pmid40315882,
year = {2025},
author = {Lou, E and Choudhry, MS and Starr, TK and Folsom, TD and Bell, J and Rathmann, B and DeFeo, AP and Kim, J and Slipek, N and Jin, Z and Sumstad, D and Klebanoff, CA and Ladner, K and Sarkari, A and McIvor, RS and Murray, TA and Miller, JS and Rao, M and Jensen, E and Ankeny, J and Khalifa, MA and Chauhan, A and Spilseth, B and Dixit, A and Provenzano, PP and Pan, W and Weber, D and Byrne-Steele, M and Henley, T and McKenna, DH and Johnson, MJ and Webber, BR and Moriarity, BS},
title = {Targeting the intracellular immune checkpoint CISH with CRISPR-Cas9-edited T cells in patients with metastatic colorectal cancer: a first-in-human, single-centre, phase 1 trial.},
journal = {The Lancet. Oncology},
volume = {26},
number = {5},
pages = {559-570},
doi = {10.1016/S1470-2045(25)00083-X},
pmid = {40315882},
issn = {1474-5488},
mesh = {Humans ; Middle Aged ; *CRISPR-Cas Systems ; Male ; Female ; *Colorectal Neoplasms/genetics/immunology/pathology/therapy ; Aged ; Adult ; *Lymphocytes, Tumor-Infiltrating/immunology/transplantation ; Gene Editing ; Young Adult ; Adolescent ; *T-Lymphocytes/immunology/transplantation ; },
abstract = {BACKGROUND: Over the past decade, immunotherapeutic strategies-mainly targeting the PD-1-PD-L1 immune checkpoint axis-have altered cancer treatment for many solid tumours, but few patients with gastrointestinal forms of cancer have benefited to date. There remains an urgent need to extend immunotherapy efficacy to more patients while addressing resistance to current immune checkpoint inhibitors. The aim of this study was to determine the safety and anti-tumour activity of knockout of CISH, which encodes cytokine-inducible SH2-containing protein, a novel intracellular immune checkpoint target and a founding member of the SOCS family of E3-ligases, using tumour infiltrating lymphocyte (TILs) genetically edited with CRISPR-Cas9 in patients with metastatic gastrointestinal epithelial cancers.

METHODS: For this first-in-human, single-centre, phase 1 trial, patients aged 18-70 years with a diagnosis of metastatic gastrointestinal epithelial cancer with progressive disease following at least one first line standard therapy, measurable disease with at least one lesion identified as resectable for TIL generation and at least one other lesion meeting RECIST criteria as measurable to serve as an indicator of disease response, and an ECOG performance status of 0 or 1 were screened and enrolled if meeting these and all other eligibility criteria. TILs procured from tumour biopsies were expanded on the basis of neoantigen reactivity, subjected to CRISPR-Cas9-mediated CISH knockout, and infused intravenously into 12 patients after non-myeloablative lymphocyte depleting chemotherapy (cyclophosphamide 60 mg/kg per dose on study days -6 and -5, and fludarabine 25 mg/m[2] per dose on days -7 to -3) followed by high-dose IL-2 (aldesleukin; 720 000 IU/kg per dose). The primary endpoint was safety of administration of neoantigen-reactive TILs with knockout of the CISH gene, and a key secondary endpoint was anti-tumour activity measured as objective radiographic response and progression-free and overall survival. This study is registered with ClinicalTrials.gov, NCT04426669, and is complete.

FINDINGS: Between May 12, 2020, and Sept 16, 2022, 22 participants were enrolled in the trial (one patient was enrolled twice owing to lack of TIL outgrowth on the first attempt); ten patients were female, and 11 were male (self-defined). One patient was Asian, the remainder were White (self-defined). We successfully manufactured CISH knockout TIL products for 19 (86%) of the patients, of whom 12 (63%) received autologous CISH knockout TIL infusion. The median follow-up time for the study was 129 days (IQR 15-283). All 12 (100%) patients had treatment-related severe adverse events. The most common grade 3-4 adverse events included haematological events (12 patients [100%]) attributable to the preparative lymphodepleting chemotherapy regimen or expected effects of IL-2, fatigue (four patients [33%]), and anorexia (three patients [25%]). Deaths of any cause for patients on study were attributed to the underlying disease under study (metastatic gastrointestinal cancer) and related complications (10 patients) or infection (grade 5 septicaemia in one patient). There were no severe (≥grade 3) cytokine release or neurotoxicity events. Six (50%) of 12 patients had stable disease by day 28, and four (33%) had stable disease ongoing at 56 days. One young adult patient with microsatellite-instability-high colorectal cancer refractory to anti-PD1/CTLA-4 therapies had a complete and ongoing response (>21 months).

INTERPRETATION: These results support the safety and potential antitumour activity of inhibiting the immune checkpoint CISH through the administration of neoantigen-reactive CISH-knockout TILs, with implications for patients with advanced metastatic cancers refractory to checkpoint inhibitor immunotherapies, and provide the first evidence that a novel intracellular checkpoint can be targeted with therapeutic effect.

FUNDING: Intima Bioscience.},
}

Humans
Middle Aged
*CRISPR-Cas Systems
Male
Female
*Colorectal Neoplasms/genetics/immunology/pathology/therapy
Aged
Adult
*Lymphocytes, Tumor-Infiltrating/immunology/transplantation
Gene Editing
Young Adult
Adolescent
*T-Lymphocytes/immunology/transplantation

@article {pmid40315825,
year = {2025},
author = {Brück, M and Randau, L},
title = {TIGR on the loose: A dual-guide RNA system for DNA targeting.},
journal = {Molecular cell},
volume = {85},
number = {9},
pages = {1712-1713},
doi = {10.1016/j.molcel.2025.04.007},
pmid = {40315825},
issn = {1097-4164},
mesh = {*RNA, Guide, CRISPR-Cas Systems/genetics/metabolism ; *DNA/genetics/metabolism ; *Gene Editing/methods ; Humans ; *CRISPR-Cas Systems ; },
abstract = {A recent study[1] unveils tandem interspaced guide RNAs (TIGRs) that simultaneously engage both strands of target DNA and direct Tas protein activity. It offers insights into the evolution of RNA-guided proteins and introduces a promising tool for genome editing.},
}

*RNA, Guide, CRISPR-Cas Systems/genetics/metabolism
*DNA/genetics/metabolism
*Gene Editing/methods
Humans
*CRISPR-Cas Systems

@article {pmid40313975,
year = {2025},
author = {Zeng, S and Ju, Y and Alam, MS and Lu, Z and Hameed, HMA and Li, L and Tian, X and Fang, C and Fang, X and Ding, J and Wang, X and Hu, J and Wang, S and Zhang, T},
title = {A CRISPR-nonhomologous end-joining-based strategy for rapid and efficient gene disruption in Mycobacterium abscessus.},
journal = {mLife},
volume = {4},
number = {2},
pages = {169-180},
pmid = {40313975},
issn = {2770-100X},
abstract = {Mycobacterium abscessus, a fast-growing, non-tuberculous mycobacterium resistant to most antimicrobial drugs, causes a wide range of serious infections in humans, posing a significant public health challenge. The development of effective genetic manipulation tools for M. abscessus is still in progress, limiting both research and therapeutic advancements. However, the clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein (Cas) systems have emerged as promising tools for generating highly specific double-strand breaks (DSBs) in its genome. One of the mechanisms that repair these DSBs is the error-prone nonhomologous end-joining (NHEJ) pathway, which facilitates targeted gene editing. In this study, we introduced a novel application of the CRISPR-NHEJ approach in M. abscessus. We demonstrated that NrgA from M. marinum plays a crucial role in repairing DSBs induced by the CRISPR-Cas system in M. abscessus. Contrary to previous findings, our study also revealed that inhibiting or overexpressing components of homologous recombination/single-strand annealing significantly reduces the efficiency of NHEJ repair in M. abscessus. This discovery challenges current perspectives and suggests that NHEJ repair in M. abscessus may involve components from both homologous recombination and single-strand annealing pathways, highlighting the complex interactions among the three DSB repair mechanisms in M. abscessus.},
}

@article {pmid40313468,
year = {2025},
author = {Zhang, X and Wu, M and Shi, H and Kim, S and Lu, S and Wang, P and Qin, J},
title = {Amplification-Free Electrochemiluminescent Biosensor for Ultrasensitive Detection of Fusobacterium nucleatum Using Tetrahedral DNA-Based CRISPR/Cas12a.},
journal = {Cyborg and bionic systems (Washington, D.C.)},
volume = {6},
number = {},
pages = {0266},
pmid = {40313468},
issn = {2692-7632},
abstract = {Fusobacterium nucleatum, a bacterium linked to colorectal cancer, possesses a specific gene called fadA that serves as an early diagnostic biomarker. The CRISPR/Cas12a system has demonstrated marked potential for nucleic acid detection due to its satisfactory selectivity and trans-cleavage ability. However, most CRISPR/Cas-based sensors suffer from problems such as probe entanglement or local aggregation, reducing the Cas enzyme efficiency. In this study, an amplification-free biosensing platform for ultrasensitive detection of F. nucleatum was developed by integrating the highly specific CRISPR/AsCas12a with an improved electrochemiluminescence (ECL) biosensor. Different from the conventional 1- or 2-dimensional probes, the platform was constructed by tetrahedral DNA nanostructure (TDN) probes conjugated with quenchers and coralliform gold (CFAu) functionalized with luminescent agents. The TDN serves as an exceptional scaffold to modulate the recognition unit, substantially enhancing the recognition and cleavage efficiency of AsCas12a toward the probes. Furthermore, the high surface area of CFAu provides extensive landing sites for the luminescent agents, thereby improving the detection sensitivity. The prepared ECL biosensor exhibited a wider linear range (10 fM to 100 nM) and was capable of detecting F. nucleatum down to 1 colony-forming unit/ml. Additionally, the high mismatch sensitivity of AsCas12a to protospacer adjacent motifs and nearby areas provides a strategy for distinguishing mutant from wild-type sequences. Finally, by designing CRISPR RNA (crRNA), this diagnostic method can also be easily modified to detect other bacteria or biomarkers for the early diagnosis of various diseases.},
}

@article {pmid40313410,
year = {2025},
author = {Liu, J and Zhou, Y and Feng, J and Cai, C and Zhang, S},
title = {Comparative metagenomic analysis reveals the adaptive evolutionary traits of siboglinid tubeworm symbionts.},
journal = {Frontiers in microbiology},
volume = {16},
number = {},
pages = {1533506},
pmid = {40313410},
issn = {1664-302X},
abstract = {Tubeworms flourish in marine cold seeps and hydrothermal vents through the establishment of symbiotic relationships with chemosynthetic bacteria. However, the environmental adaptations and evolutionary relationships of tubeworm symbionts across diverse habitats and hosts remain largely unknown. In this study, we characterized the genomes of 26 siboglinid tubeworm symbionts collected from deep-sea hydrothermal vents, cold seeps, and deep-sea mud, including two sequenced in this study and 24 previously published. Phylogenetic analysis classified the 26 symbiont genomes into five distinct clusters at the genus level. The findings highlight the remarkable diversity in symbiont classification, influenced by the habitat and species of tubeworm, with the symbiont genome characteristics of various genera revealing unique evolutionary strategies. Siboglinid symbionts exhibit functional metabolic diversity, encompassing chemical autotrophic capabilities for carbon, nitrogen, and sulfur metabolism, hydrogen oxidation, and a chemoorganotrophic ability to utilize various amino acids, cofactors, and vitamins. Furthermore, the symbiont's homeostatic mechanisms and CRISPR-Cas system are vital adaptations for survival. Overall, this study highlights the metabolic traits of siboglinid symbionts across different genera and enhances our understanding of how different habitats and hosts influence symbiont evolution, offering valuable insights into the strategies that symbionts use to adapt and thrive in extreme environments.},
}

@article {pmid40312543,
year = {2025},
author = {Karan, R and Prasannakumar, MK and Khera, HK and Harish, J and Vamsidharreddy, N and Venkateshbabu, G and Devanna, P and Manjunatha, C and Palanna, KB and Mishra, RK},
title = {Molecular virulotyping and advancing the detection of Magnaporthe oryzae using a CRISPR-Cas12a-based diagnostic tool.},
journal = {World journal of microbiology & biotechnology},
volume = {41},
number = {5},
pages = {159},
pmid = {40312543},
issn = {1573-0972},
mesh = {*Plant Diseases/microbiology ; *Oryza/microbiology ; Phylogeny ; *CRISPR-Cas Systems ; Genetic Variation ; DNA, Fungal/genetics ; Virulence/genetics ; *Ascomycota/genetics/pathogenicity/isolation & purification/classification ; *Magnaporthe/genetics/isolation & purification/pathogenicity/classification ; Plant Leaves/microbiology ; India ; Fungal Proteins/genetics ; },
abstract = {Rice blast, caused by Magnaporthe oryzae, is a major threat to global rice production. The disease affects various growth stages of rice including leaves, nodes, and panicles leading to significant yield loss. Understanding the genetic diversity and early detection of M. oryzae is essential for developing effective intervention and disease management. In current study, 30 isolates of M. oryzae were collected from major rice-growing areas in Karnataka, Tamil Nadu, Andhra Pradesh, and Telangana, revealing considerable morphological and genetic diversity. Molecular characterization using ITS, LSU, and actin primers confirmed all isolates as M. oryzae, showing 100% sequence homology to reference strains. Phylogenetic analysis revealed regional clustering of isolates, with notable diversity observed in populations from Tamil Nadu and Karnataka. Pathogenicity assays identified significant variability in disease severity, with isolates MOK1, MOTN4, and MOK10 being highly virulent. Virulotyping revealed the widespread presence of critical pathogenicity genes, with Avr-Pik, Avr-Pita, MPS1, SLP1 and TYR1 as prominent contributors. Disease severity is highly correlated with the presence of EXO 70. The tyrosinase gene remains well conserved among all the isolates and was used as a target for CRISPR cas12a based detection. A novel CRISPR-Cas12a detection system, coupled with recombinase polymerase amplification (RPA), demonstrated high sensitivity and specificity for M. oryzae DNA, detecting concentrations as low as 10[-1]/µL of DNA copies. Field testing successfully identified the pathogen in leaf, neck, and seed samples with visual confirmation via fluorescence and lateral flow assays. This integrated approach provides valuable insights into M. oryzae diversity, pathogenicity, and a robust diagnostic tool for early pathogen detection, paving the way for targeted management strategies to mitigate rice blast disease.},
}

*Plant Diseases/microbiology
*Oryza/microbiology
Phylogeny
*CRISPR-Cas Systems
Genetic Variation
DNA, Fungal/genetics
Virulence/genetics
*Ascomycota/genetics/pathogenicity/isolation & purification/classification
*Magnaporthe/genetics/isolation & purification/pathogenicity/classification
Plant Leaves/microbiology
India
Fungal Proteins/genetics

@article {pmid40311759,
year = {2025},
author = {Soltani, S and Fallah, T and Shafiei, M and Shahraki, AH and Iranbakhsh, A},
title = {Investigating the prevalence of CRISPR-Cas system and their association with antibiotic resistance genes and virulence factors in Enterococcus faecalis and Enterococcus faecium strains isolated from hospitalized patients.},
journal = {Journal of global antimicrobial resistance},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.jgar.2025.04.022},
pmid = {40311759},
issn = {2213-7173},
abstract = {OBJECTIVES: Enterococcus faecalis and Enterococcus faecium are Gram-positive opportunistic pathogens that rank among the leading causes of nosocomial infections worldwide. This study investigates the prevalence and role of CRISPR-Cas systems in modulating antimicrobial resistance and virulence factors in clinical isolates of E. faecalis and E. faecium collected from patients in Tehran, Iran.

METHODS: A total of 75 clinical isolates of E. faecalis and E. faecium were collected from various hospitals in Tehran, Iran, between January and April 2023, from adult patients with urinary tract infections (n = 55), blood infections (n = 12), and wound infections (n = 8). Conventional bacteriology tests and PCR were used to isolate and identify Enterococcus species. Phenotypic antibiotic and genotypic resistance were assessed. CRISPR-Cas repeat-spacer array were screened using PCR, and the relationship between CRISPR-Cas systems and antibiotic resistance and virulence genes was statistically analyzed. Phylogenetic, structural, and conservation analyses were performed to assess the degree of conservation in CRISPR1-Cas csn1 and CRISPR3-Cas csn1 genes, identify potential mutations, and evaluate their possible impact on Cas9 protein function.

RESULTS: 86.6% of the isolates harbored CRISPR-Cas repeat-spacer array, with a significantly higher prevalence in E. faecalis than in E. faecium (100% vs. 66.6%, p = 0.0001). CRISPR1-Cas, CRISPR2, and CRISPR3-Cas loci were identified in 76%, 82.6%, and 64% of isolates, respectively. Notably, the prevalence of CRISPR-Cas systems was significantly reduced in extensively drug-resistant (XDR) isolates (32%) compared to multidrug-resistant (MDR) isolates (68%, p = 0.0001). Conservation analyses of CRISPR1-Cas csn1 and CRISPR3-Cas csn1 genes revealed conserved regions potentially linked to functional activity. Furthermore, CRISPR-Cas repeat-spacer array were correlated with specific antimicrobial resistance phenotypes and genotypes, as well as with virulence factors.

CONCLUSIONS: These findings suggest that CRISPR-Cas systems may influence the resistance and virulence profiles of clinical Enterococcus isolates, potentially impacting their pathogenicity and adaptability.},
}

@article {pmid40311478,
year = {2025},
author = {Yang, J and Chen, J and Xia, L and Li, G},
title = {Recent progress on biosensors for detection of circulating miRNA biomarkers.},
journal = {Talanta},
volume = {294},
number = {},
pages = {128219},
doi = {10.1016/j.talanta.2025.128219},
pmid = {40311478},
issn = {1873-3573},
abstract = {Circulating miRNAs are a class of non-coding endogenous RNAs found in body fluids which typically consist of 19-24 nucleotides in length. The abnormal expression of miRNAs has been demonstrated to be associated with severe human diseases. Aiming to provide valuable insights for the further development of reliable miRNA detectors for disease early diagnosis and treatment, this work systematically summarizes the latest advancements in signal amplification strategies for miRNA analysis, based on nanomaterials, nucleic acids, enzymes, and CRISPR/Cas system. The emerging techniques for detecting circulating miRNAs in human body fluids over the past decade are highlighted, including electrochemical, optical, and dual-mode biosensors. Furthermore, the challenges of trace miRNA detection in complex samples and the development prospects of miRNA biosensors are also discussed.},
}

@article {pmid40310889,
year = {2025},
author = {Andreatta, F and Hendriks, D and Artegiani, B},
title = {Human Organoids as an Emerging Tool for Genome Screenings.},
journal = {Annual review of biomedical engineering},
volume = {27},
number = {1},
pages = {157-183},
doi = {10.1146/annurev-bioeng-103023-122327},
pmid = {40310889},
issn = {1545-4274},
mesh = {Humans ; *Organoids/metabolism/cytology ; *Gene Editing/methods ; *CRISPR-Cas Systems ; Pluripotent Stem Cells/cytology/metabolism ; *Genome, Human ; *Genetic Testing/methods ; },
abstract = {Over the last decade, a plethora of organoid models have been generated to recapitulate aspects of human development, disease, tissue homeostasis, and repair. Organoids representing multiple tissues have emerged and are typically categorized based on their origin. Tissue-derived organoids are established directly from tissue-resident stem/progenitor cells of either adult or fetal origin. Starting from pluripotent stem cells (PSCs), PSC-derived organoids instead recapitulate the developmental trajectory of a given organ. Gene editing technologies, particularly the CRISPR-Cas toolbox, have greatly facilitated gene manipulation experiments with considerable ease and scalability, revolutionizing organoid-based human biology research. Here, we review the recent adaptation of CRISPR-based screenings in organoids. We examine the strategies adopted to perform CRISPR screenings in organoids, discuss different screening scopes and readouts, and highlight organoid-specific challenges. We then discuss individual organoid-based genome screening studies that have uncovered novel genes involved in a variety of biological processes. We close by providing an outlook on how widespread adaptation of CRISPR screenings across the organoid field may be achieved, to ultimately leverage our understanding of human biology.},
}

Humans
*Organoids/metabolism/cytology
*Gene Editing/methods
*CRISPR-Cas Systems
Pluripotent Stem Cells/cytology/metabolism
*Genome, Human
*Genetic Testing/methods

@article {pmid40310292,
year = {2025},
author = {Alejandre-Sixtos, JE and Aguirre-Martínez, K and Cruz-López, J and Mares-Rivera, A and Álvarez-Martínez, SM and Zamorano-Sánchez, D},
title = {Insights on the regulation and function of the CRISPR/Cas transposition system located in the pathogenicity island VpaI-7 from Vibrio parahaemolyticus RIMD2210633.},
journal = {Infection and immunity},
volume = {},
number = {},
pages = {e0016925},
doi = {10.1128/iai.00169-25},
pmid = {40310292},
issn = {1098-5522},
abstract = {CRISPR/Cas-mediated transposition is a recently recognized strategy for horizontal gene transfer in a variety of bacterial species. However, our understanding of the factors that control their function in their natural hosts is still limited. In this work, we report our initial genetic characterization of the elements associated with the CRISPR/Cas-transposition machinery (CASTm) from Vibrio parahaemolyticus (VpaCASTm), which are encoded within the pathogenicity island VpaI-7. Our results revealed that the components of the VpaCASTm and their associated CRISPR arrays (VpaCAST system) are transcriptionally active in their native genetic context. Furthermore, we were able to detect the presence of polycistrons and several internal promoters within the loci that compose the VpaCAST system. Our results also suggest that the activity of the promoter of the atypical CRISPR array is not repressed by the baseline activity of its known regulator VPA1391 in V. parahaemolyticus. In addition, we found that the activity of the promoter of tniQ was modulated by a regulatory cascade involving ToxR, LeuO, and H-NS. Since it was previously reported that the activity of the VpaCAST system was less efficient than that of the VchCAST system at promoting transposition of a miniaturized CRISPR-associated transposon (mini-CAST) in Escherichia coli, we analyzed if the transposition efficiency mediated by the VpaCAST system could be enhanced inside its natural host V. parahaemolyticus. We provide evidence that this might be the case, suggesting that there could be host induction factors in V. parahaemolyticus that could enable more efficient transposition of CASTs.IMPORTANCEMobile genetic elements such as transposons play important roles in the evolutionary trajectories of bacterial genomes. The success of transposon dissemination depends on their ability to carry selectable markers that improve the fitness of the host cell or loci with addictive traits such as the toxin-antitoxin systems. Here we aimed to characterize a transposon from Vibrio parahaemolyticus that carries and could disseminate multiple virulence factors. This transposon belongs to a recently discovered family of transposons whose transposition is guided by crRNA. We showed that the transposition machinery of this transposon is transcribed in V. parahaemolyticus and that there are likely host-associated factors that favor transposition in the natural host V. parahaemolyticus over transposition in Escherichia coli.},
}

@article {pmid40309116,
year = {2025},
author = {Kinde, MZ and Kerisew, B and Eshetu, T and Gessese, AT},
title = {Genomic analysis of Listeria monocytogenes strains from dairy products in Ethiopia.},
journal = {Frontiers in bioinformatics},
volume = {5},
number = {},
pages = {1572241},
pmid = {40309116},
issn = {2673-7647},
abstract = {This study explored virulence genes, antibiotic resistance genes, and mobile genetic elements in 14 Listeria monocytogenes strains from milk and dairy products collected from different regions of Ethiopia. The strains were classified into two Multilocus Sequence Typing sequence types (ST2 and ST45) and further grouped into clonal complexes (CC2) and different cgMLST types. Twenty-nine virulence genes were identified across all 14 strains, with lplA1 detected at higher levels in all strains except SAMN28661660. All L. monocytogenes strains also carried four antibiotic resistance genes (fosX, lin, norB, mprF), contributing to their ability to withstand multiple antimicrobial agents. Notably, no plasmids or mobile genetic elements were detected. Stress resistance genes, including stress survival islet 1 (SSI1_lmo0447), lmo 1800, and lmo1799, were identified in all strains. However, genes encoding for disinfectant resistance were not identified from all strains. LGI-2 was found in all the strains and none of the studied strains harbored LGI-1 and LGI-3. Conserved CRISPR-Cas systems were found in some strains. KEGG pathway analysis revealed that inlA and inlB genes facilitate bacterial internalization through host actin polymerization. Overall, the study provided crucial insights into the genomic features of L. monocytogenes in the Ethiopian dairy chain. It is crucial to establish continuous monitoring of L. monocytogenes in dairy products, improve sanitation, enforce stricter antibiotic usage and food safety regulations, and raise public awareness of associated risks.},
}

@article {pmid40307869,
year = {2025},
author = {Sitara, A and Hocq, R and Lu, AJ and Pflügl, S},
title = {Hi-TARGET: a fast, efficient and versatile CRISPR type I-B genome editing tool for the thermophilic acetogen Thermoanaerobacter kivui.},
journal = {Biotechnology for biofuels and bioproducts},
volume = {18},
number = {1},
pages = {49},
pmid = {40307869},
issn = {2731-3654},
support = {Y-1513//Austrian Science Fund/ ; },
abstract = {BACKGROUND: Due to its ability to grow fast on CO2, CO and H2 at high temperatures and with high energy efficiency, the thermophilic acetogen Thermoanaerobacter kivui could become an attractive host for industrial biotechnology. In a circular carbon economy, diversification and upgrading of C1 platform feedstocks into value-added products (e. g., ethanol, acetone and isopropanol) could become crucial. To that end, genetic and bioprocess engineering tools are required to facilitate the development of bioproduction scenarios. Currently, the genome editing tools available for T. kivui present some limitations in speed and efficiency, thus restricting the development of a powerful strain chassis for industrial applications.

RESULTS: In this study, we developed the versatile genome editing tool Hi-TARGET, based on the endogenous CRISPR Type I-B system of T. kivui. Hi-TARGET demonstrated 100% efficiency for gene knock-out (from both purified plasmid and cloning mixture) and knock-in, and 49% efficiency for creating point mutations. Furthermore, we optimized the transformation and plating protocol and increased transformation efficiency by 245-fold to 1.96 × 10[4] ± 8.7 × 10[3] CFU μg[-1]. Subsequently, Hi-TARGET was used to demonstrate gene knock-outs (pyrE, rexA, hrcA), a knock-in (ldh::pFAST), a single nucleotide mutation corresponding to PolC[C629Y], and knock-down of the fluorescent protein pFAST. Analysis of the ∆rexA deletion mutant created with Hi-TARGET revealed that the transcriptional repressor rexA is likely involved in the regulation of the expression of lactate dehydrogenase (ldh). Following genome engineering, an optimized curing procedure for edited strains was devised. In total, the time required from DNA to a clean, edited strain is 12 days, rendering Hi-TARGET a fast, robust and complete method for engineering T. kivui.

CONCLUSIONS: The CRISPR-based genome editing tool Hi-TARGET developed for T. kivui can be used for scarless deletion, insertion, point mutation and gene knock-down, thus fast-tracking the generation of industrially-relevant strains for the production of carbon-negative chemicals and fuels as well as facilitating studies of acetogen metabolism and physiology.},
}

@article {pmid40307455,
year = {2025},
author = {Chen, G and Hou, L and Li, Z and Xie, B and Liu, Y},
title = {A new strategy for Cas protein recognition based on graph neural networks and SMILES encoding.},
journal = {Scientific reports},
volume = {15},
number = {1},
pages = {15236},
pmid = {40307455},
issn = {2045-2322},
mesh = {*Neural Networks, Computer ; *CRISPR-Cas Systems ; *CRISPR-Associated Proteins/chemistry/genetics/metabolism ; *Computational Biology/methods ; Databases, Protein ; Graph Neural Networks ; },
abstract = {The CRISPR-Cas system, an adaptive immune mechanism found in bacteria and archaea, has evolved into a promising genomic editing tool, with various types of Cas proteins playing a crucial role. In this study, we developed a set of strategies for mining and identifying Cas1 proteins. Firstly, we analyzed the characteristic differences of 14 types of Cas proteins in the protein large language model embedding space in detail; then converted proteins into the Simplified Molecular Input Line Entry System (SMILES) format, thereby constructing graph data representing atom and bond features. Next, based on the characteristic differences of different Cas proteins, we designed and trained an ensemble model composed of two Directed Message Passing Neural Network (DMPNN) models for high-precision identification of Cas1 proteins. This ensemble model performed excellently on both training data and newly designed datasets. The comparison of this method with other methods, such as CRISPRCasFinder, has demonstrated its effectiveness. Finally, the ensemble model was successfully employed to identify potential Cas1 proteins in the Ensemble database, further highlighting its robustness and practicality. The strategies and models from this research may potentially be extended to other types of Cas proteins, though this would require further investigation and validation. Moreover, our work highlights SMILES encoding as a versatile tool for studying biological macromolecules, enabling efficient structural representation and advanced computational applications in protein research and beyond.},
}

*Neural Networks, Computer
*CRISPR-Cas Systems
*CRISPR-Associated Proteins/chemistry/genetics/metabolism
*Computational Biology/methods
Databases, Protein
Graph Neural Networks

@article {pmid40307212,
year = {2025},
author = {Wang, YY and Lin, JF and Wu, WW and Fu, Z and Cao, F and Chen, YX and Mo, HY and Sheng, H and Liu, ZX and Zeng, ZL and Guan, XY and Ju, HQ and Liao, K and Xu, RH},
title = {Inhibition of MBTPS1 enhances antitumor immunity and potentiates anti-PD-1 immunotherapy.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {4047},
pmid = {40307212},
issn = {2041-1723},
support = {82303306//National Natural Science Foundation of China (National Science Foundation of China)/ ; 82173128//National Natural Science Foundation of China (National Science Foundation of China)/ ; },
mesh = {Animals ; Mice ; *Immunotherapy/methods ; Humans ; *Programmed Cell Death 1 Receptor/antagonists & inhibitors/immunology ; Cell Line, Tumor ; *Colorectal Neoplasms/immunology/therapy/genetics ; STAT1 Transcription Factor/metabolism/genetics ; CD8-Positive T-Lymphocytes/immunology ; Mice, Inbred C57BL ; CRISPR-Cas Systems ; Immune Checkpoint Inhibitors/pharmacology ; Female ; },
abstract = {Despite advances in cancer immunotherapy, colorectal cancer patients exhibit limited therapeutic responses. Therefore, the exploration of strategies combining immunotherapy with adjuvant approaches to enhance adaptive immune responses is in demand. Here, we perform a customized in vivo CRISPR-Cas9 screen to target genes encoding membrane and secreted proteins in CRC mouse models with different immune characteristics. We observe that loss of membrane-bound transcription factor site-1 protease (MBTPS1) in tumor cells enhances antitumor immunity and potentiates anti-PD-1 therapy. Mechanistic studies reveal that tumor cell-intrinsic MBTPS1 competes with USP13 for binding to STAT1, thereby disrupting the USP13-dependent deubiquitination-mediated STAT1 stabilization. The upregulated STAT1-transcribed chemokines including CXCL9, CXCL10, and CXCL11, promote CXCR3[+]CD8[+] T cell infiltration. Notably, the regulatory role of MBTPS1 in antitumor immunity operates independently of its classic function in cleaving membrane-bound transcription factors. Collectively, our results provide a theoretical basis for MBTPS1 as a potential immunotherapy target.},
}

Animals
Mice
*Immunotherapy/methods
Humans
*Programmed Cell Death 1 Receptor/antagonists & inhibitors/immunology
Cell Line, Tumor
*Colorectal Neoplasms/immunology/therapy/genetics
STAT1 Transcription Factor/metabolism/genetics
CD8-Positive T-Lymphocytes/immunology
Mice, Inbred C57BL
CRISPR-Cas Systems
Immune Checkpoint Inhibitors/pharmacology
Female

@article {pmid40306516,
year = {2025},
author = {Bina, F and Bani, F and Khalilzadeh, B and Gheit, T and Karimi, A},
title = {Advancements in fluorescent nanobiosensors for HPV detection: from integrating nanomaterials to DNA nanotechnology.},
journal = {International journal of biological macromolecules},
volume = {311},
number = {Pt 1},
pages = {143619},
doi = {10.1016/j.ijbiomac.2025.143619},
pmid = {40306516},
issn = {1879-0003},
abstract = {Human papillomavirus (HPV) is a leading cause of cervical cancer and other malignancies, necessitating the development of highly sensitive and specific detection tools. This review explores recent advancements in fluorescent nanobiosensors (FNBS) for HPV detection, focusing on the integration of nanomaterials and DNA nanotechnology, highlighting their contributions to improving sensitivity, specificity, and point-of-care (POC) usability. The review critically evaluates a range of nanomaterial-based FNBS, including those employing quantum and carbon dots, nanoclusters, nanosheets, and nanoparticles, discussing their underlying signal amplification mechanisms, target recognition strategies, and limitations related to toxicity, stability, and reproducibility. Furthermore, it examines the application of diverse DNA nanotechnology, such as DNA origami, DNAzyme, catalytic hairpin assembly (CHA), hybridization chain reaction (HCR), and DNA hydrogel in improving FNBS performance. It also addresses the current challenges in clinical translation, emphasizing the necessity for large-scale production methods and thorough clinical validation to ensure biosafety. It also outlines the potential of innovative technologies, such as CRISPR-Cas-based diagnostics and artificial intelligence, to further revolutionize HPV detection and enable accessible, cost-effective screening, particularly in resource-limited settings. This review provides a valuable resource for researchers and clinicians seeking to develop next-generation FNBS for improved HPV diagnostics and cervical cancer prevention.},
}

@article {pmid40305594,
year = {2025},
author = {Sun, J and Xiong, X and Lai, W and Wu, Z and Wang, H and Yang, L and Xue, N and Yao, Q and Song, G and Zhao, Y and Li, L and Wang, F and Fan, C and Pei, H},
title = {Implementing complex nucleic acid circuits in living cells.},
journal = {Science advances},
volume = {11},
number = {18},
pages = {eadv6512},
pmid = {40305594},
issn = {2375-2548},
mesh = {Humans ; CRISPR-Cas Systems ; Gene Editing ; *Nucleic Acids/genetics/chemistry ; RNA, Messenger/genetics ; HEK293 Cells ; Synthetic Biology ; },
abstract = {Synthetic nucleic acid-based computing has demonstrated complex computational capabilities in vitro. However, translating these circuits into living cells remains challenging because of instability and cellular interference. We introduce an allosteric strand exchange (ASE) strategy for complex intracellular computing. Leveraging conformational cooperativity to regulate strand exchange, ASE offers a modular platform for designing intracellular circuits with flexible programmability. We engineer a scalable circuit architecture based on ASE that can execute AND and OR logic and scale to an eight-input expression. We demonstrate ASE-based circuits can detect messenger RNAs with high specificity in mammalian cells via AND logic computation. The capacity of ASE-based circuits to accept messenger RNAs as inputs enables integration of endogenous cellular information for efficient multi-input information processing, demonstrated by a multi-input molecular classifier monitoring key cell reprogramming events. Reprogramming ASE-based circuit to interface with CRISPR-Cas9 enables programmable control of Cas9-targeting activity for gene editing, highlighting their potential for advancing intracellular biocomputation.},
}

Humans
CRISPR-Cas Systems
Gene Editing
*Nucleic Acids/genetics/chemistry
RNA, Messenger/genetics
HEK293 Cells
Synthetic Biology

@article {pmid40305308,
year = {2025},
author = {Nayyab, S and Gervasi, MG and Tourzani, DA and Shamailova, Y and Akizawa, H and Taghavi, M and Cui, W and Fissore, R and Salicioni, AM and Georg, GI and Snyder, E and Visconti, PE},
title = {Identification of TSSK1 and TSSK2 as Novel Targets for Male Contraception.},
journal = {Biomolecules},
volume = {15},
number = {4},
pages = {},
pmid = {40305308},
issn = {2218-273X},
support = {1R01HD038082-21A1/NH/NIH HHS/United States ; },
mesh = {Animals ; Male ; Mice ; Female ; Testis/metabolism ; Mice, Knockout ; Spermatogenesis/genetics ; *Protein Serine-Threonine Kinases/genetics/metabolism ; CRISPR-Cas Systems ; Spermatozoa/metabolism ; Sperm Motility ; *Contraception ; Infertility, Male/genetics ; },
abstract = {The testis-specific serine kinases (TSSKs) are post-meiotically expressed in testicular germ cells. Their testis-specific expression, together with their putative role in phosphorylation pathways, suggests that TSSKs have relevant roles in spermiogenesis, sperm function, or both. Independent Tssk3 and Tssk6 knockout mice, as well as the double Tssk1/Tssk2 KO males, are sterile. However, the double KO results are silent regarding the individual roles of TSSK1 and TSSK2. The aim of this study was to develop independent mutant mouse models of Tssk1 and Tssk2, using CRISPR/Cas9, to evaluate their independent roles in reproduction. Male heterozygous pups were used to establish one Tssk1 and two independent Tssk2 mutant lines. Natural mating mutant Tssk1 and Tssk2 homozygous males but not females were found to be sterile. Additionally, homozygous males have lower sperm numbers and decreased motility, and were infertile in vitro. Anti-TSSK2 antibodies were validated against Tssk2 mutants and used in Western blot and immunofluorescence experiments. TSSK2 is localized to the sperm head; importantly, it is present in the testes and sperm from Tssk1 mutant mice, confirming individual mutation. Our results indicate that both TSSK1 and TSSK2 are individually essential for male reproduction and support both kinases as suitable nonhormonal male contraceptive targets.},
}

Animals
Male
Mice
Female
Testis/metabolism
Mice, Knockout
Spermatogenesis/genetics
*Protein Serine-Threonine Kinases/genetics/metabolism
CRISPR-Cas Systems
Spermatozoa/metabolism
Sperm Motility
*Contraception
Infertility, Male/genetics

@article {pmid40303110,
year = {2024},
author = {Jin, K and Huang, P and Li, B and Cao, Z and Huang, Z and Zhang, Z and Liu, M and Li, H and Niu, L and Zhang, T and Li, Y and Li, X and Wang, H and Zhang, H},
title = {A Single-Copy Sensitive and Field-Deployable One-Pot RT-RPA CRISPR/Cas12a Assay for the Specific Visual Detection of the Nipah Virus.},
journal = {Transboundary and emerging diseases},
volume = {2024},
number = {},
pages = {4118007},
pmid = {40303110},
issn = {1865-1682},
mesh = {*Nipah Virus/isolation & purification/genetics ; Animals ; *CRISPR-Cas Systems ; Sensitivity and Specificity ; *Henipavirus Infections/virology/diagnosis/veterinary ; *Nucleic Acid Amplification Techniques/veterinary/methods ; Humans ; },
abstract = {Nipah virus (NiV) is an emerging bat-borne zoonotic virus that can be transmitted to humans and other animals through infected bats or contaminated foods. The disease is highly lethal in humans (40%-75%) and has the potential for human-to-human transmission. Currently, there are no approved treatments or vaccines for NiV infection in humans or animals. Consequently, there is a pressing need for a highly sensitive, precise, and visually detectable assay to enable early intervention and mitigate the transmission of NiV infection. Here, we report a single-copy sensitive, field-deployable, one-pot visual reverse transcription-recombinase polymerase amplification (RT-RPA)-clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR associate system (Cas)12 for the detection of NiV. The assay works by targeting the N gene of NiV, and the results are directly visible to the naked eye. The assay has demonstrated the ability to detect as few as 5.5 copies/μl of positive plasmids or 5.5 × 10[1] copies/μl of RNA transcripts when reacted at constant temperature for 40 min. It showed high specificity for NiV and had no cross-reaction with other pathogens, including rabies virus (RABV), Japanese encephalitis virus (JEV), herpes simplex virus type 1 (HSV-1), Hendra virus (HeV), and Streptococcus suis (S. suis), that can cause clinical symptoms similar to those of NiV infection. Moreover, this assay had a 100% coincidence rate with the reverse transcription quantitative polymerase chain reaction (RT-qPCR) method recommended by the World Organization for Animal Health (WOAH) for the detection of simulated clinical samples, indicating that it has great potential as an ultrasensitive, simple, and portable novel assay for the onsite diagnosis of NiV infection.},
}

*Nipah Virus/isolation & purification/genetics
Animals
*CRISPR-Cas Systems
Sensitivity and Specificity
*Henipavirus Infections/virology/diagnosis/veterinary
*Nucleic Acid Amplification Techniques/veterinary/methods
Humans

@article {pmid40303064,
year = {2024},
author = {Wu, M and Chen, M and Qiu, R and Ge, L and Fan, Z and Hu, B and Wei, H and Li, Y and Wang, F and Song, Y},
title = {Specific Detection of RHDV GI.1 and GI.2 by RT-LAMP-CRISPR/Cas12a Platform.},
journal = {Transboundary and emerging diseases},
volume = {2024},
number = {},
pages = {3881457},
pmid = {40303064},
issn = {1865-1682},
mesh = {Animals ; *Caliciviridae Infections/veterinary/virology/diagnosis/epidemiology ; *Nucleic Acid Amplification Techniques/veterinary/methods ; *Hemorrhagic Disease Virus, Rabbit/isolation & purification/genetics/classification ; Rabbits ; Sensitivity and Specificity ; *Molecular Diagnostic Techniques/veterinary/methods ; CRISPR-Cas Systems ; China/epidemiology ; },
abstract = {Rabbit hemorrhagic disease is a highly contagious and acute fatal disease caused by rabbit hemorrhagic disease virus (RHDV). The first outbreak of RHDV2 in 2020 has posed a serious threat to the rabbit breeding industry in China. An effective and specific detection strategy for RHDV GI.1 (RHDV1) and GI.2 (RHDV2) is urgently needed. In this study, we established a reverse transcription loop-mediated isothermal amplification (RT-LAMP)-CRISPR/Cas12a-based dual readout portable detection platform. The platform showed excellent specificity to identify RHDV1 and RHDV2 strains and no cross-reaction with other prevalent pathogens of rabbit. The detection limit for RHDV1 and RHDV2 by RT-LAMP-CRISPR/Cas12a could reach 10 copies/μl of the VP60 gene per reaction. Furthermore, 74 clinical samples were detected for both RHDV1 and RHDV2. RT-LAMP-CRISPR/Cas12a-based dual readout portable detection platform showed 25.68% (19/74) RHDV1-positive samples, 43.24% (32/74) RHDV2-positive samples, and 8.11% (6/74) RHDV1/RHDV2 double positive samples, respectively. The coincidence rates of detection RHDV1 and RHDV2 between RT-LAMP-CRISPR/Cas12a and quantitative real-time-polymerase chain reaction (qPCR) were both 97.30%. RT-LAMP-CRISPR/Cas12a showed higher sensitivity and detection rate compared with qPCR. Moreover, the results were visible to the naked eye within 1.5 h combined with lateral flow strips (LFSs) and visual fluorescence. The RT-LAMP-CRISPR/Cas12a portable platform has the advantages of high sensitivity, specificity, fast, low equipment requirements, which can be used in clinical practice in rural areas and resource-limited settings.},
}

Animals
*Caliciviridae Infections/veterinary/virology/diagnosis/epidemiology
*Nucleic Acid Amplification Techniques/veterinary/methods
*Hemorrhagic Disease Virus, Rabbit/isolation & purification/genetics/classification
Rabbits
Sensitivity and Specificity
*Molecular Diagnostic Techniques/veterinary/methods
CRISPR-Cas Systems
China/epidemiology

@article {pmid40303012,
year = {2024},
author = {Kan, X and Wu, Y and Zhang, X and Zeng, Z and Lou, C and Wu, L and Huang, X and Wang, X and Huang, B},
title = {Palm Multidiagnostic of Mycoplasma pneumoniae, Chlamydia pneumoniae, Haemophilus influenzae, and Streptococcus pneumoniae Using One-Tube CRISPR/Cas12a.},
journal = {Transboundary and emerging diseases},
volume = {2024},
number = {},
pages = {5002521},
pmid = {40303012},
issn = {1865-1682},
mesh = {*Mycoplasma pneumoniae/isolation & purification/genetics ; *Haemophilus influenzae/isolation & purification/genetics ; *Streptococcus pneumoniae/isolation & purification/genetics ; Humans ; *Chlamydophila pneumoniae/isolation & purification/genetics ; *CRISPR-Cas Systems ; *Pneumonia, Mycoplasma/diagnosis/microbiology ; *Haemophilus Infections/diagnosis/microbiology ; Respiratory Tract Infections/diagnosis/microbiology ; },
abstract = {The recent high incidence of Mycoplasma pneumoniae (Mp) infections has raised widespread public health concerns. Therefore, rapid and accurate diagnosis of respiratory pathogenic microbial infections is of paramount importance to provide clinicians with accurate diagnostic insights and guide clinical medication. In response to this urgent need, we developed a one-tube Palm CRISPR/Cas12a Diagnostic (PaCD) method. This method facilitates the rapid detection of Mp infections, as well as three other prevalent respiratory pathogens, Chlamydia pneumoniae (Cp), Haemophilus influenzae (Hi), and Streptococcus pneumoniae (SP). In addition, 3D printing was employed to fabricate a compact detection device that includes a temperature control module set at 39°C and a blue light irradiation module, significantly enhancing the feasibility of point-of-care testing. The PaCD diagnostic process takes only 30 min with a detection limit of 50 copies/test, making it suitable for analysis of sputum and throat swab samples. PaCD demonstrated 100% concordance (72/72) with next-generation sequencing and exhibited high concordance with computed tomography test results. These findings demonstrate the clinical feasibility of PaCD for the rapid and accurate diagnosis of infections caused by four prevalent respiratory pathogens, offering theoretical insights into the versatile application of point-of-care tests for the detection of other respiratory pathogens in various clinical scenarios.},
}

*Mycoplasma pneumoniae/isolation & purification/genetics
*Haemophilus influenzae/isolation & purification/genetics
*Streptococcus pneumoniae/isolation & purification/genetics
Humans
*Chlamydophila pneumoniae/isolation & purification/genetics
*CRISPR-Cas Systems
*Pneumonia, Mycoplasma/diagnosis/microbiology
*Haemophilus Infections/diagnosis/microbiology
Respiratory Tract Infections/diagnosis/microbiology

@article {pmid40302337,
year = {2025},
author = {Sobah, ML and Liongue, C and Ward, AC},
title = {Socs3a is Dispensable for Zebrafish Hematopoiesis and is Required for Neuromast Formation.},
journal = {Frontiers in bioscience (Landmark edition)},
volume = {30},
number = {4},
pages = {36537},
doi = {10.31083/FBL36537},
pmid = {40302337},
issn = {2768-6698},
mesh = {Animals ; *Zebrafish/genetics/embryology/metabolism ; *Hematopoiesis/genetics ; *Suppressor of Cytokine Signaling 3 Protein/genetics/metabolism ; *Zebrafish Proteins/genetics/metabolism ; CRISPR-Cas Systems ; Gene Knockout Techniques ; },
abstract = {BACKGROUND: Suppressor of cytokine signaling (SOCS)3 is a regulatory protein that participates in an important negative feedback loop downstream of several critical cytokines, especially members of the interleukin-6 (IL-6) family. As a result, SOCS3 has been shown to impact the development and function of blood and immune cells. Zebrafish harbor duplicates of SOCS3, Socs3a and Socs3b, both of which possess conserved functional domains.

METHODS: This study explored the role of zebrafish Socs3a by creating a whole genome knockout using CRISPR/Cas9, with a focus on hematopoiesis and neuromast formation.

RESULTS: A zebrafish Socs3a knockout mutant was successfully generated. Characterization of this mutant revealed that normal hematopoiesis was not impacted nor was neutrophils lacking Socs3a displayed normal responses to injury or their production during emergency granulopoiesis. Neuromast formation was severely impacted in Socs3a knockout zebrafish.

CONCLUSIONS: Zebrafish Socs3a mutants display normal hematopoiesis and myeloid function, but the formation of the lateral line neuromast was affected by the absence of Socs3a.},
}

Animals
*Zebrafish/genetics/embryology/metabolism
*Hematopoiesis/genetics
*Suppressor of Cytokine Signaling 3 Protein/genetics/metabolism
*Zebrafish Proteins/genetics/metabolism
CRISPR-Cas Systems
Gene Knockout Techniques

@article {pmid40302206,
year = {2025},
author = {Drebes Dörr, NC and Lemopoulos, A and Blokesch, M},
title = {Exploring Mobile Genetic Elements in Vibrio cholerae.},
journal = {Genome biology and evolution},
volume = {},
number = {},
pages = {},
doi = {10.1093/gbe/evaf079},
pmid = {40302206},
issn = {1759-6653},
abstract = {Members of the bacterial species Vibrio cholerae are known both as prominent constituents of marine environments and as the causative agents of cholera, a severe diarrheal disease. While strains responsible for cholera have been extensively studied over the past century, less is known about their environmental counterparts, despite their contributions to the species' pangenome. This study analyzed the genome compositions of 46 V. cholerae strains, including pandemic and non-pandemic, toxigenic, and environmental variants, to investigate the diversity of mobile genetic elements (MGEs), embedded bacterial defense systems, and phage-associated signatures. Our findings include both conserved and novel MGEs across strains, pointing to shared evolutionary pathways and ecological niches. The defensome analysis revealed a wide array of antiphage/anti-plasmid mechanisms, extending well beyond the traditional CRISPR-Cas and restriction-modification systems. This underscores the dynamic arms race between V. cholerae and MGEs and suggests that non-pandemic strains may act as reservoirs for emerging defense strategies. Moreover, the study showed that MGEs are integrated into genomic hotspots, which may serve as critical platforms for the exchange of defense systems, thereby enhancing V. cholerae's adaptive capabilities against phage attacks and other invading MGEs. Overall, this research offers new insights into V. cholerae's genetic complexity and potential adaptive strategies, offering a better understanding of the differences between environmental strains and their pandemic counterparts, as well as the possible evolutionary pathways that led to the emergence of pandemic strains.},
}

@article {pmid40302137,
year = {2025},
author = {Cañizares, E and Giovannini, L and Gumus, BO and Fotopoulos, V and Balestrini, R and González-Guzmán, M and Arbona, V},
title = {Seeds of Change: exploring the transformative effects of seed priming in sustainable agriculture.},
journal = {Physiologia plantarum},
volume = {177},
number = {3},
pages = {e70226},
doi = {10.1111/ppl.70226},
pmid = {40302137},
issn = {1399-3054},
support = {CNS2023-143759//Agencia Estatal de Investigación/ ; PCI2021-121920//Agencia Estatal de Investigación/ ; PID2020-118126RB-I00//Agencia Estatal de Investigación/ ; PID2023-150080OB-I00//Agencia Estatal de Investigación/ ; EXCELLENCE/0421/0462//research and innovation foundation of cyprus/ ; CIGRIS/2021/014//Conselleria de Innovación, Universidades, Ciencia y Sociedad Digital, Generalitat Valenciana/ ; DD 16302//Ministero dell'Università e della Ricerca/ ; },
mesh = {*Seeds/physiology/genetics ; *Agriculture/methods ; Germination/physiology ; Climate Change ; Crops, Agricultural/genetics ; Plants, Genetically Modified ; },
abstract = {The threats posed by climate change on agriculture at a global scale have fostered researchers to explore new and efficient strategies to ensure stable and safe food production. These new strategies must not only be efficient in reducing yield loss but also comply with environmental and consumer safety regulations, which particularly refer to restrictions to pesticide application as well as the implementation of genetically modified organisms, including CRISPR/Cas edited lines. Among other approaches, priming constitutes an easier and relatively cheaper strategy to cope with the effects of abiotic and biotic stresses by boosting plants' endogenous potential. Particularly, pre-sowing seed priming has proven effective in improving germination and seedling establishment as well as tolerance to environmental and biotic factors throughout the plant's life cycle, exhibiting clear long-lasting effects. This tolerance response to a wide range of adverse factors is associated with physiological, metabolic and genetic mechanisms and responses at the seed level and subsequently in the established plant. The genetic and epigenetic mechanisms enabling this tolerance response in plants and their subsequent generation, as a transgenerational effect, will be reviewed. Finally, the potential of the different seed priming approaches contributing to an ecologically and economically more sustainable agriculture will be discussed.},
}

*Seeds/physiology/genetics
*Agriculture/methods
Germination/physiology
Climate Change
Crops, Agricultural/genetics
Plants, Genetically Modified

@article {pmid40301471,
year = {2025},
author = {Shin, KC and Hasan, W and Ali, G and Abdelrahman, D and Abuarja, T and Stanton, LW and Da'as, SI and Park, Y},
title = {Seizure-like behavior and hyperactivity in napb knockout zebrafish as a model for autism and epilepsy.},
journal = {Scientific reports},
volume = {15},
number = {1},
pages = {14579},
pmid = {40301471},
issn = {2045-2322},
mesh = {Animals ; Zebrafish/genetics ; *Autistic Disorder/genetics/physiopathology ; Disease Models, Animal ; *Epilepsy/genetics/physiopathology ; *Seizures/genetics/physiopathology ; *Zebrafish Proteins/genetics ; Behavior, Animal ; CRISPR-Cas Systems ; Gene Knockout Techniques ; Humans ; },
abstract = {We identified N-ethylmaleimide-sensitive factor attachment protein beta (NAPB) as a potential risk gene for autism and epilepsy. Notably, Qatari monozygotic triplets with loss of function mutations in NAPB exhibit early onset epileptic encephalopathy and varying degrees of autism. In this study, we generated NAPB zebrafish model using CRISPR-Cas9-sgRNAs technology for gene editing of the two orthologs napba and napbb. We observed that napb crispants (CR) show shorter motor neuron axons length together with altered locomotion behavior, including significant increases in larvae total distance traveled, swimming velocity, and rotation frequency, indicating that these behavioral changes effectively mimic the human epileptic phenotype. We applied microelectrode array (MEA) technology to monitor neural activity and hyperactivity in the zebrafish model. The napb CR shows hyperexcitability in the brain region. By combining behavioral tests with electrophysiological MEA assays, the established NAPB zebrafish model can be employed to study the pathophysiological mechanisms of ASD and epilepsy to screen potential therapeutic drugs.},
}

Animals
Zebrafish/genetics
*Autistic Disorder/genetics/physiopathology
Disease Models, Animal
*Epilepsy/genetics/physiopathology
*Seizures/genetics/physiopathology
*Zebrafish Proteins/genetics
Behavior, Animal
CRISPR-Cas Systems
Gene Knockout Techniques
Humans

@article {pmid40301362,
year = {2025},
author = {Vlasova, KY and Kerr, A and Pennock, ND and Jozic, A and Sahel, DK and Gautam, M and Murthy, NTV and Roberts, A and Ali, MW and MacDonald, KD and Walker, JM and Luxenhofer, R and Sahay, G},
title = {Synthesis of ionizable lipopolymers using split-Ugi reaction for pulmonary delivery of various size RNAs and gene editing.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {4021},
pmid = {40301362},
issn = {2041-1723},
support = {R01 CA270783/CA/NCI NIH HHS/United States ; R01CA270783-01//U.S. Department of Health & Human Services | NIH | National Heart, Lung, and Blood Institute (NHLBI)/ ; R01HL146736//U.S. Department of Health & Human Services | NIH | National Heart, Lung, and Blood Institute (NHLBI)/ ; SAHAY19XX0//Cystic Fibrosis Foundation (CF Foundation)/ ; R01 HL146736/HL/NHLBI NIH HHS/United States ; },
mesh = {Animals ; *Gene Editing/methods ; Mice ; Humans ; *Lung/metabolism ; Nanoparticles/chemistry ; RNA, Messenger/administration & dosage/genetics/chemistry ; Mice, Knockout ; Cystic Fibrosis Transmembrane Conductance Regulator/genetics/metabolism ; *Gene Transfer Techniques ; *Polymers/chemistry/chemical synthesis ; Mice, Inbred C57BL ; Genetic Therapy/methods ; Lipids/chemistry ; CRISPR-Cas Systems ; },
abstract = {We present an efficient method for synthesizing cationic poly(ethylene imine) derivatives using the multicomponent split-Ugi reaction to create a library of functional ionizable lipopolymers. Here we show 155 polymers, formulated into polyplexes, to establish structure-activity relationships essential for endosomal escape and transfection. A lead structure is identified, and lipopolymer-lipid hybrid nanoparticles are developed to deliver mRNA to lung endothelium and immune cells, including T cells, with low in vivo toxicity. These nanoparticles show significant improvements in mRNA delivery to the lung compared to in vivo-JetPEI® and demonstrate effective delivery of therapeutic mRNA(s) of various sizes. IL-12 mRNA-loaded nanoparticles delay Lewis Lung cancer progression, while human CFTR mRNA restores CFTR protein function in CFTR knockout mice. Additionally, we demonstrate in vivo CRISPR-Cas9 mRNA delivery, achieving gene editing in lung tissue and successful PD-1 knockout in T cells in mice. These results highlight the platform's potential for systemic gene therapy delivery.},
}

Animals
*Gene Editing/methods
Mice
Humans
*Lung/metabolism
Nanoparticles/chemistry
RNA, Messenger/administration & dosage/genetics/chemistry
Mice, Knockout
Cystic Fibrosis Transmembrane Conductance Regulator/genetics/metabolism
*Gene Transfer Techniques
*Polymers/chemistry/chemical synthesis
Mice, Inbred C57BL
Genetic Therapy/methods
Lipids/chemistry
CRISPR-Cas Systems

@article {pmid40301361,
year = {2025},
author = {Xing, C and Zhang, C and Xu, Z and Wang, Y and Lu, W and Liu, X and Zhang, Y and Ma, J and Yang, S and Du, Y and Xu, G and Liu, Y},
title = {Genome-wide CRISPR screening identifies LRP1 as an entry factor for SFTSV.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {4036},
pmid = {40301361},
issn = {2041-1723},
support = {81772203//National Natural Science Foundation of China (National Science Foundation of China)/ ; 82302524//National Natural Science Foundation of China (National Science Foundation of China)/ ; },
mesh = {Animals ; *Low Density Lipoprotein Receptor-Related Protein-1/genetics/metabolism/antagonists & inhibitors ; *Virus Internalization/drug effects ; Mice ; Humans ; Male ; *Phlebovirus/physiology/genetics ; Fibroblasts/virology/metabolism ; Antibodies, Neutralizing/pharmacology ; HEK293 Cells ; CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {Severe fever with thrombocytopenia syndrome (SFTS) is an emerging tick-borne disease caused by the SFTS virus (SFTSV), which has high mortality rates and poses a significant threat to public health. To identify potential therapeutic targets against SFTSV, we conduct genome-wide knockout screening, which identifies the previously known host factor CCR2, and reveals prolow-density lipoprotein receptor-related protein 1 (LRP1) as an entry factor for SFTSV. Knockdown or knockout of LRP1 significantly attenuate SFTSV infection in mouse embryonic fibroblasts (MEFs). Additionally, inhibition of LRP1 suppresses SFTSV pseudovirus infection in MEFs, suggesting its role in viral entry. The interaction between the SFTSV glycoprotein Gn and LRP1 via the CLI and CLII domains is revealed by co-IP and surface plasmon resonance (SPR). Moreover, LRP1 antagonists and neutralizing antibodies effectively attenuate SFTSV infection in MEFs. Administration of an LRP1-neutralizing antibody in a lethal male mouse model reduces the viral load, mitigates tissue damage, and improves survival. This study identifies LRP1 as a host entry receptor for SFTSV, providing a target for therapeutic strategy development.},
}

Animals
*Low Density Lipoprotein Receptor-Related Protein-1/genetics/metabolism/antagonists & inhibitors
*Virus Internalization/drug effects
Mice
Humans
Male
*Phlebovirus/physiology/genetics
Fibroblasts/virology/metabolism
Antibodies, Neutralizing/pharmacology
HEK293 Cells
CRISPR-Cas Systems
Clustered Regularly Interspaced Short Palindromic Repeats

@article {pmid40301355,
year = {2025},
author = {Liang, X and Gupta, D and Xie, J and Van Wonterghem, E and Van Hoecke, L and Hean, J and Niu, Z and Ghaeidamini, M and Wiklander, OPB and Zheng, W and Wiklander, RJ and He, R and Mamand, DR and Bost, J and Zhou, G and Zhou, H and Roudi, S and Estupiñán, HY and Rädler, J and Zickler, AM and Görgens, A and Hou, VWQ and Slovak, R and Hagey, DW and de Jong, OG and Uy, AG and Zong, Y and Mäger, I and Perez, CM and Roberts, TC and Carter, D and Vader, P and Esbjörner, EK and de Fougerolles, A and Wood, MJA and Vandenbroucke, RE and Nordin, JZ and El Andaloussi, S},
title = {Engineering of extracellular vesicles for efficient intracellular delivery of multimodal therapeutics including genome editors.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {4028},
pmid = {40301355},
issn = {2041-1723},
mesh = {*Extracellular Vesicles/metabolism/genetics ; Animals ; *Gene Editing/methods ; Mice ; Humans ; Integrases/genetics/metabolism ; CRISPR-Cas Systems ; CRISPR-Associated Protein 9/metabolism ; Inflammation/chemically induced/therapy ; HEK293 Cells ; NF-kappa B/metabolism ; *Drug Delivery Systems/methods ; },
abstract = {Intracellular delivery of protein and RNA therapeutics represents a major challenge. Here, we develop highly potent engineered extracellular vesicles (EVs) by incorporating bio-inspired attributes required for effective delivery. These comprise an engineered mini-intein protein with self-cleavage activity for active cargo loading and release, and fusogenic VSV-G protein for endosomal escape. Combining these components allows high efficiency recombination and genome editing in vitro following EV-mediated delivery of Cre recombinase and Cas9/sgRNA RNP cargoes, respectively. In vivo, infusion of a single dose Cre loaded EVs into the lateral ventricle in brain of Cre-LoxP R26-LSL-tdTomato reporter mice results in greater than 40% and 30% recombined cells in hippocampus and cortex respectively. In addition, we demonstrate therapeutic potential of this platform by showing inhibition of LPS-induced systemic inflammation via delivery of a super-repressor of NF-ĸB activity. Our data establish these engineered EVs as a platform for effective delivery of multimodal therapeutic cargoes, including for efficient genome editing.},
}

*Extracellular Vesicles/metabolism/genetics
Animals
*Gene Editing/methods
Mice
Humans
Integrases/genetics/metabolism
CRISPR-Cas Systems
CRISPR-Associated Protein 9/metabolism
Inflammation/chemically induced/therapy
HEK293 Cells
NF-kappa B/metabolism
*Drug Delivery Systems/methods

@article {pmid40298457,
year = {2025},
author = {Zhang, Y and Liu, J and Zhou, Y and Hu, X and Geng, B and Yang, S},
title = {Establishment of a RecET-Assisted CRISPR-Cas12a System for Large Deoxyribonucleic Acid-Fragment Manipulation in Zymomonas mobilis.},
journal = {ACS synthetic biology},
volume = {},
number = {},
pages = {},
doi = {10.1021/acssynbio.4c00863},
pmid = {40298457},
issn = {2161-5063},
abstract = {The lack of effective and efficient genome-editing toolkits for large DNA-fragment manipulation impedes the development of robust cell factories to meet the needs of fast-growing biomanufacturing. Zymomonas mobilis is an important nonmodel polyploid industrial strain with excellent industrial characteristics. Although diverse CRISPR-Cas systems have been established in Z. mobilis for gene deletion, replacement, and ssDNA recombination, it is still challenging to achieve large DNA-fragment manipulation due to its low recombination and repair efficiencies for double-strand DNA breaks. In this study, a RecET-assisted CRISPR-Cas12a genome editing system was developed using a chromosome-borne cas12a and recET-encoded recombinase, as well as an all-in-one editing plasmid. Different promoters were used for recET and cas12a to determine optimal expression. The combination of PB-cas12a_Pt-recET had the highest efficiency of 97.92 ± 2.95% for 9-kb DNA-fragment deletion, which also had efficiencies about 100%, 80%, and 5%, respectively, for the deletion of 9-16, 20-25, and 30 kb DNA fragments. The RecET-assisted CRISPR-Cas12a was further applied for deletions of different large gene clusters and had the potential for efficient pathway knock-in. This study highlights the importance of the Cas12a nuclease expression levels and the combination of the RecET system in improving the double-strand DNA repair capability for large DNA-fragment manipulation in Z. mobilis. The RecET-assisted CRISPR-Cas12a system established in this study provides a versatile and powerful tool for large DNA-fragment manipulation in Z. mobilis, which is beneficial for functional genomic research, strain improvement, as well as the development of synthetic microbial chassis.},
}

@article {pmid40298107,
year = {2025},
author = {Sun, J and Yu, X and Tang, G and Chen, M and Zheng, Y and Hu, Y and Li, Q and Li, X and Li, N and Li, Z and Li, Y and Lu, N and Tan, W and Yang, Y and Lyu, X and Zhao, G and Wang, H and Dai, L and Zhao, GP and Ai, L and Zhao, W},
title = {A CRISPR-SpCas9M-reporting system for efficient and rapid genome editing in Caulobacter crescentus.},
journal = {Nucleic acids research},
volume = {53},
number = {8},
pages = {},
pmid = {40298107},
issn = {1362-4962},
support = {2024YFA0917100//National Key R&D Program of China/ ; XDB0480000//Chinese Academy of Sciences/ ; 32025029//National Natural Science Foundation of China/ ; 2023A1515030069//Guangdong Basic and Applied Basic Research Foundation/ ; },
mesh = {*Caulobacter crescentus/genetics ; *CRISPR-Cas Systems ; *Gene Editing/methods ; Genome, Bacterial ; Bacterial Proteins/genetics ; *CRISPR-Associated Protein 9/genetics/metabolism ; Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {As members of the α-proteobacteria group, Caulobacter crescentus and its relatives are wildly studied for their unique asymmetric life cycle and versatile applications in industry, agriculture, and biomedicine. However, genetic manipulation in these bacteria remains challenging, typically requiring time-consuming and labor-intensive procedures. Here, we report a practical CRISPR-SpCas9M-reporting system that overcomes the limitations of SpCas9 expression and CRISPR escape, enabling efficient, markerless, and rapid genome editing in C. crescentus. Two genes encoding for a pair of scaffold proteins were knocked out individually or iteratively, demonstrating their direct involvements in cellular signaling asymmetry. Key components, including the Cas protein, Cas inducer, sgRNA, homologous arms, and reporter, were systematically analyzed and optimized in the system, finally achieving the apparent editing efficiency up to 80% in C. crescentus. Furthermore, we applied the CRISPR-SpCas9M-reporting system to two C. crescentus relatives, Agrobacterium fabrum and Sinorhizobium meliloti, establishing it as an efficient and general editing strategy. We anticipate that this system could be applied to other CRISPR-Cas-recalcitrant organisms, accelerating both basic and applied research in α-proteobacteria.},
}

*Caulobacter crescentus/genetics
*CRISPR-Cas Systems
*Gene Editing/methods
Genome, Bacterial
Bacterial Proteins/genetics
*CRISPR-Associated Protein 9/genetics/metabolism
Clustered Regularly Interspaced Short Palindromic Repeats

@article {pmid40295218,
year = {2025},
author = {Jeong, SO and Kim, HJ and Lee, SJ},
title = {Adaptive Evolution of GatC, a Component of the Galactitol Phosphotransferase System, for Glucose Transport in Escherichia coli.},
journal = {Journal of microbiology and biotechnology},
volume = {35},
number = {},
pages = {e2502021},
doi = {10.4014/jmb.2502.02021},
pmid = {40295218},
issn = {1738-8872},
mesh = {*Escherichia coli/genetics/metabolism/enzymology ; *Glucose/metabolism ; *Escherichia coli Proteins/genetics/metabolism ; *Phosphoenolpyruvate Sugar Phosphotransferase System/genetics/metabolism ; Directed Molecular Evolution ; Fermentation ; CRISPR-Cas Systems ; Biological Transport ; Mutation, Missense ; Metabolic Engineering ; Mutation ; Succinic Acid/metabolism ; },
abstract = {Microbial adaptive laboratory evolution is a powerful approach for uncovering novel gene functions within metabolic pathways. Building on our previous discovery of ExuT as a glucose transporter in ptsG-deficient Escherichia coli, this study investigates strains lacking recognized glucose transporters (ptsG, manX, and exuT). Successive rounds of experimental evolution revealed key genetic adaptations, including loss-of-function mutations in malI and nagC, which encode repressors of the maltose and N-acetylglucosamine phosphotransferase systems (PTS), respectively. Additionally, a gain-of-function mutation in gatC, a component of the galactitol PTS EIIC, was identified. The functional significance of these mutations was validated through transcript analysis, genetic knockouts, and CRISPR-Cas9-mediated site-specific genome mutagenesis, with a particular focus on the gatC missense mutation (F340C). The resulting modifications were examined for their effects on sugar specificity and metabolic flux. Furthermore, our findings identified succinate as the predominant fermentation product in engineered strains utilizing alternative glucose transport pathways, including the maltose, N-acetylglucosamine, and galactitol PTS. This study advances our understanding of sugar transport mechanisms in E. coli and offers insights into regulatory networks, fermentative metabolism, and substrate specificity, which can be leveraged for evolutionary engineering in biotechnological applications.},
}

*Escherichia coli/genetics/metabolism/enzymology
*Glucose/metabolism
*Escherichia coli Proteins/genetics/metabolism
*Phosphoenolpyruvate Sugar Phosphotransferase System/genetics/metabolism
Directed Molecular Evolution
Fermentation
CRISPR-Cas Systems
Biological Transport
Mutation, Missense
Metabolic Engineering
Mutation
Succinic Acid/metabolism

@article {pmid40295092,
year = {2025},
author = {Dipalo, LL and Mikkelsen, JG and Gijsbers, R and Carlon, MS},
title = {Trojan Horse-Like Vehicles for CRISPR-Cas Delivery: Engineering Extracellular Vesicles and Virus-Like Particles for Precision Gene Editing in Cystic Fibrosis.},
journal = {Human gene therapy},
volume = {},
number = {},
pages = {},
doi = {10.1089/hum.2024.258},
pmid = {40295092},
issn = {1557-7422},
abstract = {The advent of genome editing has kindled the hope to cure previously uncurable, life-threatening genetic diseases. However, whether this promise can be ultimately fulfilled depends on how efficiently gene editing agents can be delivered to therapeutically relevant cells. Over time, viruses have evolved into sophisticated, versatile, and biocompatible nanomachines that can be engineered to shuttle payloads to specific cell types. Despite the advances in safety and selectivity, the long-term expression of gene editing agents sustained by viral vectors remains a cause for concern. Cell-derived vesicles (CDVs) are gaining traction as elegant alternatives. CDVs encompass extracellular vesicles (EVs), a diverse set of intrinsically biocompatible and low-immunogenic membranous nanoparticles, and virus-like particles (VLPs), bioparticles with virus-like scaffold and envelope structures, but devoid of genetic material. Both EVs and VLPs can efficiently deliver ribonucleoprotein cargo to the target cell cytoplasm, ensuring that the editing machinery is only transiently active in the cell and thereby increasing its safety. In this review, we explore the natural diversity of CDVs and their potential as delivery vectors for the clustered regularly interspaced short palindromic repeats (CRISPR) machinery. We illustrate different strategies for the optimization of CDV cargo loading and retargeting, highlighting the versatility and tunability of these vehicles. Nonetheless, the lack of robust and standardized protocols for CDV production, purification, and quality assessment still hinders their widespread adoption to further CRISPR-based therapies as advanced "living drugs." We believe that a collective, multifaceted effort is urgently needed to address these critical issues and unlock the full potential of genome-editing technologies to yield safe, easy-to-manufacture, and pharmacologically well-defined therapies. Finally, we discuss the current clinical landscape of lung-directed gene therapies for cystic fibrosis and explore how CDVs could drive significant breakthroughs in in vivo gene editing for this disease.},
}

@article {pmid40293975,
year = {2025},
author = {Deragon, MA and Sharifi, HJ and LaRocca, TJ},
title = {Generation of a RIP1 Knockout U937 Cell Line Using the CRISPR-Cas9 System.},
journal = {Journal of visualized experiments : JoVE},
volume = {},
number = {218},
pages = {},
doi = {10.3791/66077},
pmid = {40293975},
issn = {1940-087X},
mesh = {Humans ; *CRISPR-Cas Systems ; U937 Cells ; *Gene Knockout Techniques/methods ; *Receptor-Interacting Protein Serine-Threonine Kinases/genetics ; *Nuclear Pore Complex Proteins/genetics ; },
abstract = {This protocol outlines a procedure for knocking out the RIP1 gene using CRISPR/Cas9 in the human monocyte U937 cell line. The method utilizes designated guide RNA plasmids and lentiviral packaging plasmids to achieve RIP1 gene knockout. The protocol addresses challenges and improvements to traditional CRISPR methods, enabling its replication for future cell death studies. The resulting mutant cells can be used to investigate mechanistic changes in cell death, where functional RIP1 proteins would otherwise play a role. Viability assays demonstrated a significant reduction in cell death in knockout cells following necroptosis induction. Fluorescence microscopy revealed a marked decrease in mitochondrial reactive oxygen species (ROS) in knockout cells under the same conditions. Together, these functional assays confirm the loss of RIP1 protein. Optimized for use with U937 human monocytes, this procedure may also be adapted to target other key cell death regulators, yielding functional, non-lethal mutants. Potential pitfalls are addressed throughout to provide insights into challenges that may arise during mutant generation.},
}

Humans
*CRISPR-Cas Systems
U937 Cells
*Gene Knockout Techniques/methods
*Receptor-Interacting Protein Serine-Threonine Kinases/genetics
*Nuclear Pore Complex Proteins/genetics

@article {pmid40292247,
year = {2025},
author = {Suthar, MK and Mittal, MK},
title = {Genome-wide identification and characterization of dicer-like genes in Andrographis paniculata (Burm. f.) Wall. ex Nees and their expression response to methyl jasmonate elicitation.},
journal = {3 Biotech},
volume = {15},
number = {5},
pages = {141},
pmid = {40292247},
issn = {2190-572X},
abstract = {Andrographis paniculata, commonly known as the "King of Bitters," is a medicinal plant valued for its bioactive diterpenoids, particularly andrographolides. Dicer-like (DCL) proteins are central to the miRNA pathway, processing precursor miRNAs into mature miRNAs that regulate gene expression. While miRNAs influence plant metabolism, their role in secondary metabolite biosynthesis in A. paniculata remains unexplored. This study identified and characterized five ApDCL genes, mapped to distinct chromosomes in A. paniculata. The structural analysis revealed that ApDCL3 contained the highest number of introns (24), whereas ApDCL2 had the fewest (10). Conserved RNA-processing domains were confirmed, and phylogenetic analysis revealed evolutionary conservation, showing that ApDCLs are closely related to the DCLs of Sesamum indicum and Salvia miltiorrhiza. Expression analysis showed ApDCL1 and ApDCL2 were predominantly expressed in roots, whereas ApDCL3, ApDCL4a, and ApDCL4b were more abundant in leaves. Methyl jasmonate treatment upregulated ApDCL3 (~ 3.5-fold) and ApDCL4a (~ 1.5-fold), but further research is needed to determine whether this response directly influences secondary metabolism or results from MeJ-induced stress. These findings provide a foundation for functional validation through gene knockdown, overexpression, and CRISPR-Cas-based genome editing to elucidate DCL-mediated regulatory mechanisms. Future research leveraging these insights could aid in modulating RNA silencing pathways to enhance the biosynthesis of pharmacologically significant metabolites in A. paniculata through biotechnological interventions.},
}

@article {pmid40288310,
year = {2025},
author = {Qian, J and Zhang, B and Liu, C and Xue, Y and Zhou, H and Huang, L and Zheng, S and Chen, M and Fu, YQ},
title = {Reconfigurable acoustic tweezer for precise tracking and in-situ sensing of trace miRNAs in tumor cells.},
journal = {Biosensors & bioelectronics},
volume = {282},
number = {},
pages = {117505},
doi = {10.1016/j.bios.2025.117505},
pmid = {40288310},
issn = {1873-4235},
abstract = {MicroRNAs (miRNAs) have emerged as critical biomarkers for early cancer diagnosis and monitoring. However, their isolation from clinical samples typically yields only trace amounts, significantly limiting the sensitivity and efficiency of cancer detection. To address this challenge, we present a octangular reconfigurable acoustic tweezer (ORAT) as an integrated platform for precise tumor cell tracking and in-situ detection of trace miRNAs. By simultaneously modulating multidirectional acoustic signals and parameters, the ORAT dynamically reshapes the acoustic field, enabling precise control over manipulation areas, particle spacing, array angles, distribution patterns, and node rotation. This device allows selective particle manipulation across entire regions or specific areas through adaptive adjustments of the microchamber boundary. Notably, the ORAT achieves rapid and accurate localization and labeling of rare tumor cells within a large population of normal cells. Furthermore, it enhances the sensitivity of CRISPR/Cas-based miRNA detection in digital microdroplets by three orders of magnitude, if compared to that of the conventional tube-based method. With its versatile capabilities, the ORAT holds remarkable promise for advancing nucleic acid analysis in a wide range of cancers and related diseases.},
}

@article {pmid40285901,
year = {2025},
author = {Lin, W and Huang, M and Fu, H and Yu, L and Chen, Y and Chen, L and Lin, Y and Wen, T and Luo, X and Cong, Y},
title = {An EXPAR-CRISPR/Cas12a Assay for Rapid Detection of Salmonella.},
journal = {Current microbiology},
volume = {82},
number = {6},
pages = {262},
pmid = {40285901},
issn = {1432-0991},
support = {20231800500552//Dongguan Science and Technology Commissioner Project/ ; 81974299//National Natural Science Foundation of China/ ; 2023A1515010938//Basic and Applied Basic Research Foundation of Guangdong Province/ ; },
mesh = {*Salmonella/genetics/isolation & purification ; *CRISPR-Cas Systems ; *Nucleic Acid Amplification Techniques/methods ; *Molecular Diagnostic Techniques/methods ; Bacterial Proteins/genetics ; Sensitivity and Specificity ; DNA, Bacterial/genetics ; Salmonella Infections/diagnosis/microbiology ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {Salmonella is considered as one of the primary pathogens associated with foodborne diseases globally. The effective treatment of these illnesses depends on the rapid and accurate identification of this organism. Traditional culture methods, however, necessitate extended testing periods, while many alternative techniques often lack precision. This research presents an innovative detection system that employs CRISPR-Cas12a for the detection of Salmonella. The detection system specifically targets the yfiR gene, which is amplified through isothermal exponential amplification (EXPAR). Target DNA hybridizes with the hairpin probe to form the DNA strand. The DNA strand was nicked to generate a nick by nicking endonuclease owing to its recognition sequence toward the hairpin probe. DNA polymerase can extend the 3'-end of the nicked site, which simultaneously displaces the newly synthesized strand. Thus, a large number of single-stranded DNA (ssDNA) were produced in the circle of nicking, polymerization, and strand displacement to achieve exponential amplification. The resultant amplified ssDNA products are subsequently recognized by CRISPR/Cas12a, resulting in the emission of a fluorescence signal. The detection system demonstrates a limit of detection of 10 fM for synthetic DNA and exhibits a strong linear relationship between 10 fM and 100 nM. Furthermore, the EXPAR-CRISPR/Cas12a detection system successfully identifies extracted genomic DNA samples containing Salmonella strains less than one hour, achieving a detection threshold of 1 pg/μL. This assay not only offers rapid results, requiring less than one hour for sample-to-answer outcomes, but is also cost-effective, minimizes aerosol risks, and provides exceptional specificity and sensitivity for the detection of Salmonella.},
}

*Salmonella/genetics/isolation & purification
*CRISPR-Cas Systems
*Nucleic Acid Amplification Techniques/methods
*Molecular Diagnostic Techniques/methods
Bacterial Proteins/genetics
Sensitivity and Specificity
DNA, Bacterial/genetics
Salmonella Infections/diagnosis/microbiology
Endodeoxyribonucleases
CRISPR-Associated Proteins

@article {pmid40285354,
year = {2025},
author = {John, T and Czechowicz, A},
title = {Clinical Hematopoietic Stem Cell-Based Gene Therapy.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.ymthe.2025.04.029},
pmid = {40285354},
issn = {1525-0024},
abstract = {Hematopoietic stem cell (HSC)-based gene therapies have seen extraordinary progress since their initial conception, now fundamentally transforming the treatment paradigms for various inherited hematologic, immunologic and metabolic conditions - with additional use cases under exploration. Decades worth of work with advances in viral vector technologies and cell manufacturing have paved the way for HSC gene therapy with marked improvement in the safety and efficiency of gene delivery into HSCs. These have been augmented by the recent rise of innovative genome editing techniques, particularly using clustered regularly interspaced short palindromic repeats - CRISPR-associated proteins (CRISPR-Cas)-based technologies, which have enabled more precise and reproducible genome alterations in HSCs and fostered opportunities for targeted gene modification or gene correction. These breakthroughs have led to the development of many active clinical trials and culminated in the recent federal regulatory agency approvals of multiple clinical HSC gene therapies for various indications that are now becoming available across different geographies. These treatments aim to offer significant, long-lasting benefits to patients world-wide without the toxicities of alternative treatment approaches. This review explores the history and advancements in HSC gene therapies and provides a comprehensive overview of the latest clinical innovations and cell therapy products. Further, it concludes with a discussion of the persistent challenges that have limited adoption and potential future opportunities that aspire to enable curative treatment of many different patients through such personalized medicines.},
}

@article {pmid40284926,
year = {2025},
author = {Yin, J and Cui, J and Zheng, H and Guo, T and Wei, R and Sha, Z and Gu, S and Ni, B},
title = {Implementation of RT-RAA and CRISPR/Cas13a for an NiV Point-of-Care Test: A Promising Tool for Disease Control.},
journal = {Viruses},
volume = {17},
number = {4},
pages = {},
pmid = {40284926},
issn = {1999-4915},
support = {2021YFD1800301-04//National Key R&D Program for the 14th Five-Year Plan/ ; },
mesh = {Animals ; Swine ; *CRISPR-Cas Systems ; *Swine Diseases/diagnosis/virology ; Sensitivity and Specificity ; *Nipah Virus/genetics/isolation & purification ; *Point-of-Care Testing ; *Nucleic Acid Amplification Techniques/methods ; Point-of-Care Systems ; },
abstract = {Nipah virus (NiV) is a severe zoonotic pathogen that substantially threatens public health. Pigs are the natural hosts of NiV and can potentially transmit this disease to humans. Establishing a rapid, sensitive, and accurate point-of-care detection method is critical in the timely identification of infected pig herds. In this study, we developed an NiV detection method based on reverse transcription-recombinase polymerase amplification (RT-RAA) and the clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 13a (Cas13a) system for the precise detection of NiV. The highly conserved region of the NiV gene was selected as the detection target. We first designed eleven pairs of RT-RAA primers, and the optimal primer combination and reaction temperature were identified on the basis of RT-RAA efficiency. Additionally, the most efficient crRNA sequence was selected on the basis of the fluorescence signal intensity. The results revealed that the optimal reaction temperature for the developed method was 37 °C. The detection limit was as low as 1.565 copies/μL. Specificity testing revealed no cross-reactivity with nucleic acids from six common swine viruses, including Seneca virus A (SVA), foot-and-mouth disease virus (FMDV), classical swine fever virus (CSFV), porcine epidemic diarrhea virus (PEDV), African swine fever virus (ASFV), and pseudorabies virus (PRV). A validation test using simulated clinical samples revealed a 100% concordance rate. The detection results can be visualized via a fluorescence reader or lateral flow strips (LFSs). Compared with conventional detection methods, this RT-RAA-CRISPR/Cas13a-based method is rapid and simple and does not require scientific instruments. Moreover, the reagents can be freeze-dried for storage, eliminating the need for cold-chain transportation. This detection technology provides a convenient and efficient new tool for the point-of-care diagnosis of NiV and for preventing and controlling outbreaks.},
}

Animals
Swine
*CRISPR-Cas Systems
*Swine Diseases/diagnosis/virology
Sensitivity and Specificity
*Nipah Virus/genetics/isolation & purification
*Point-of-Care Testing
*Nucleic Acid Amplification Techniques/methods
Point-of-Care Systems

@article {pmid40284749,
year = {2025},
author = {Hu, M and Chua, SL},
title = {Antibiotic-Resistant Pseudomonas aeruginosa: Current Challenges and Emerging Alternative Therapies.},
journal = {Microorganisms},
volume = {13},
number = {4},
pages = {},
pmid = {40284749},
issn = {2076-2607},
abstract = {Antibiotic-resistant Pseudomonas aeruginosa is a pathogen notorious for its resilience in clinical settings due to biofilm formation, efflux pumps, and the rapid acquisition of resistance genes. With traditional antibiotic therapy rendered ineffective against Pseudomonas aeruginosa infections, we explore alternative therapies that have shown promise, including antimicrobial peptides, nanoparticles and quorum sensing inhibitors. While these approaches offer potential, they each face challenges, such as specificity, stability, and delivery, which require careful consideration and further study. We also delve into emerging alternative strategies, such as bacteriophage therapy and CRISPR-Cas gene editing that could enhance targeted treatment for personalized medicine. As most of them are currently in experimental stages, we highlight the need for clinical trials and additional research to confirm their feasibility. Hence, we offer insights into new therapeutic avenues that could help address the pressing issue of antibiotic-resistant Pseudomonas aeruginosa, with an eye toward practical applications in future healthcare.},
}

@article {pmid40282900,
year = {2025},
author = {Ganesh, I and Karthiga, I and Murugan, M and Rangarajalu, K and Ballambattu, VB and Ravikumar, S},
title = {CRISPR/Cas-Based Prenatal Screening for Aneuploidy: Challenges and Opportunities for Early Diagnosis.},
journal = {Medicina (Kaunas, Lithuania)},
volume = {61},
number = {4},
pages = {},
pmid = {40282900},
issn = {1648-9144},
mesh = {Humans ; Female ; Pregnancy ; *Aneuploidy ; *Prenatal Diagnosis/methods/trends ; *CRISPR-Cas Systems/genetics ; Early Diagnosis ; Pregnancy Trimester, First ; Genetic Testing/methods ; },
abstract = {Aneuploidy is increasingly recognized globally as a common cause of miscarriage among expectant mothers. The existing prenatal screening techniques for detecting aneuploidy have several limitations. The ability to diagnose aneuploidy early in a non-invasive manner is not feasible with the current screening methods, as they may produce false positive or false negative results. Recently, the widely used gene editing tool CRISPR/Cas has shown great promise in diagnostics. This review summarizes the prenatal screening tests used in the first trimester to assess aneuploidy conditions. Additionally, we discuss the advantages and disadvantages of molecular diagnostic tests, including the benefits and challenges of CRISPR/Cas-based trisomy detection. Thus, the proposed prenatal screening using CRISPR/Cas could provide significant benefits to expectant mothers by potentially enabling the early diagnosis of trisomy, helping to prevent miscarriage and birth defects. Furthermore, it opens new avenues for research, allowing clinicians and researchers to develop, optimize, and implement CRISPR/Cas-based prenatal screening assays in the future.},
}

Humans
Female
Pregnancy
*Aneuploidy
*Prenatal Diagnosis/methods/trends
*CRISPR-Cas Systems/genetics
Early Diagnosis
Pregnancy Trimester, First
Genetic Testing/methods

@article {pmid40282347,
year = {2025},
author = {Zheng, Y and Zou, Q and Li, J and Yang, Y},
title = {CRISPR-MFH: A Lightweight Hybrid Deep Learning Framework with Multi-Feature Encoding for Improved CRISPR-Cas9 Off-Target Prediction.},
journal = {Genes},
volume = {16},
number = {4},
pages = {},
pmid = {40282347},
issn = {2073-4425},
support = {32471860//National Natural Science Foundation of China/ ; Y202352570//scientific research project of the education department of Zhejiang province/ ; },
mesh = {*Deep Learning ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; RNA, Guide, CRISPR-Cas Systems/genetics ; Humans ; },
abstract = {BACKGROUND: The CRISPR-Cas9 system has emerged as one of the most promising gene-editing technologies in biology. However, off-target effects remain a significant challenge. While recent advances in deep learning have led to the development of models for off-target prediction, these models often fail to fully leverage sequence pair information. Furthermore, as the models' parameter sizes increase, so do their complexities, limiting their practical applicability.

METHODS: In this study, we introduce a novel multi-feature independent encoding method, which encodes the gRNA-DNA sequence pair into three distinct feature matrices to minimize information loss. Additionally, we propose a lightweight hybrid deep learning framework, CRISPR-MFH, that integrates multi-scale separable convolutions and hybrid attention mechanisms for efficient and accurate off-target prediction.

RESULTS: Extensive experiments across multiple benchmark datasets demonstrate that the proposed encoding method effectively captures critical features and that CRISPR-MFH outperforms or matches state-of-the-art models with significantly fewer parameters across multiple evaluation metrics.

CONCLUSIONS: This study offers a novel perspective for advancing deep learning technology in the realm of CRISPR-Cas9 off-target detection.},
}

*Deep Learning
*CRISPR-Cas Systems/genetics
*Gene Editing/methods
RNA, Guide, CRISPR-Cas Systems/genetics
Humans

@article {pmid40282329,
year = {2025},
author = {Rathe, SK and Marko, TA and Edwards, EN and Ridder, PH and Varshney, J and Williams, KB and Johnson, JE and Moriarity, BS and Largaespada, DA},
title = {Techniques for Validating CRISPR Changes Using RNA-Sequencing Data.},
journal = {Genes},
volume = {16},
number = {4},
pages = {},
pmid = {40282329},
issn = {2073-4425},
support = {MSTP grant T32 GM008224/NH/NIH HHS/United States ; R01-CA113636/NH/NIH HHS/United States ; #123939//ACS Research Professorship/ ; NA//Zach Sobiech Osteosarcoma Fund of the Children's Cancer Research Fund, Minneapolis MN/ ; NA//Children's Cancer Research Fund Emerging Scientist Award/ ; NA//Children's Tumor Foundation Young Investigator Award/ ; 5T32CA009138-40//Children's Tumor Foundation/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; Humans ; *Sequence Analysis, RNA/methods ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Transcriptome/genetics ; *RNA-Seq/methods ; Gene Editing/methods ; Gene Knockout Techniques/methods ; },
abstract = {UNLABELLED: The use of CRISPR to knockdown or knockout genes is a powerful tool for understanding the specific role of a gene in disease development. However, it can cause many unanticipated changes to the transcriptome that are not detected by DNA amplification and Sanger sequencing of the target site. Various RNA-sequencing techniques can be used to identify these changes and effectively gauge the full impact of the CRISPR knockout, thereby providing a means of selecting appropriate clones for further experimentation.

BACKGROUND/OBJECTIVES: RNA-seq data from 4 CRISPR knockout experiments were analyzed and techniques developed to both confirm the success of the CRISPR modifications and identify potential issues.

METHODS: A broad-based analysis of RNA-sequencing data identified many CRISPR-based changes not identified by PCR amplification of DNA around the CRISPR target site. These changes included an inter-chromosomal fusion event, exon skipping, chromosomal truncation, and the unintentional transcriptional modification and amplification of a neighboring gene.

CONCLUSIONS: The inadvertent modifications identified by the evaluation of 4 CRISPR experiments highlight the value of using RNA-seq to identify transcriptional changes to cells altered by CRISPR, many of which cannot be recognized by evaluating DNA alone. Specific guidelines are presented for designing and analyzing CRISPR experiments using RNA-seq data.},
}

*CRISPR-Cas Systems/genetics
Humans
*Sequence Analysis, RNA/methods
*Clustered Regularly Interspaced Short Palindromic Repeats/genetics
Transcriptome/genetics
*RNA-Seq/methods
Gene Editing/methods
Gene Knockout Techniques/methods

@article {pmid40280947,
year = {2025},
author = {Chen, H and Song, F and Wang, B and Huang, H and Luo, Y and Han, X and He, H and Lin, S and Wan, L and Huang, Z and Fu, Z and Ledesma-Amaro, R and Yin, D and Mao, H and He, L and Yang, T and Chen, Z and Ma, Y and Xue, EY and Wan, Y and Mao, C},
title = {Ultrasensitive detection of clinical pathogens through a target-amplification-free collateral-cleavage-enhancing CRISPR-CasΦ tool.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {3929},
pmid = {40280947},
issn = {2041-1723},
support = {42376184//National Science Foundation of China | National Natural Science Foundation of China-Yunnan Joint Fund (NSFC-Yunnan Joint Fund)/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; Humans ; Limit of Detection ; RNA, Guide, CRISPR-Cas Systems/genetics ; *Bacteria/isolation & purification/genetics ; DNA, Bacterial/genetics ; },
abstract = {Clinical pathogen diagnostics detect targets by qPCR (but with low sensitivity) or blood culturing (but time-consuming). Here we leverage a dual-stem-loop DNA amplifier to enhance non-specific collateral enzymatic cleavage of an oligonucleotide linker between a fluophore and its quencher by CRISPR-CasΦ, achieving ultrasensitive target detection. Specifically, the target pathogens are lysed to release DNA, which binds its complementary gRNA in CRISPR-CasΦ to activate the collateral DNA-cleavage capability of CasΦ, enabling CasΦ to cleave the stem-loops in the amplifier. The cleavage product binds its complementary gRNA in another CRISPR-CasΦ to activate more CasΦ. The activated CasΦ collaterally cleaves the linker, releasing the fluophore to recover its fluorescent signal. The cycle of stem-loop-cleavage/CasΦ-activation/fluorescence-recovery amplifies the detection signal. Our target amplification-free collateral-cleavage-enhancing CRISPR-CasΦ method (TCC), with a detection limit of 0.11 copies/μL, demonstrates enhanced sensitivity compared to qPCR. It can detect pathogenic bacteria as low as 1.2 CFU/mL in serum within 40 min.},
}

*CRISPR-Cas Systems/genetics
Humans
Limit of Detection
RNA, Guide, CRISPR-Cas Systems/genetics
*Bacteria/isolation & purification/genetics
DNA, Bacterial/genetics

@article {pmid40280080,
year = {2025},
author = {Xiong, Q and Zhu, C and Yin, X and Zhu, L},
title = {CRISPR/Cas and Argonaute-based biosensors for nucleic acid detection.},
journal = {Talanta},
volume = {294},
number = {},
pages = {128210},
doi = {10.1016/j.talanta.2025.128210},
pmid = {40280080},
issn = {1873-3573},
abstract = {Nowadays, nucleic acid detection technology has been applied to disease diagnosis, prevention, food safety, environmental testing and many other aspects. However, traditional methods still have shortcomings. Therefore, there is an urgent need for a simple, rapid, sensitive, and specific new method to supersede traditional nucleic acid detection technology. CRISPR/Cas(Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated) system and Argonaute (Ago) system play an important role in microbial immune defense. Their targeting specificity, programmability and special trans-cleavage activity make it possible to develop some new platforms for nucleic acid detection in combination with a variety of biosensors. We introduce the origins of these two systems and the biosensors developed based on CRISPR/Cas system and Ago system, respectively, especially the prospects for the future development of Cascade Amplification biosensors. This review is expected to provide useful guidance for researchers in related fields and provide inspiration for the development of Cascade Amplification biosensors in the future.},
}

@article {pmid40278504,
year = {2025},
author = {Lv, J and Jin, J and Ding, L and Xiang, L and Xie, B and Wu, K and Chen, Q},
title = {Directed Evolution of OgeuIscB With Enhanced Activity in Human Cells.},
journal = {FASEB journal : official publication of the Federation of American Societies for Experimental Biology},
volume = {39},
number = {8},
pages = {e70570},
doi = {10.1096/fj.202500082R},
pmid = {40278504},
issn = {1530-6860},
support = {Y20220148//Science and Technology Project of Wenzhou City/ ; Y2023784//Science and Technology Project of Wenzhou City/ ; ZCLTGD24H1202//MOST | NSFC | NSFC-Zhejiang Joint Fund | | Natural Science Foundation of Zhejiang Province (ZJNSF)/ ; LTGD23H120001//MOST | NSFC | NSFC-Zhejiang Joint Fund | | Natural Science Foundation of Zhejiang Province (ZJNSF)/ ; LTGC23H120001//MOST | NSFC | NSFC-Zhejiang Joint Fund | | Natural Science Foundation of Zhejiang Province (ZJNSF)/ ; 82101169//National Natural Science Foundation of China/ ; },
mesh = {Humans ; *Gene Editing/methods ; *Directed Molecular Evolution/methods ; HEK293 Cells ; Protein Engineering/methods ; Mutation ; CRISPR-Cas Systems ; },
abstract = {The miniature RNA-guided endonuclease IscB, as the evolutionary progenitor of Cas9, is attracting increased attention for genome editing due to its compact size and suitability for in vivo delivery. However, the poor editing efficiency of IscB in eukaryotic cells presents a significant challenge to its widespread application in precise site-specific human genome editing. In this study, we employed structure-guided rational design and protein engineering to optimize OgeuIscB, resulting in the identification of enIscB-F138R, which further enhanced editing activity up to 3.49-fold in mammalian cells compared to the high-activity OgeuIscB variant enIscB. Furthermore, we engineered an enIscB-F138R nickase-based adenine base editor, termed miABE-F138R, exhibiting enhanced base editing efficiency relative to miABE. To illustrate the practical applications of miABE-F138R, we applied it to rectify the prevalent R560C mutation in Pde6β associated with autosomal recessive retinitis pigmentosa, resulting in a significant improvement in activity compared to miABE. In conclusion, enIscB-F138R and miABE-F138R offer adaptable platforms for genome editing with potential significance in future biomedical applications.},
}

Humans
*Gene Editing/methods
*Directed Molecular Evolution/methods
HEK293 Cells
Protein Engineering/methods
Mutation
CRISPR-Cas Systems

@article {pmid40277901,
year = {2025},
author = {Plümers, R and Jelinek, S and Lindenkamp, C and Osterhage, MR and Knabbe, C and Hendig, D},
title = {Investigation on ABCC6-Deficient Human Hepatocytes Generated by CRISPR-Cas9 Genome Editing.},
journal = {Cells},
volume = {14},
number = {8},
pages = {},
pmid = {40277901},
issn = {2073-4409},
mesh = {Humans ; *Hepatocytes/metabolism ; *CRISPR-Cas Systems/genetics ; *Multidrug Resistance-Associated Proteins/deficiency/genetics/metabolism ; *Gene Editing/methods ; Pseudoxanthoma Elasticum/genetics/pathology/metabolism ; },
abstract = {Patients affected by the rare disease pseudoxanthoma elasticum (PXE) exhibit the calcification of elastic fibers in ocular, dermal, and vascular tissues. These symptoms are triggered by mutations in the ATP-binding cassette transporter subfamily C member 6 (ABCC6), whose substrate remains unknown. Interestingly, ABCC6 is predominantly expressed in the liver tissue, leading to the hypothesis that PXE is a metabolic disorder. We developed a genome-editing system targeting ABCC6 in human immortalized hepatocytes (HepIms) for further investigations. The HepIms were transfected with an ABCC6-specific clustered regulatory interspaced short palindromic repeat (CRISPR-Cas9) genome-editing plasmid, resulting in the identification of a heterozygous (ht[ABCC6]HepIm) and a compound heterozygous (cht[ABCC6]HepIm) clone. These clones were analyzed for key markers associated with the PXE pathobiochemistry. Hints of impaired lipid trafficking, defects in the extracellular matrix remodeling, the induction of calcification inhibitor expression, and the down regulation of senescence and inflammatory markers in ABCC6-deficienct HepIms were found. Our ABCC6 knock-out model of HepIms provides a valuable tool for studying the metabolic characteristics of PXE in vitro. The initial analysis of the clones mirrors various features of the PXE pathobiochemistry and provides an outlook on future research approaches.},
}

Humans
*Hepatocytes/metabolism
*CRISPR-Cas Systems/genetics
*Multidrug Resistance-Associated Proteins/deficiency/genetics/metabolism
*Gene Editing/methods
Pseudoxanthoma Elasticum/genetics/pathology/metabolism

@article {pmid40277547,
year = {2025},
author = {Xue, B and Qiao, B and Jia, L and Chi, J and Su, M and Song, Y and Du, J},
title = {A Sensitive and Fast microRNA Detection Platform Based on CRlSPR-Cas12a Coupled with Hybridization Chain Reaction and Photonic Crystal Microarray.},
journal = {Biosensors},
volume = {15},
number = {4},
pages = {},
pmid = {40277547},
issn = {2079-6374},
support = {2021YFA0805100//National Key Research and Development Program of China/ ; 82070407//National Natural Science Foundation of China/ ; 82200459//National Natural Science Foundation of China/ ; },
mesh = {*MicroRNAs/analysis ; Nucleic Acid Hybridization ; CRISPR-Cas Systems ; *Biosensing Techniques ; Oligonucleotide Array Sequence Analysis ; Humans ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {Changes in microRNA (miRNA) levels are closely associated with the pathological processes of many diseases. The sensitive and fast detection of miRNAs is critical for diagnosis and prognosis. Here, we report a platform employing CRISPR/Cas12a to recognize and report changes in miRNA levels while avoiding complex multi-thermal cycling procedures. A non-enzyme-dependent hybridization chain reaction (HCR) was used to convert the miRNA signal into double-stranded DNA, which contained a Cas12a activation sequence. The target sequence was amplified simply and isothermally, enabling the test to be executed at a constant temperature of 37 °C. The detection platform had the capacity to measure concentrations down to the picomolar level, and the target miRNA could be distinguished at the nanomolar level. By using photonic crystal microarrays with a stopband-matched emission spectrum of the fluorescent-quencher modified reporter, the fluorescence signal was moderately enhanced to increase the sensitivity. With this enhancement, analyzable fluorescence results were obtained in 15 min. The HCR and Cas12a cleavage processes could be conducted in a single tube by separating the two procedures into the bottom and the cap. We verified the sensitivity and specificity of this one-pot system, and both were comparable to those of the two-step method. Overall, our study produced a fast and sensitive miRNA detection platform based on a CRISPR/Cas12a system and enzyme-free HCR amplification. This platform may serve as a potential solution for miRNA detection in clinical practice.},
}

*MicroRNAs/analysis
Nucleic Acid Hybridization
CRISPR-Cas Systems
*Biosensing Techniques
Oligonucleotide Array Sequence Analysis
Humans
Bacterial Proteins
Endodeoxyribonucleases
CRISPR-Associated Proteins

@article {pmid40277544,
year = {2025},
author = {Chao, A and Hu, Q and Yin, K},
title = {A Label-Free CRISPR/Cas12a-G4 Biosensor Integrated with FTA Card for Detection of Foodborne Pathogens.},
journal = {Biosensors},
volume = {15},
number = {4},
pages = {},
pmid = {40277544},
issn = {2079-6374},
support = {ZDYF2022SHFZ321//Hainan Province Science and Technology Special Fund/ ; X2020-02-08-00-12-F01110//Shanghai Agriculture Applied Technology Development Program/ ; 202340069//Project of Shanghai Municipal Health Commission/ ; 22104090//National Natural Science Foundation of China/ ; 22ZR1436200//Natural Science Foundation of Shanghai/ ; NA//Shanghai Oriental Talent Program (Youth Project)/ ; 22474076//National Natural Science Foundation of China/ ; },
mesh = {*Biosensing Techniques ; *Escherichia coli O157/isolation & purification ; *CRISPR-Cas Systems ; *Food Microbiology ; G-Quadruplexes ; Limit of Detection ; },
abstract = {CRISPR/Cas-based diagnostics offer unparalleled specificity, but their reliance on fluorescently labeled probes and complex nucleic acid extraction limits field applicability. To tackle this problem, we have developed a label-free, equipment-free platform integrating FTA card-based extraction, CRISPR/Cas12a, and pre-folded G-quadruplex (G4)-Thioflavin T (ThT) signal reporter. This system eliminates costly fluorescent labeling by leveraging G4-ThT structural binding for visible fluorescence output, while FTA cards streamline nucleic acid isolation without centrifugation. Achieving a limit of detection (LOD) to 10[1] CFU/mL for Escherichia coli O157:H7 in spiked food samples, the platform demonstrated 100% concordance with qPCR and standard fluorescent probe-based CRISPR/Cas12a system. Its simplicity, minimal equipment (portable heating/imaging), and cost-effectiveness make it a revolutionary tool for detecting foodborne pathogens in resource-limited environments.},
}

*Biosensing Techniques
*Escherichia coli O157/isolation & purification
*CRISPR-Cas Systems
*Food Microbiology
G-Quadruplexes
Limit of Detection

@article {pmid40277382,
year = {2025},
author = {Xu, D and Wu, Q and Yang, F and Zhang, Q and Jiang, Q and Zeng, X and Zhang, Y and Lv, T and Wang, J and Li, F},
title = {Fast-Flu: RT-RPA-CRISPR/Cas12a assisted one-step platform for rapid influenza B virus detection.},
journal = {Microbiology spectrum},
volume = {},
number = {},
pages = {e0036525},
doi = {10.1128/spectrum.00365-25},
pmid = {40277382},
issn = {2165-0497},
abstract = {UNLABELLED: Influenza B virus (Flu B) is a prevalent respiratory pathogen responsible for seasonal influenza epidemics. Despite its clinical significance, there remains a lack of rapid and accurate diagnostic methods for Flu B detection. In this study, we developed a novel Flu B detection system, named Fast-Flu, by integrating reverse transcription recombinase polymerase amplification (RT-RPA) with the clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein (Cas) system (CRISPR/Cas). Through optimization of reaction temperature and adjustment of Cas12a concentrations, we successfully balanced RPA amplification and CRISPR/Cas12a trans-cleavage activity, enabling the establishment of a one-step detection system. The one-step Fast-Flu system demonstrated the ability to specifically identify Flu B within 45 min, with a limit of detection of 58 copies per test. It eliminates the need for uncapping operations and minimizes the risk of cross-contamination, without cross-reactivity with other pathogens. When evaluated using 101 clinical throat swab samples, the one-step Fast-Flu system achieved a sensitivity of 56.25% and a specificity of 100% compared to the PCR-based method, with an overall concordance rate of 93.06% (94/101). The development of this one-step RT-RPA-CRISPR/Cas12a system represents a significant advancement in the rapid, convenient, and accurate detection of Flu B, highlighting its potential for clinical diagnosis. Furthermore, with future technical improvements to enhance sensitivity, this one-step RT-RPA-CRISPR assay holds promise as a versatile tool for the rapid nucleic acid detection of other RNA viruses.

IMPORTANCE: Influenza B virus (Flu B) is a significant global health concern, and rapid, accurate pathogen diagnosis is crucial for effective influenza prevention and control. The integration of isothermal amplification methods with the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) system has achieved high sensitivity and specificity for nucleic acid detection. Although CRISPR/Cas-based systems have been developed for influenza detection, existing platforms require the transfer of amplified products into the CRISPR/Cas12a detection system through uncapping operations, which increases the risk of cross-contamination. In this study, we developed a one-step reverse transcription recombinase polymerase amplification-CRISPR/Cas12a Flu B detection method using a one-pot detection system. By optimizing the reaction temperature and Cas12a concentration, we achieved a streamlined and contamination-free workflow. This innovative approach not only improves Flu B detection but also serves as a valuable reference for constructing CRISPR/Cas systems for the detection of other pathogens and targets, paving the way for broader applications in molecular diagnostics.},
}

@article {pmid40277083,
year = {2025},
author = {Dillard, KE and Zhang, H and Dubbs, LZ and Chou, CW and Terrace, C and Javanmardi, K and Kim, W and Forsberg, KJ and Finkelstein, IJ},
title = {Mechanism of Cas9 inhibition by AcrIIA11.},
journal = {Nucleic acids research},
volume = {53},
number = {8},
pages = {},
pmid = {40277083},
issn = {1362-4962},
support = {F-1808//Welch Foundation/ ; R01GM124141/GF/NIH HHS/United States ; //Searle Scholars Award/ ; //University of Texas Southwestern Medical Center/ ; //Howard Hughes Medical Institute Emerging Pathogens Initiative/ ; },
mesh = {*CRISPR-Associated Protein 9/metabolism/antagonists & inhibitors/genetics/chemistry ; Humans ; *CRISPR-Cas Systems/genetics ; Staphylococcus aureus/genetics/enzymology ; Streptococcus pyogenes/genetics/enzymology ; DNA Cleavage ; *Bacterial Proteins/metabolism/genetics/antagonists & inhibitors ; Francisella/genetics/enzymology ; DNA/metabolism ; HEK293 Cells ; },
abstract = {Mobile genetic elements evade CRISPR-Cas adaptive immunity by encoding anti-CRISPR proteins (Acrs). Acrs inactivate CRISPR-Cas systems via diverse mechanisms but generally coevolve with a narrow subset of Cas effectors that share high sequence similarity. Here, we demonstrate that AcrIIA11 inhibits Streptococcus pyogenes (Sp), Staphylococcus aureus (Sa), and Francisella novicida (Fn) Cas9s in vitro and in human cells. Single-molecule imaging reveals that AcrIIA11 hinders SaCas9 target search by reducing its diffusion on nonspecific DNA. DNA cleavage is inhibited because the AcrIIA11:SaCas9 complex binds to protospacer adjacent motif (PAM)-rich off-target sites, preventing SaCas9 from reaching its target. AcrIIA11 also greatly slows down DNA cleavage after SaCas9 reaches its target site. A negative-stain electron microscopy reconstruction of an AcrIIA11:SaCas9 RNP complex reveals that the heterodimer assembles with a 1:1 stoichiometry. Physical AcrIIA11-Cas9 interactions across type IIA and IIB Cas9s correlate with nuclease inhibition and support its broad-spectrum activity. These results add a kinetic inhibition mechanism to the phage-CRISPR arms race.},
}

*CRISPR-Associated Protein 9/metabolism/antagonists & inhibitors/genetics/chemistry
Humans
*CRISPR-Cas Systems/genetics
Staphylococcus aureus/genetics/enzymology
Streptococcus pyogenes/genetics/enzymology
DNA Cleavage
*Bacterial Proteins/metabolism/genetics/antagonists & inhibitors
Francisella/genetics/enzymology
DNA/metabolism
HEK293 Cells

@article {pmid40275527,
year = {2025},
author = {Chen, Y and Li, M and Liu, X and Duan, Q and Xiao, L and Wang, L and Huang, C and Song, H and Cao, Y},
title = {Establishment of CRISPR-STAR System to Realise Simultaneous Transcriptional Activation and Repression in Yarrowia lipolytica.},
journal = {Microbial biotechnology},
volume = {18},
number = {4},
pages = {e70151},
pmid = {40275527},
issn = {1751-7915},
support = {2021YFC2104400//National Key Research and Development Program of China/ ; NSFC22478294//the National Natural Science Foundation of China/ ; NSFC22078240//the National Natural Science Foundation of China/ ; },
mesh = {*Yarrowia/genetics/metabolism ; *Transcriptional Activation ; *Gene Expression Regulation, Fungal ; *CRISPR-Cas Systems ; Promoter Regions, Genetic ; Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {The ability to regulate gene expression in multiple directions is crucial to maximise the production of microbial cell factories. However, the lack of a regulatory tool that can simultaneously activate and repress multiple genes restricts the manipulation diversity of Yarrowia lipolytica, which is an industrial workhorse for bioproduction. To address this issue, we developed a CRISPR scaffold RNAs (scRNAs)-mediated transcriptional activation and repression (CRISPR-STAR) platform. Firstly, we evaluated different methods for bidirectional regulation using CRISPR on both endogenous and synthetic promoters in Y. lipolytica, and chose the utilisation of orthogonal scRNAs to recruit activation and inhibition domains. Secondly, CRISPR-STAR was optimised by the introduction of alternative dCas proteins, scRNA structures and activators. 2.6-fold and 54.9-fold activation were achieved for synthetic and endogenous promoters, respectively, when the VPR transcriptional activator was recruited via MS2 hairpin. The repression of several genes was successfully achieved, with repression levels ranging from 3% to 32%, when the MXI1 transcriptional repressor was recruited via PP7 hairpin. Finally, CRISPR-STAR was applied to enhance fatty alcohol production by activating the FAR gene (encodes fatty acyl-CoA reductase) and repression of the PEX10 gene (encodes an integral membrane protein required for peroxisome biogenesis and matrix protein import). Compared to the non-targeting control, the bidirectionally regulated strain showed a 55.7% increase in yield to 778.8 mg/L. Our findings demonstrate that the CRISPR-STAR platform enables multi-mode regulation of genes, offering engineering opportunities to improve the productive performance of Y. lipolytica.},
}

*Yarrowia/genetics/metabolism
*Transcriptional Activation
*Gene Expression Regulation, Fungal
*CRISPR-Cas Systems
Promoter Regions, Genetic
Clustered Regularly Interspaced Short Palindromic Repeats

@article {pmid40275470,
year = {2025},
author = {Kelly, G and Plesser, E and Bdolach, E and Arroyave, M and Belausov, E and Doron-Faigenboim, A and Rozen, A and Zemach, H and Zach, YY and Goldenberg, L and Arad, T and Yaniv, Y and Sade, N and Sherman, A and Eyal, Y and Carmi, N},
title = {In planta genome editing in citrus facilitated by co-expression of CRISPR/Cas and developmental regulators.},
journal = {The Plant journal : for cell and molecular biology},
volume = {122},
number = {2},
pages = {e70155},
pmid = {40275470},
issn = {1365-313X},
mesh = {*Citrus/genetics/growth & development ; *Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; Plants, Genetically Modified/genetics ; *Genome, Plant/genetics ; Agrobacterium tumefaciens/genetics ; Plant Shoots/genetics/growth & development ; },
abstract = {Recent advances in the field of genome editing offer a promising avenue for targeted trait improvements in fruit trees. However, the predominant method taken for genome editing in citrus (and other fruit trees) involves the time-consuming tissue culture approach, thereby prolonging the overall citrus breeding process and subjecting it to the drawbacks associated with somaclonal variation. In this study, we introduce an in planta approach for genome editing in soil-grown citrus plants via direct transformation of young seedlings. Our editing system, abbreviated here as IPGEC (in planta genome editing in citrus), is designed to transiently co-express three key gene groups in citrus tissue via Agrobacterium tumefaciens: (i) a genome-editing catalytic group, (ii) a shoot induction and regeneration group, and (iii) a T-DNA enhanced delivery group. This integrated system significantly improves de novo shoot induction and regeneration efficiency of edited tissue. By incorporating single-guides RNA's (sgRNA's) targeting the carotenoid biosynthetic gene PHYTOENE DESATURASE (CsPDS), the IPGEC system effectively produced mutated albino shoots, confirming its ability to generate homozygous/biallelic genome-edited plants. By using high throughput screening, we provide evidence that transgene-free genome-edited plants could be obtained following the IPGEC approach. Our findings further suggest that the efficiency of specific developmental regulators in inducing transformation and regeneration rates may be cultivar-specific and therefore needs to be optimized per cultivar. Finally, targeted breeding for specific trait improvements in already successful cultivars is likely to revolutionize fruit tree breeding and will pave the way for accelerating the development of high-quality citrus cultivars.},
}

*Citrus/genetics/growth & development
*Gene Editing/methods
*CRISPR-Cas Systems/genetics
Plants, Genetically Modified/genetics
*Genome, Plant/genetics
Agrobacterium tumefaciens/genetics
Plant Shoots/genetics/growth & development

@article {pmid40275435,
year = {2025},
author = {Yang, Z and Bai, W and Guo, G and Huang, S and Wang, Y and Zhou, Y and Zhang, Y and Sun, J},
title = {The Q-interacted protein QIP3 recruits TaTPL to regulate spike architecture in wheat.},
journal = {The Plant journal : for cell and molecular biology},
volume = {122},
number = {2},
pages = {e70149},
doi = {10.1111/tpj.70149},
pmid = {40275435},
issn = {1365-313X},
support = {32472096//National Natural Science Foundation of China Grant/ ; 2023YFF1000402//National Key Research and Development Program of China/ ; //Innovation Program of the Chinese Academy of Agricultural Sciences/ ; },
mesh = {*Triticum/genetics/metabolism/anatomy & histology/growth & development ; *Plant Proteins/metabolism/genetics ; *Transcription Factors/metabolism/genetics ; Gene Expression Regulation, Plant ; CRISPR-Cas Systems ; },
abstract = {Spike architecture is a critical determinant of grain yield in wheat; yet the regulatory mechanisms remain poorly understood. Here, we demonstrate that the AP2 transcription factor Q directly represses the expression of TaMYB30-6A, a gene associated with spike length in wheat. We further identify QIP3 as a Q-interacting protein harboring an N-terminal EAR motif. Simultaneously, we reveal that QIP3 exhibits transcriptional repression activity, dependent on the EAR motif, and physically interacts with the transcriptional corepressor TaTPL. Importantly, the clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9)-generated qip3-aabbdd mutants exhibit reduced plant height and increased spike length phenotypes. Furthermore, RNA-seq and RT-qPCR assays show that QIP3 negatively regulates the expression of the Q target gene TaMYB30-6A in wheat. Collectively, we propose that the EAR motif-containing QIP3 interacts with Q to regulate spike architecture by recruiting the transcriptional corepressor TaTPL in wheat.},
}

*Triticum/genetics/metabolism/anatomy & histology/growth & development
*Plant Proteins/metabolism/genetics
*Transcription Factors/metabolism/genetics
Gene Expression Regulation, Plant
CRISPR-Cas Systems

@article {pmid40274952,
year = {2025},
author = {Zhang, Z and Abreu, B and Brothwood, JL and Alexander, J and Sims, MJ and Lyons, JF and Munck, JM and Hindley, CJ},
title = {The identification of functional regions of MEK1 using CRISPR tiling screens.},
journal = {Communications biology},
volume = {8},
number = {1},
pages = {656},
pmid = {40274952},
issn = {2399-3642},
mesh = {*MAP Kinase Kinase 1/genetics/antagonists & inhibitors/metabolism/chemistry ; Humans ; *CRISPR-Cas Systems ; Protein Kinase Inhibitors/pharmacology ; Cell Line, Tumor ; Drug Resistance, Neoplasm/genetics ; Mutation ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Drug Discovery/methods ; Cell Survival/drug effects ; },
abstract = {CRISPR tiling screen is a powerful tool to identify protein regions relevant to its biological function. Understanding the functional relevance of the regions of target protein is of great help for structure-based drug discovery. Studying the drug resistance mechanisms of small-molecule inhibitors is important for the development and clinical application of the compounds. Using MEK1 and MEK inhibitors as example here, we demonstrate the utility of CRISPR tiling to identify regions essential for cancer cell viability and regions where mutations are resistant to MEK inhibitors. We study the drug resistance mechanisms of the regions and discussed the potential, as well as limitations, of applying the technology to drug development. Our findings demonstrate the value and prompt the utilization of CRISPR tiling technology in structure-based drug discovery.},
}

*MAP Kinase Kinase 1/genetics/antagonists & inhibitors/metabolism/chemistry
Humans
*CRISPR-Cas Systems
Protein Kinase Inhibitors/pharmacology
Cell Line, Tumor
Drug Resistance, Neoplasm/genetics
Mutation
*Clustered Regularly Interspaced Short Palindromic Repeats
Drug Discovery/methods
Cell Survival/drug effects

@article {pmid40274871,
year = {2025},
author = {Chai, R and Guo, J and Yang, C and Zhu, D and Li, T and Yang, W and Liu, X and Chen, X and Huang, S and Wang, H and Yao, X and Gao, Y and Qiu, L},
title = {Enhanced chemotaxis and degradation of nonylphenol in Pseudoxanthomonas mexicana via CRISPR-mediated receptor modification.},
journal = {Scientific reports},
volume = {15},
number = {1},
pages = {14296},
pmid = {40274871},
issn = {2045-2322},
support = {25B610014//the Key scientific research projects of colleges and universities in Henan Province/ ; GTTXM202412//the Consortium Construction Research Project of China Biotechnology Vocational Education Teaching Steering Committee/ ; 2403107//the Kaifeng Science and Technology Development Project/ ; 252102320066//the Key Research and Development Special Project of Henan Provincial Science and Technology/ ; },
mesh = {*Chemotaxis/genetics ; *Phenols/metabolism ; Biodegradation, Environmental ; Phylogeny ; Bacterial Proteins/genetics/metabolism ; Wastewater/microbiology ; Sewage/microbiology ; *CRISPR-Cas Systems ; },
abstract = {In this study, a novel nonylphenol (NP)-degrading bacterium, Pseudoxanthomonas mexicana CH, was isolated from wastewater treatment plant effluent. Phylogenetic analysis showed its close relationship to P. mexicana AMX 26BT. The strain displayed chemotaxis toward NP, with Mcp24 as the key chemoreceptor. The Mcp24 deletion mutant (CH- 1) had weaker chemotaxis and NP degradation (over 30% lower in solution and 8% lower in sludge than the wild type). In vitro, Mcp15's C-terminal pentapeptide DWQEF was methylated by CheR. Using CRISPR, this pentapeptide was added to Mcp24 to create CH- 2. CH- 2 showed better NP chemotaxis (17% higher in plate assays and 39% higher in capillary assays) and higher NP degradation rates (23.5% and 24.2% higher in solution and sludge, respectively). These findings demonstrate that NP acts as a bacterial chemoattractant, with Mcp24 as the receptor. Enhancing Mcp24's C-terminal pentapeptide improves chemotaxis and degradation efficiency, representing a significant advancement in bioremediation by strengthening bacterial responses to pollutants.},
}

*Chemotaxis/genetics
*Phenols/metabolism
Biodegradation, Environmental
Phylogeny
Bacterial Proteins/genetics/metabolism
Wastewater/microbiology
Sewage/microbiology
*CRISPR-Cas Systems

@article {pmid40274854,
year = {2025},
author = {Balakrishnan, A and Hunziker, M and Tiwary, P and Pandey, V and Drew, D and Billker, O},
title = {A CRISPR homing screen finds a chloroquine resistance transporter-like protein of the Plasmodium oocyst essential for mosquito transmission of malaria.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {3895},
pmid = {40274854},
issn = {2041-1723},
support = {LT000131/2020-L//Human Frontier Science Program (HFSP)/ ; LT000131/2020-L//Human Frontier Science Program (HFSP)/ ; 895744//EC | EU Framework Programme for Research and Innovation H2020 | H2020 Priority Excellent Science | H2020 Marie Skłodowska-Curie Actions (H2020 Excellent Science - Marie Skłodowska-Curie Actions)/ ; 788516//EC | EC Seventh Framework Programm | FP7 Ideas: European Research Council (FP7-IDEAS-ERC - Specific Programme: "Ideas" Implementing the Seventh Framework Programme of the European Community for Research, Technological Development and Demonstration Activities (2007 to 2013))/ ; 895744//EC | Horizon 2020 Framework Programme (EU Framework Programme for Research and Innovation H2020)/ ; P2SKP3_187635//Schweizerischer Nationalfonds zur Förderung der Wissenschaftlichen Forschung (Swiss National Science Foundation)/ ; },
mesh = {*Plasmodium berghei/genetics/drug effects/metabolism ; Animals ; *Malaria/transmission/parasitology ; *Oocysts/metabolism/genetics/drug effects/growth & development ; *Protozoan Proteins/genetics/metabolism ; CRISPR-Cas Systems ; Gene Editing/methods ; Mice ; Mosquito Vectors/parasitology ; Drug Resistance/genetics ; Chloroquine/pharmacology ; Female ; Anopheles/parasitology ; },
abstract = {Genetic screens with barcoded PlasmoGEM vectors have identified thousands of Plasmodium berghei gene functions in haploid blood stages, gametocytes and liver stages. However, the formation of diploid cells by fertilisation has hindered similar research on the parasites' mosquito stages. In this study, we develop a scalable genetic system that uses barcoded gene targeting vectors equipped with a CRISPR-mediated homing mechanism to generate homozygous loss-of-function mutants after one parent introduces a modified allele into the zygote. To achieve this, we use vectors additionally expressing a target gene specific gRNA. When integrated into one of the parental alleles it directs Cas9 to the intact allele after fertilisation, leading to its disruption. This homing strategy is 90% effective at generating homozygous gene editing of a fluorescence-tagged reporter locus in the oocyst. A pilot screen identifies PBANKA_0916000 as a chloroquine resistance transporter-like protein (CRTL) essential for oocyst growth and sporogony, pointing to an unexpected importance for malaria transmission of the poorly understood digestive vacuole of the oocyst that contains hemozoin granules. Homing screens provide a method for the systematic discovery of malaria transmission genes whose first essential functions are after fertilisation in the bloodmeal, enabling their potential as targets for transmission-blocking interventions to be assessed.},
}

*Plasmodium berghei/genetics/drug effects/metabolism
Animals
*Malaria/transmission/parasitology
*Oocysts/metabolism/genetics/drug effects/growth & development
*Protozoan Proteins/genetics/metabolism
CRISPR-Cas Systems
Gene Editing/methods
Mice
Mosquito Vectors/parasitology
Drug Resistance/genetics
Chloroquine/pharmacology
Female
Anopheles/parasitology

@article {pmid40274667,
year = {2025},
author = {Mukherjee, A and Samanta, S and Das, S and Haque, MZ and Jana, PS and Samanta, I and Kar, I and Das, S and Nanda, PK and Thomas, P and Dandapat, P},
title = {Leveraging CRISPR-Cas-Enhanced Isothermal Amplification Tools for Quick Identification of Pathogens Causing Livestock Diseases.},
journal = {Current microbiology},
volume = {82},
number = {6},
pages = {260},
pmid = {40274667},
issn = {1432-0991},
support = {STBT-11012(27)/5/2024-ST SEC//Department of Science and Technology, Government of West Bengal/ ; },
mesh = {Animals ; *Nucleic Acid Amplification Techniques/methods ; *Livestock/microbiology/virology ; *CRISPR-Cas Systems ; *Molecular Diagnostic Techniques/methods ; *Animal Diseases/diagnosis/microbiology/virology ; *Bacteria/genetics/isolation & purification ; },
abstract = {Prompt and accurate diagnosis of infectious pathogens of livestock origin is of utmost importance for epidemiological surveillance and effective therapeutic strategy formulation. Among various methods, nucleic acid-based detection of pathogens is the most sensitive and specific; but the majority of these assays need expensive equipment and skilled workers. Due to the rapid advancement of clustered regularly interspaced short palindromic repeats-CRISPR-associated protein (CRISPR-Cas)-based nucleic acid detection methods, these are now being widely used for pathogen detection. CRISPR-Cas is a bacterial counterpart of "adaptive immunity", generally used for editing genome. Many CRISPR systems have been modified for nucleic acid detection due to their excellent selectivity in detecting DNA and RNA sequences. The combination of CRISPR with suitable isothermal amplification technologies has made it more sensitive, specific, versatile, and reproducible for the detection of pathogen nucleic acids at the point of care. Amplification of pathogen nucleic acid by isothermal amplification followed by CRISPR-Cas-based detection has several advantages, including short sample-to-answer times and no requirement for laboratory set-up. They are also significantly less expensive than the existing nucleic acid detection methods. This review focuses on the recent trends in the use of this precision diagnostic method for diagnosis of a wide range of animal pathogens with or without zoonotic potential, particularly various isothermal amplification strategies, and visualization methods for sensing bacteria, viruses, and parasites of veterinary and public health importance.},
}

Animals
*Nucleic Acid Amplification Techniques/methods
*Livestock/microbiology/virology
*CRISPR-Cas Systems
*Molecular Diagnostic Techniques/methods
*Animal Diseases/diagnosis/microbiology/virology
*Bacteria/genetics/isolation & purification

@article {pmid40273916,
year = {2025},
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 = {Molecular cell},
volume = {85},
number = {9},
pages = {1730-1742.e9},
doi = {10.1016/j.molcel.2025.03.024},
pmid = {40273916},
issn = {1097-4164},
mesh = {*CRISPR-Cas Systems ; *Gene Editing/methods ; *RNA, Guide, CRISPR-Cas Systems/genetics/metabolism ; *Streptococcus pyogenes/genetics/enzymology ; Humans ; *CRISPR-Associated Protein 9/genetics/metabolism ; HEK293 Cells ; *Bacterial Proteins/genetics/metabolism ; *DNA/genetics/metabolism ; Nucleotide Motifs ; },
abstract = {RNA-guided CRISPR-Cas enzymes initiate programmable genome editing by recognizing a ∼20-base-pair DNA sequence next 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 Streptococcus pyogenes Cas9 (SpyCas9) 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.},
}

*CRISPR-Cas Systems
*Gene Editing/methods
*RNA, Guide, CRISPR-Cas Systems/genetics/metabolism
*Streptococcus pyogenes/genetics/enzymology
Humans
*CRISPR-Associated Protein 9/genetics/metabolism
HEK293 Cells
*Bacterial Proteins/genetics/metabolism
*DNA/genetics/metabolism
Nucleotide Motifs

@article {pmid40273800,
year = {2025},
author = {Zheng, M and Bao, N and Wang, Z and Song, C and Jin, Y},
title = {Alternative splicing in autism spectrum disorder: Recent insights from mechanisms to therapy.},
journal = {Asian journal of psychiatry},
volume = {108},
number = {},
pages = {104501},
doi = {10.1016/j.ajp.2025.104501},
pmid = {40273800},
issn = {1876-2026},
abstract = {Alternative splicing (AS) is a vital and highly dynamic RNA regulatory mechanism that allows a single gene to generate multiple mRNA and protein isoforms. Dysregulation of AS has been identified as a key contributor to the pathogenesis of autism spectrum disorders (ASD). A comprehensive understanding of aberrant splicing mechanisms and their functional consequences in ASD can help uncover the molecular basis of the disorder and facilitate the development of therapeutic strategies. This review focuses on the major aberrant splicing events and key splicing regulators associated with ASD, highlighting their roles in linking defective splicing to ASD pathogenesis. In addition, a discussion of how emerging technologies, such as long-read sequencing, single-cell sequencing, spatial transcriptomics and CRISPR-Cas systems are offering novel insights into the role and mechanisms of AS in ASD is presented. Finally, the RNA splicing-based therapeutic strategies are evaluated, emphasizing their potential to address unmet clinical needs in ASD treatment.},
}

@article {pmid40273258,
year = {2025},
author = {Hayes, VM and Zhang, JT and Katz, MA and Li, Y and Kocsis, B and Brinkley, DM and Jia, N and Meeske, AJ},
title = {RNA-mediated CRISPR-Cas13 inhibition through crRNA structural mimicry.},
journal = {Science (New York, N.Y.)},
volume = {388},
number = {6745},
pages = {387-391},
doi = {10.1126/science.adr3656},
pmid = {40273258},
issn = {1095-9203},
mesh = {*Bacteriophages/genetics ; *CRISPR-Associated Proteins/chemistry/genetics ; *CRISPR-Cas Systems ; Cryoelectron Microscopy ; *Listeria/virology/genetics ; *Molecular Mimicry ; Nucleic Acid Conformation ; Plasmids/genetics ; *RNA, Viral/chemistry/genetics ; *RNA, Small Untranslated/chemistry/genetics ; },
abstract = {To circumvent CRISPR-Cas immunity, phages express anti-CRISPR factors that inhibit the expression or activities of Cas proteins. Whereas most anti-CRISPRs described to date are proteins, recently described small RNAs called RNA anti-CRISPRs (rAcrs) have sequence homology to CRISPR RNAs (crRNAs) and displace them from cognate Cas nucleases. In this work, we report the discovery of rAcrVIA1-a plasmid-encoded small RNA that inhibits the RNA-targeting CRISPR-Cas13 system in its natural host, Listeria seeligeri. We solved the cryo-electron microscopy structure of the Cas13-rAcr complex, which revealed that rAcrVIA1 adopts a fold nearly identical to crRNA despite sharing negligible sequence similarity. Collectively, our findings expand the diversity of rAcrs and reveal an example of immune antagonism through RNA structural mimicry.},
}

*Bacteriophages/genetics
*CRISPR-Associated Proteins/chemistry/genetics
*CRISPR-Cas Systems
Cryoelectron Microscopy
*Listeria/virology/genetics
*Molecular Mimicry
Nucleic Acid Conformation
Plasmids/genetics
*RNA, Viral/chemistry/genetics
*RNA, Small Untranslated/chemistry/genetics

@article {pmid40273189,
year = {2025},
author = {Akiyama, C and Sakata, S and Ono, F},
title = {Normal locomotion in zebrafish lacking the sodium channel NaV1.4 suggests that the need for muscle action potentials is not universal.},
journal = {PLoS biology},
volume = {23},
number = {4},
pages = {e3003137},
pmid = {40273189},
issn = {1545-7885},
mesh = {Animals ; *Zebrafish/physiology/genetics ; *NAV1.4 Voltage-Gated Sodium Channel/genetics/metabolism/physiology ; *Action Potentials/physiology ; *Locomotion/physiology/genetics ; Muscle Contraction/physiology ; *Zebrafish Proteins/genetics/metabolism ; Muscle, Skeletal/physiology/metabolism ; Animals, Genetically Modified ; CRISPR-Cas Systems ; Tetrodotoxin/pharmacology ; Calcium/metabolism ; },
abstract = {Extensive studies over decades have firmly established the concept that action potentials (APs) in muscles are indispensable for muscle contraction. To re-examine the significance of APs, we generated zebrafish lacking APs by editing the scn4aa and scn4ab genes, which together encode NaV1.4 (NaVDKO), using the CRISPR-Cas9 system. Surprisingly, the escape response of NaVDKOs to tactile stimuli, both in the embryonic and adult stages, was indistinguishable from that of wild-type (WT) fish. Ca2+ imaging using the calcium indicator protein GCaMP revealed that myofibers isolated from WT fish could be excited by the application of acetylcholine (ACh), even in the presence of tetrodotoxin (TTX) indicating that NaVs are dispensable for skeletal muscle contraction in zebrafish. Mathematical simulations showed that the end-plate potential was able to elicit a change in membrane potential large enough to activate the dihydropyridine receptors of the entire muscle fiber owing to the small fiber size and the disseminated distribution of neuromuscular synapses in both adults and embryos. Our data demonstrate that NaVs are not essential for muscle contraction in zebrafish and that the physiological significance of NaV1.4 in muscle is not uniform across vertebrates.},
}

Animals
*Zebrafish/physiology/genetics
*NAV1.4 Voltage-Gated Sodium Channel/genetics/metabolism/physiology
*Action Potentials/physiology
*Locomotion/physiology/genetics
Muscle Contraction/physiology
*Zebrafish Proteins/genetics/metabolism
Muscle, Skeletal/physiology/metabolism
Animals, Genetically Modified
CRISPR-Cas Systems
Tetrodotoxin/pharmacology
Calcium/metabolism